Proteopedia - User contributions [en]

Sandbox WWC9

2016-05-13T17:20:48Z

Dana Emmert:

== The µ-opioid Receptor (4DKL) ==

Opioids are highly specific endogenous and exogenous ligands in function as a means of analgesia and bind to a family of receptor proteins known as the opioid receptors (ORs). The three types of opioid receptors (µ-, κ-, δ-, respectively) belong to the protein superfamily G-protein coupled receptors (GCPRs); transmembrane proteins consisting of 7 membrane-spanning alpha-helical structures in which the extracellular ligand binding pocket exists. The µ-OR, exists primarily in the brain, spinal cord, and in the periphery. (1)

<Structure load='4dkl' size='450' frame='true' align='right' caption='4dkl' scene='Insert optional scene name here' />
<scene name='72/727893/C_to_n_rainbow/1'>See N to C sequence</scene>
{{Template:ColorKey_Amino2CarboxyRainbow}}

Here we can see a <scene name='72/727893/Hydrophobicity/1'>hydrophobicity display</scene> for this protein. Hydrophobic portions of the protein appear in grey, whereas polar potions appear in pink.
{{Template:ColorKey_Hydrophobic}}{{Template:ColorKey_Polar}}

== Structure ==
=== Architecture ===
The µ-OR crystalized is that of the Mus musculus and has been given the protein database (PDB) identification 4DKL. This protein was crystalized on a 2.8 Å resolution with a bound irreversible morphinan antagonist β-funaltrexamine (β-FNA). The crystal structure of the protein shows the seven transmembrane helices characteristic of GPCRs that are connected by three extracellular loops and three intracellular loops. The receptors are arranges in dimers closely associated with transmembrane (TM) helices 5 and 6 (See image). The receptor also displays alternating aqueous and lipidic layers corresponding to transmembrane domains. (2)
=== Binding site ===
The µ-OR, unlike most GPCRs that possess a buried binding site, contains an open solvent filled binding site of approximately 2173 Å3 and an accessible pocket area of 237Å2. (2,3)

== Function ==

== Ligand Subtypes ==

== References ==
1. Julian, R., Advokat, C., & Comaty, J. (2011). A Primer of Drug Action (Twelfth Edition). Worth Publishers.
2. Manglic, A.; Kruse , A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Crystal Structure Of the µ-Opioid Receptor Bound to a Morphinan Antagonist. Nature. 2012, 485, 321–326.

3. Cui, X.; Yeliseev, A.; Liu, R. Ligand Interaction, Binding Site And G Protein Activation of the Mu Opioid Receptor. Eur J Pharmacol. 2013, 702, 309–315.

4. Al-Hasani, R.; Bruchas, M. R. Molecular Mechanisms Of Opioid Receptor-Dependent Signaling and Behavior. Anesthesiology . 2011, 115, 1363–1381.

Sandbox WWC9

2016-05-13T17:18:33Z

Dana Emmert:

== The µ-opioid Receptor (4DKL) ==

Opioids are highly specific endogenous and exogenous ligands in function as a means of analgesia and bind to a family of receptor proteins known as the opioid receptors (ORs). The three types of opioid receptors (µ-, κ-, δ-, respectively) belong to the protein superfamily G-protein coupled receptors (GCPRs); transmembrane proteins consisting of 7 membrane-spanning alpha-helical structures in which the extracellular ligand binding pocket exists. The µ-OR, exists primarily in the brain, spinal cord, and in the periphery. (1)

<Structure load='4dkl' size='450' frame='true' align='right' caption='4dkl' scene='Insert optional scene name here' />
<scene name='72/727893/C_to_n_rainbow/1'>See N to C sequence</scene>
{{Template:ColorKey_Amino2CarboxyRainbow}}

Here we can see a <scene name='72/727893/Hydrophobicity/1'>hydrophobicity display</scene> for this protein. Hydrophobic portions of the protein appear in grey, whereas polar potions appear in pink.
{{Template:ColorKey_Hydrophobic}}{{Template:ColorKey_Polar}}

== Structure ==
=== Architecture ===
The µ-OR crystalized is that of the Mus musculus and has been given the protein database (PDB) identification 4DKL. This protein was crystalized on a 2.8 Å resolution with a bound irreversible morphinan antagonist β-funaltrexamine (β-FNA). The crystal structure of the protein shows the seven transmembrane helices characteristic of GPCRs that are connected by three extracellular loops and three intracellular loops. The receptors are arranges in dimers closely associated with transmembrane (TM) helices 5 and 6 (See image). The receptor also displays alternating aqueous and lipidic layers corresponding to transmembrane domains. (2)
Binding site:
The µ-OR, unlike most GPCRs that possess a buried binding site, contains an open solvent filled binding site of approximately 2173 Å3 and an accessible pocket area of 237Å2. (2,3)

== References ==
1. Julian, R., Advokat, C., & Comaty, J. (2011). A Primer of Drug Action (Twelfth Edition). Worth Publishers.
2. Manglic, A.; Kruse , A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Crystal Structure Of the µ-Opioid Receptor Bound to a Morphinan Antagonist. Nature. 2012, 485, 321–326.

3. Cui, X.; Yeliseev, A.; Liu, R. Ligand Interaction, Binding Site And G Protein Activation of the Mu Opioid Receptor. Eur J Pharmacol. 2013, 702, 309–315.

4. Al-Hasani, R.; Bruchas, M. R. Molecular Mechanisms Of Opioid Receptor-Dependent Signaling and Behavior. Anesthesiology . 2011, 115, 1363–1381.

Sandbox WWC9

2016-05-13T17:12:17Z

Dana Emmert:

== The µ-opioid Receptor (4DKL) ==

== Introduction ==
Opioids are highly specific endogenous and exogenous ligands in function as a means of analgesia and bind to a family of receptor proteins known as the opioid receptors (ORs). The three types of opioid receptors (µ-, κ-, δ-, respectively) belong to the protein superfamily G-protein coupled receptors (GCPRs); transmembrane proteins consisting of 7 membrane-spanning alpha-helical structures in which the extracellular ligand binding pocket exists. The µ-OR, exists primarily in the brain, spinal cord, and in the periphery. (1)

<Structure load='4dkl' size='450' frame='true' align='right' caption='4dkl' scene='Insert optional scene name here' />
<scene name='72/727893/C_to_n_rainbow/1'>See N to C sequence</scene>
{{Template:ColorKey_Amino2CarboxyRainbow}}

Here we can see a <scene name='72/727893/Hydrophobicity/1'>hydrophobicity display</scene> for this protein. Hydrophobic portions of the protein appear in grey, whereas polar potions appear in pink.
{{Template:ColorKey_Hydrophobic}}{{Template:ColorKey_Polar}}

== Structure ==
=== Architecture ===
The µ-OR crystalized is that of the Mus musculus and has been given the protein database (PDB) identification 4DKL. This protein was crystalized on a 2.8 Å resolution with a bound irreversible morphinan antagonist β-funaltrexamine (β-FNA). The crystal structure of the protein shows the seven transmembrane helices characteristic of GPCRs that are connected by three extracellular loops and three intracellular loops. The receptors are arranges in dimers closely associated with transmembrane (TM) helices 5 and 6 (See image). The receptor also displays alternating aqueous and lipidic layers corresponding to transmembrane domains. (2)
Binding site:
The µ-OR, unlike most GPCRs that possess a buried binding site, contains an open solvent filled binding site of approximately 2173 Å3 and an accessible pocket area of 237Å2. (2,3)

== References ==
1. Julian, R., Advokat, C., & Comaty, J. (2011). A Primer of Drug Action (Twelfth Edition). Worth Publishers.
2. Manglic, A.; Kruse , A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Crystal Structure Of the µ-Opioid Receptor Bound to a Morphinan Antagonist. Nature. 2012, 485, 321–326.

3. Cui, X.; Yeliseev, A.; Liu, R. Ligand Interaction, Binding Site And G Protein Activation of the Mu Opioid Receptor. Eur J Pharmacol. 2013, 702, 309–315.

4. Al-Hasani, R.; Bruchas, M. R. Molecular Mechanisms Of Opioid Receptor-Dependent Signaling and Behavior. Anesthesiology . 2011, 115, 1363–1381.

Sandbox WWC9

2016-05-13T16:52:31Z

Dana Emmert:

== The µ-opioid Receptor (4DKL) ==

== Introduction ==
Opioids are highly specific endogenous and exogenous ligands in function as a means of analgesia and bind to a family of receptor proteins known as the opioid receptors (ORs). The three types of opioid receptors (µ-, κ-, δ-, respectively) belong to the protein superfamily G-protein coupled receptors (GCPRs); transmembrane proteins consisting of 7 membrane-spanning alpha-helical structures in which the extracellular ligand binding pocket exists. The µ-OR, exists primarily in the brain, spinal cord, and in the periphery. (1)

<Structure load='4dkl' size='450' frame='true' align='right' caption='4dkl' scene='Insert optional scene name here' />
<scene name='72/727893/C_to_n_rainbow/1'>See N to C sequence</scene>
{{Template:ColorKey_Amino2CarboxyRainbow}}

Here we can see a <scene name='72/727893/Hydrophobicity/1'>hydrophobicity display</scene> for this protein. Hydrophobic portions of the protein appear in grey, whereas polar potions appear in pink.
{{Template:ColorKey_Hydrophobic}}{{Template:ColorKey_Polar}}

== Structure ==
Architecture:
The µ-OR crystalized is that of the Mus musculus and has been given the protein database (PDB) identification 4DKL. This protein was crystalized on a 2.8 Å resolution with a bound irreversible morphinan antagonist β-funaltrexamine (β-FNA). The crystal structure of the protein shows the seven transmembrane helices characteristic of GPCRs that are connected by three extracellular loops and three intracellular loops. The receptors are arranges in dimers closely associated with transmembrane (TM) helices 5 and 6 (See image). The receptor also displays alternating aqueous and lipidic layers corresponding to transmembrane domains. (2)
Binding site:
The µ-OR, unlike most GPCRs that possess a buried binding site, contains an open solvent filled binding site of approximately 2173 Å3 and an accessible pocket area of 237Å2. (2,3)

== References ==
1. Julian, R., Advokat, C., & Comaty, J. (2011). A Primer of Drug Action (Twelfth Edition). Worth Publishers.
2. Manglic, A.; Kruse , A. C.; Kobilka, T. S.; Thian, F. S.; Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B. K.; Granier, S. Crystal Structure Of the µ-Opioid Receptor Bound to a Morphinan Antagonist. Nature. 2012, 485, 321–326.

3. Cui, X.; Yeliseev, A.; Liu, R. Ligand Interaction, Binding Site And G Protein Activation of the Mu Opioid Receptor. Eur J Pharmacol. 2013, 702, 309–315.

4. Al-Hasani, R.; Bruchas, M. R. Molecular Mechanisms Of Opioid Receptor-Dependent Signaling and Behavior. Anesthesiology . 2011, 115, 1363–1381.

CYP3A4

2016-05-13T15:08:15Z

Dana Emmert:

== Overview ==

<Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Cytochrome P450 3A4 (CYP3A4) is in the heme-thiolate monooxygenase enzyme family meaning the protein contains a heme group with an iron atom. Enzymes in Cytochrome P450 are oxidizing enzymes and CYP3A4 works in the body oxidizing foreign molecules such as toxins and drugs<ref name="a">PMID:16389357</ref><ref name="b">PMID:15603755</ref>. It appears almost half of the marketed pharmaceutical drugs are metabolized by CYP3A4 [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687] All Cytochrome P450 are essential enzymes for metabolism and the enzyme CYP3A4 is the most important. [http://www.medsafe.govt.nz/profs/PUArticles/March2014DrugMetabolismCytochromeP4503A4.htm] Primarily found in the liver and intestine, CYP3A4 is localized in the endoplasmic reticulum membrane [http://www.uniprot.org/uniprot/P08684] and are present in all eukaryotic organisms as well as some prokaryotes<ref name="c">PMID: 15352783</ref>. While CYP3A4's role in drug metabolism are numerous, they are often aiding deactivation through facilitated excretion from the system or by direct inactivation. [https://en.wikipedia.org/wiki/CYP3A4#Tissue_distribution]

== Mechanism ==

Because water molecules are stripped away when substrate binds, this causes an entropy change of 26 kcal/mol. This entropy driven reaction has a spontaneous free energy change of -7.7kcal/mol at 21ºC <ref name="d">PMID: 15352783</ref>.
The cycle begins with the binding of the substrate to the ferric heme. With the addition of an electron, the heme gets reduced. This can only happen once the substrate is bound because the reduction potential is -300V which is more electronegative. After substrate binding, the induced conformational shift causes the reduction potential to be more positive at -230V making this a more thermodynamically favorable reaction. Oxygen next binds to the heme and oxidizes the iron. The addition of a second electron cleaves the oxygen-oxygen bond allowing one atom to bind two protons producing water. The second oxygen joins the substrate in what is called a monoxygenation reaction <ref> Devlin, Thomas M., ed. Textbook of Biochemistry with Clinical Correlations. 6th ed. Hoboken: John Wiley, 2006. Print.</ref>.

[[Image:Proteo.gif]]

== Structure and Sequence ==

The gene encoding CYP3A4 is located on chromosome 7 in the human genome<ref name="e">PMID: 1391968</ref> and it has been found that there are significant variants of the protein correlating to race <ref name="f"> PMID: 11714865</ref>. This finding is relevant due to the proteins altered ability to react with substrates such as testosterone <ref name="g">PMID: 11714865</ref>.

The <Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><scene name='72/728174/N_to_c_terminus/1'>alpha helices </scene> can be seen colored in rainbow succession from the N to C terminus. The protein pocket, where the reactions are catalyzed, more specifically, the <scene name='72/728174/Heme_binding_site/1'>heme binding site</scene> contains twenty-two residues [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=4NY4] but the main interaction is with a Cysteine at position 442, which coordinates with iron. [http://www.uniprot.org/uniprot/P08684].

== Drug Metabolism ==

CYP3A4 is thought to affect the metabolism of 30-60% of the drugs on the market [https://www.pharmgkb.org/gene/PA130?tabType=tabVip#tabview=tab3&subtab=32] [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687]. An orally prescribed drug can take a while to arrive at its target cell and along the way the body has enzymes that filter out the drug dampening its efficacy. Of those enzymes, CYP3A4 is a main player and still a lot more needs to be learned. In many cases, like that of the ß2 receptor, there is only a fraction of the drug remaining after what is known as the first-pass metabolism so that by the time the drug gets to the circulatory system where it can be delivered to the tissues its potency has already been compromised <ref name="h"> PMCID: PMC2045626</ref>.
One way to work around this is by intravenous administration so as to bypass the biological filters of the gut and go right into the bloodstream.

[[Image:First-pass metabolism.jpg]]

CYP3A4

2016-05-13T15:05:32Z

Dana Emmert:

== Overview ==

<Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Cytochrome P450 3A4 (CYP3A4) is in the heme-thiolate monooxygenase enzyme family meaning the protein contains a heme group with an iron atom. Enzymes in Cytochrome P450 are oxidizing enzymes and CYP3A4 works in the body oxidizing foreign molecules such as toxins and drugs<ref name="a">PMID:16389357</ref><ref name="b">PMID:15603755</ref>. It appears almost half of the marketed pharmaceutical drugs are metabolized by CYP3A4 [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687] All Cytochrome P450 are essential enzymes for metabolism and the enzyme CYP3A4 is the most important. [http://www.medsafe.govt.nz/profs/PUArticles/March2014DrugMetabolismCytochromeP4503A4.htm] Primarily found in the liver and intestine, CYP3A4 is localized in the endoplasmic reticulum membrane [http://www.uniprot.org/uniprot/P08684] and are present in all eukaryotic organisms as well as some prokaryotes<ref name="c">PMID: 15352783</ref>. While CYP3A4's role in drug metabolism are numerous, they are often aiding deactivation through facilitated excretion from the system or by direct inactivation. [https://en.wikipedia.org/wiki/CYP3A4#Tissue_distribution]

== Mechanism ==

Because water molecules are stripped away when substrate binds, this causes an entropy change of 26 kcal/mol. This entropy driven reaction has a spontaneous free energy change of -7.7kcal/mol at 21ºC <ref name="d">PMID: 15352783</ref>.
The cycle begins with the binding of the substrate to the ferric heme. With the addition of an electron, the heme gets reduced. This can only happen once the substrate is bound because the reduction potential is -300V which is more electronegative. After substrate binding, the induced conformational shift causes the reduction potential to be more positive at -230V making this a more thermodynamically favorable reaction. Oxygen next binds to the heme and oxidizes the iron. The addition of a second electron cleaves the oxygen-oxygen bond allowing one atom to bind two protons producing water. The second oxygen joins the substrate in what is called a monoxygenation reaction <ref> Devlin, Thomas M., ed. Textbook of Biochemistry with Clinical Correlations. 6th ed. Hoboken: John Wiley, 2006. Print.</ref>.

[[Image:Proteo.gif]]

== Structure and Sequence ==

The gene encoding CYP3A4 is located on chromosome 7 in the human genome<ref name="e">PMID: 1391968</ref> and it has been found that there are significant variants of the protein correlating to race <ref name="f"> PMID: 11714865</ref>. This finding is relevant due to the proteins altered ability to react with substrates such as testosterone <ref name="g">PMID: 11714865</ref>.

The <Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><scene name='72/728174/N_to_c_terminus/1'>alpha helices </scene> can be seen colored in rainbow succession from the N to C terminus. The protein pocket, where the reactions are catalyzed, more specifically, the <scene name='72/728174/Heme_binding_site/1'>heme binding site</scene> contains twenty-two residues [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=4NY4] but the main interaction is with a Cysteine at position 442, which coordinates with iron. [http://www.uniprot.org/uniprot/P08684].

== Relevance ==

CYP3A4 is thought to affect the metabolism of 30-60% of the drugs on the market [https://www.pharmgkb.org/gene/PA130?tabType=tabVip#tabview=tab3&subtab=32] [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687]. An orally prescribed drug can take a while to arrive at its target cell and along the way the body has enzymes that filter out the drug dampening its efficacy. In certain cases, like that of the ß2 receptor, there is a fraction of the drug remaining after what is known as the first-pass metabolism so that by the time the drug gets to the circulatory system where it can be delivered to the tissues its potency has already been compromised <ref name="h"> PMCID: PMC2045626</ref>.
One way to work around this is by intravenous administration so as to bypass the biological filters of the gut and go right into the bloodstream.

[[Image:First-pass metabolism.jpg]]

CYP3A4

2016-05-13T14:52:45Z

Dana Emmert:

== Overview ==

<Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Cytochrome P450 3A4 (CYP3A4) is in the heme-thiolate monooxygenase enzyme family meaning the protein contains a heme group with an iron atom. Enzymes in Cytochrome P450 are oxidizing enzymes and CYP3A4 works in the body oxidizing foreign molecules such as toxins and drugs<ref name="a">PMID:16389357</ref><ref name="b">PMID:15603755</ref>. It appears almost half of the marketed pharmaceutical drugs are metabolized by CYP3A4 [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687] All Cytochrome P450 are essential enzymes for metabolism and the enzyme CYP3A4 is the most important. [http://www.medsafe.govt.nz/profs/PUArticles/March2014DrugMetabolismCytochromeP4503A4.htm] Primarily found in the liver and intestine, CYP3A4 is localized in the endoplasmic reticulum membrane [http://www.uniprot.org/uniprot/P08684] and are present in all eukaryotic organisms as well as some prokaryotes<ref name="c">PMID: 15352783</ref>. While CYP3A4's role in drug metabolism are numerous, they are often aiding deactivation through facilitated excretion from the system or by direct inactivation. [https://en.wikipedia.org/wiki/CYP3A4#Tissue_distribution]

== Mechanism ==

Because water molecules are stripped away when substrate binds, this causes an entropy change of 26 kcal/mol. This entropy driven reaction has a spontaneous free energy change of -7.7kcal/mol at 21ºC <ref name="d">PMID: 15352783</ref>.
The cycle begins with the binding of the substrate to the ferric heme. With the addition of an electron, the heme gets reduced. This can only happen once the substrate is bound because the reduction potential is -300V which is more electronegative. After substrate binding, the induced conformational shift causes the reduction potential to be more positive at -230V making this a more thermodynamically favorable reaction. Oxygen next binds to the heme and oxidizes the iron. The addition of a second electron cleaves the oxygen-oxygen bond allowing one atom to bind two protons producing water. The second oxygen joins the substrate in what is called a monoxygenation reaction <ref> Devlin, Thomas M., ed. Textbook of Biochemistry with Clinical Correlations. 6th ed. Hoboken: John Wiley, 2006. Print.</ref>.

[[Image:Proteo.gif]]

== Structure and Sequence ==

The gene encoding CYP3A4 is located on chromosome 7 in the human genome<ref name="e">PMID: 1391968</ref> and it has been found that there are significant variants of the protein correlating to race <ref name="f"> PMID: 11714865</ref>. This finding is relevant due to the proteins altered ability to react with substrates such as testosterone <ref name="g">PMID: 11714865</ref>.

The <Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><scene name='72/728174/N_to_c_terminus/1'>alpha helices </scene> can be seen colored in rainbow succession from the N to C terminus. The protein pocket, where the reactions are catalyzed, more specifically, the <scene name='72/728174/Heme_binding_site/1'>heme binding site</scene> contains twenty-two residues [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=4NY4] but the main interaction is with a Cysteine at position 442, which coordinates with iron. [http://www.uniprot.org/uniprot/P08684].

== Relevance ==

CYP3A4 is thought to affect the metabolism of 30-60% of the drugs on the market [https://www.pharmgkb.org/gene/PA130?tabType=tabVip#tabview=tab3&subtab=32] [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687]. An orally prescribed drug can take a while to arrive at its target cell and along the way the body has enzymes that filter out the drug dampening its efficacy. In certain cases, like that of the ß2 receptor, there is a fraction of the drug remaining after what is known as the first-pass metabolism so that by the time the drug gets to the circulatory system where it can be delivered to the tissues its potency has already been compromised <ref name="h"> PMCID: PMC2045626</ref>.

[[Image:First-pass metabolism.jpg]]

One way to work around !!!!!! is by intravenous administration so as to bypass the biological filters of the gut and go right into the bloodstream.

CYP3A4

2016-05-13T14:40:50Z

Dana Emmert:

== Overview ==

<Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Cytochrome P450 3A4 (CYP3A4) is in the heme-thiolate monooxygenase enzyme family meaning the protein contains a heme group with an iron atom. Enzymes in Cytochrome P450 are oxidizing enzymes and CYP3A4 works in the body oxidizing foreign molecules such as toxins and drugs<ref name="a">PMID:16389357</ref><ref name="b">PMID:15603755</ref>. It appears almost half of the marketed pharmaceutical drugs are metabolized by CYP3A4 [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687] All Cytochrome P450 are essential enzymes for metabolism and the enzyme CYP3A4 is the most important. [http://www.medsafe.govt.nz/profs/PUArticles/March2014DrugMetabolismCytochromeP4503A4.htm] Primarily found in the liver and intestine, CYP3A4 is localized in the endoplasmic reticulum membrane [http://www.uniprot.org/uniprot/P08684] and are present in all eukaryotic organisms as well as some prokaryotes<ref name="c">PMID: 15352783</ref>. While CYP3A4's role in drug metabolism are numerous, they are often aiding deactivation through facilitated excretion from the system or by direct inactivation. [https://en.wikipedia.org/wiki/CYP3A4#Tissue_distribution]

== Mechanism ==

Because water molecules are stripped away when substrate binds, this causes an entropy change of 26 kcal/mol. This entropy driven reaction has a spontaneous free energy change of -7.7kcal/mol at 21ºC <ref name="d">PMID: 15352783</ref>.
The cycle begins with the binding of the substrate to the ferric heme. With the addition of an electron, the heme gets reduced. This can only happen once the substrate is bound because the reduction potential is -300V which is more electronegative. After substrate binding, the induced conformational shift causes the reduction potential to be more positive at -230V making this a more thermodynamically favorable reaction. Oxygen next binds to the heme and oxidizes the iron. The addition of a second electron cleaves the oxygen-oxygen bond allowing one atom to bind two protons producing water. The second oxygen joins the substrate in what is called a monoxygenation reaction <ref> Devlin, Thomas M., ed. Textbook of Biochemistry with Clinical Correlations. 6th ed. Hoboken: John Wiley, 2006. Print.</ref>.

[[Image:Proteo.gif]]

== Structure and Sequence ==

The gene encoding CYP3A4 is located on chromosome 7 in the human genome<ref name="e">PMID: 1391968</ref> and it has been found that there are significant variants of the protein correlating to race <ref name="f"> PMID: 11714865</ref>. This finding is relevant due to the proteins altered ability to react with substrates such as testosterone <ref name="g">PMID: 11714865</ref>.

The <Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><scene name='72/728174/N_to_c_terminus/1'>alpha helices </scene> can be seen colored in rainbow succession from the N to C terminus. The protein pocket, where the reactions are catalyzed, more specifically, the <scene name='72/728174/Heme_binding_site/1'>heme binding site</scene> contains twenty-two residues [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=4NY4] but the main interaction is with a Cysteine at position 442, which coordinates with iron. [http://www.uniprot.org/uniprot/P08684].

== Relevance ==

CYP3A4 is thought to affect the metabolism of 30-60% of the drugs on the market [https://www.pharmgkb.org/gene/PA130?tabType=tabVip#tabview=tab3&subtab=32] [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687]. Because of this, the continual study of this enzyme is valued. A orally prescribed drug can take a while to arrive at its target cell and along the way the body has enzymes that filter out the drug dampening its efficacy.

[[Image:First-pass metabolism.jpg]]

== External Sources ==
Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes [http://pubs.acs.org/doi/full/10.1021/cr020443g]
<references/>

CYP3A4

2016-05-13T14:39:10Z

Dana Emmert:

== Overview ==

<Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />

Cytochrome P450 3A4 (CYP3A4) is in the heme-thiolate monooxygenase enzyme family meaning the protein contains a heme group with an iron atom. Enzymes in Cytochrome P450 are oxidizing enzymes and CYP3A4 works in the body oxidizing foreign molecules such as toxins and drugs<ref name="a">PMID:16389357</ref><ref name="b">PMID:15603755</ref>. It appears almost half of the marketed pharmaceutical drugs are metabolized by CYP3A4 [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687] All Cytochrome P450 are essential enzymes for metabolism and the enzyme CYP3A4 is the most important. [http://www.medsafe.govt.nz/profs/PUArticles/March2014DrugMetabolismCytochromeP4503A4.htm] Primarily found in the liver and intestine, CYP3A4 is localized in the endoplasmic reticulum membrane [http://www.uniprot.org/uniprot/P08684] and are present in all eukaryotic organisms as well as some prokaryotes<ref name="c">PMID: 15352783</ref>. While CYP3A4's role in drug metabolism are numerous, they are often aiding deactivation through facilitated excretion from the system or by direct inactivation. [https://en.wikipedia.org/wiki/CYP3A4#Tissue_distribution]

== Mechanism ==

Because water molecules are stripped away when substrate binds, this causes an entropy change of 26 kcal/mol. This entropy driven reaction has a spontaneous free energy change of -7.7kcal/mol at 21ºC <ref name="d">PMID: 15352783</ref>.
The cycle begins with the binding of the substrate to the ferric heme. With the addition of an electron, the heme gets reduced. This can only happen once the substrate is bound because the reduction potential is -300V which is more electronegative. After substrate binding, the induced conformational shift causes the reduction potential to be more positive at -230V making this a more thermodynamically favorable reaction. Oxygen next binds to the heme and oxidizes the iron. The addition of a second electron cleaves the oxygen-oxygen bond allowing one atom to bind two protons producing water. The second oxygen joins the substrate in what is called a monoxygenation reaction <ref> Devlin, Thomas M., ed. Textbook of Biochemistry with Clinical Correlations. 6th ed. Hoboken: John Wiley, 2006. Print.</ref>.

[[Image:Proteo.gif]]

== Structure and Sequence ==

The gene encoding CYP3A4 is located on chromosome 7 in the human genome<ref name="e">PMID: 1391968</ref> and it has been found that there are significant variants of the protein correlating to race <ref name="f"> PMID: 11714865</ref>. This finding is relevant due to the proteins altered ability to react with substrates such as testosterone <ref name="g">PMID: 11714865</ref>.

The <Structure load='4NY4' size='350' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' /><scene name='72/728174/N_to_c_terminus/1'>alpha helices </scene> can be seen colored in rainbow succession from the N to C terminus. The protein pocket, where the reactions are catalyzed, more specifically, the <scene name='72/728174/Heme_binding_site/1'>heme binding site</scene> contains twenty-two residues [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=4NY4] but the main interaction is with a Cysteine at position 442, which coordinates with iron. [http://www.uniprot.org/uniprot/P08684].

== Relevance ==

CYP3A4 is thought to affect the metabolism of 30-60% of the drugs on the market [https://www.pharmgkb.org/gene/PA130?tabType=tabVip#tabview=tab3&subtab=32] [http://www.pharmacytimes.com/publications/issue/2008/2008-09/2008-09-8687]. Because of this, the continual study of this enzyme is valued. A orally prescribed drug can take a while to arrive at its target cell and along the way the body has enzymes that filter out the drug dampening its efficacy.

== External Sources ==
Mechanism of Oxidation Reactions Catalyzed by Cytochrome P450 Enzymes [http://pubs.acs.org/doi/full/10.1021/cr020443g]
<references/>

File:First-pass metabolism.jpg

2016-05-13T14:38:25Z

Dana Emmert:

Sandbox WWC6

2016-05-13T13:15:37Z

Dana Emmert:

[[Hemolysins]] are a lipid or protein toxins secreted by pathogens that lyse erythrocyte and some bacterial cell membranes. These toxins belong to a family of microbial exotoxins called cytolysins, which act on a broad number of cells<ref name ="cyt">http://www.uniprot.org/uniprot/P09616</ref>. The primary function of peptide hemolysins is pore formation at the cell membranes creating acytolytic effect, and is achieved by the release of cytosolic ions and small molecules through the hydrophilic, transmembrane portion of the beta-barrel pore <ref name ="bar>http://www.sciencedirect.com/science/article/pii/S0041010101001532</ref>.

<Structure load='7AHL' size='350' frame='true' align='right' caption='Stapholococcal alpha-hemolysin' scene='Insert optional scene name here' />

== Function ==

Hemolysins are most commonly proteins found in red blood cells that allow for the rapid release of small molecules and ions across the membrane. <ref name ="hem">https://en.wikipedia.org/wiki/Hemolysin#cite_note-pmid20692229-3</ref> or lipid biosurfactants that disrupt membrane composition resulting in cell lysis. Hemolysins act through disruption of the cell membrane. <ref>http://www.sciencedirect.com/science/article/pii/S0005273610002610</ref> Pore formation is the olgomerization of the pore sunbunits within the membrane. The pore is quickly filled with water, ions, and small molecules that rapidly exit the cell, dissipating ionic gradients and membrane potential. Osmotic pressure causes a rapid swelling of the cell, leading to total rupture of the membrane <ref name ="ion>http://www.ks.uiuc.edu/Research/hemolysin/</ref>. These proteins are important for some erythrocyte nutrient accession, but cause massive erythrocyte destruction in bacterial infection, specifically responsible forhemolytic anemia, which causes fatigue, pain, arrythmias, and even heart failure in affected individuals. <ref>http://www.nhlbi.nih.gov/health/health-topics/topics/ha/</ref>

==Structure==
Hemolysins have three structural variations: alpha, beta, and gamma. These hemolysin types are comprised of di-, hepta- or octomeric subunits.

*Alpha-hemolysin

<scene name='69/696302/Alpha-hemolysin/1'>Alpha-hemolysin</scene>

[http://proteopedia.org/wiki/index.php/Pore_forming_toxin,_%CE%B1-hemolysin Alpha hemolysin] causes a partial lysis of red blood cells. The heptameric pore assembles from water-soluble subunits. The alpha subunit, depicted right, consists seven identical monomeric units that exhibit rotational symmetry in oligomerized form. Each distinct subunit is differently colored for easy identification. The beta-barrel transmembrane domain is 50 Å in length. <ref>http://www.ks.uiuc.edu/Research/hemolysin/</ref>

<scene name='69/696302/Alpha-hemolysin_polar_residues/1'>Alpha-hemolysin residue polarity</scene>

{{Template:ColorKey_Hydrophobic}}

{{Template:ColorKey_Polar}}

*Beta-hemolysin

<scene name='69/696302/Beta-hemolysin/2'>Beta-hemolysin</scene>

Beta-hemolysins cause a total lysis of red blood cells.

*Gamma-hemolysin

<scene name='69/696302/Beta-hemolysin/1'>Gamma-hemolysin</scene>

[http://proteopedia.org/wiki/index.php/3b07 Gamma-hemolysin] is both hemolytic and leukotoxic.

<scene name='69/696302/Gamma-hemolysin_residue_polar/1'>Gamma-hemolysin residue polarity</scene>

{{Template:ColorKey_Hydrophobic}}

{{Template:ColorKey_Polar}}

==Pathogenic microorganisms==
Pore-forming toxins have been shown to closely relate to the pathogenicity of the toxin-producing organism. <ref>http://www.ncbi.nlm.nih.gov/pubmed/1930675</ref> Both gram positive and gram negative bacteria are producers of hemolysins, as well as some clinically relevant fungi. Toxin secretion facillitates the availability of water, ions, and small molecules like sugar for the secreting pathogen. Hemolysin producing pathogen are identified by their ability to lyse cells in vitro. <ref>https://en.wikipedia.org/wiki/Hemolysin</ref>

==Mechanism==

Four of each of the two subunits assemble in an alternating, circular pattern in the γ-HL pore, whereas seven distinct α-HL protomers assemble in a circular arrangement in the α-HL pore. These typically are comprised of three domains: the cap, rim and stem domains, named for the structural resemblance to a mushroom. The cap domain contains β-sandwiches from each protomer, while just below, the rim domain contains four looping β-strands. The stem domain takes on the antiparallel β-barrel, a portion of which becomes the transmembrane structure. <ref name ="nat">doi:10.1038/ncomms5897</ref>

===Pore formation===
Pore formation of hemolysins is believed to be a conserved process across subtypes. <ref name ="nat"/>
Studies suggest that pore formation is achieved through a nonlytic intermediate oligomer, known as a prepore. The prepore model proposal suggests that the monomeric components assemble on the cell membrane surfacte into a prepore with prestem subunits packed inside. The formed prepore then goes through a conformational change prestem, forming the β-barrel pore. Several issues with the proposed pore formation mechanism have been identified including steric hindrance of the packed prestem structure.

[[Image:Ncomms5897-f5.jpg]]

This image shows the proposed mechanism of pore formation in the cell membrane. <ref name ="nat"/>

==Medical Implications==

===Role in infection===
Hemolysin lysis of red blood cells is a marker for many kinds of pathogenic infection characterized by death of red blood cells. <ref name="hem"/>

===Oncology===
Thermostable direct hemoslysin (TDH) is one type of hemolysin, secreted by ''Vibrio parahaemolyticus'', that may be linked to the down regulation of colon carcinoma cell proliferation. The presence of this hemolysin is responsible for the influx of calcium ions from the extracellular space, activating protein kinase C, an inhibitor of tyrosine kinase activity on a key growth factor of this cancer.<ref>http://www.sciencedirect.com/science/article/pii/S030441651200116X</ref>

===Hemolytic anemia===
Hemolytic anemia occurs when lysis of red blood cells occurs at rates faster than they can be replaced by bone marrow. Hypoxia due to this condition can result in dizziness, shortness of breath, poor maintenance of body temperature, and jaundice.<ref>https://www.nhlbi.nih.gov/health/health-topics/topics/ha</ref>

[[Image:Hemolysis3-147A9D970590BADE656.jpg]]

==References==
<references/>

Sandbox WWC6

2016-05-13T13:12:12Z

Dana Emmert:

[[Hemolysins]] are a lipid or protein toxins secreted by pathogens that lyse erythrocyte and some bacterial cell membranes. These toxins belong to a family of microbial exotoxins called cytolysins, which act on a broad number of cells<ref name ="cyt">http://www.uniprot.org/uniprot/P09616</ref>. The primary function of peptide hemolysins is pore formation at the cell membranes creating acytolytic effect, and is achieved by the release of cytosolic ions and small molecules through the hydrophilic, transmembrane portion of the beta-barrel pore <ref name ="bar>http://www.sciencedirect.com/science/article/pii/S0041010101001532</ref>.

<Structure load='7AHL' size='350' frame='true' align='right' caption='Stapholococcal alpha-hemolysin' scene='Insert optional scene name here' />

== Function ==

Hemolysins are most commonly proteins found in red blood cells that allow for the rapid release of small molecules and ions across the membrane. <ref name ="hem">https://en.wikipedia.org/wiki/Hemolysin#cite_note-pmid20692229-3</ref> or lipid biosurfactants that disrupt membrane composition resulting in cell lysis. Hemolysins act through disruption of the cell membrane. <ref>http://www.sciencedirect.com/science/article/pii/S0005273610002610</ref> Pore formation is the olgomerization of the pore sunbunits within the membrane. The pore is quickly filled with water, ions, and small molecules that rapidly exit the cell, dissipating ionic gradients and membrane potential. Osmotic pressure causes a rapid swelling of the cell, leading to total rupture of the membrane <ref name ="ion>http://www.ks.uiuc.edu/Research/hemolysin/</ref>. These proteins are important for some erythrocyte nutrient accession, but cause massive erythrocyte destruction in bacterial infection, specifically responsible forhemolytic anemia, which causes fatigue, pain, arrythmias, and even heart failure in affected individuals. <ref>http://www.nhlbi.nih.gov/health/health-topics/topics/ha/</ref>

==Structure==
Hemolysins have three structural variations: alpha, beta, and gamma. These hemolysin types are comprised of di-, hepta- or octomeric subunits.

*Alpha-hemolysin

<scene name='69/696302/Alpha-hemolysin/1'>Alpha-hemolysin</scene>

[http://proteopedia.org/wiki/index.php/Pore_forming_toxin,_%CE%B1-hemolysin Alpha hemolysin] causes a partial lysis of red blood cells. The heptameric pore assembles from water-soluble subunits. The alpha subunit, depicted right, consists seven identical monomeric units that exhibit rotational symmetry in oligomerized form. Each distinct subunit is differently colored for easy identification. The beta-barrel transmembrane domain is 50 Å in length. <ref>http://www.ks.uiuc.edu/Research/hemolysin/<ref>

<scene name='69/696302/Alpha-hemolysin_polar_residues/1'>Alpha-hemolysin residue polarity</scene>

{{Template:ColorKey_Hydrophobic}}

{{Template:ColorKey_Polar}}

*Beta-hemolysin

<scene name='69/696302/Beta-hemolysin/2'>Beta-hemolysin</scene>

Beta-hemolysins cause a total lysis of red blood cells.

*Gamma-hemolysin

<scene name='69/696302/Beta-hemolysin/1'>Gamma-hemolysin</scene>

[http://proteopedia.org/wiki/index.php/3b07 Gamma-hemolysin] is both hemolytic and leukotoxic.

<scene name='69/696302/Gamma-hemolysin_residue_polar/1'>Gamma-hemolysin residue polarity</scene>

{{Template:ColorKey_Hydrophobic}}

{{Template:ColorKey_Polar}}

==Pathogenic microorganisms==
Pore-forming toxins have been shown to closely relate to the pathogenicity of the toxin-producing organism. <ref>http://www.ncbi.nlm.nih.gov/pubmed/1930675<ref> Both gram positive and gram negative bacteria are producers of hemolysins, as well as some clinically relevant fungi. Toxin secretion facillitates the availability of water, ions, and small molecules like sugar for the secreting pathogen. Hemolysin producing pathogen are identified by their ability to lyse cells in vitro. <ref>https://en.wikipedia.org/wiki/Hemolysin<ref>

==Mechanism==

Four of each of the two subunits assemble in an alternating, circular pattern in the γ-HL pore, whereas seven distinct α-HL protomers assemble in a circular arrangement in the α-HL pore. These typically are comprised of three domains: the cap, rim and stem domains, named for the structural resemblance to a mushroom. The cap domain contains β-sandwiches from each protomer, while just below, the rim domain contains four looping β-strands. The stem domain takes on the antiparallel β-barrel, a portion of which becomes the transmembrane structure. <ref name ="nat">doi:10.1038/ncomms5897</ref>

===Pore formation===
Pore formation of hemolysins is believed to be a conserved process across subtypes. <ref name ="nat"/>
Studies suggest that pore formation is achieved through a nonlytic intermediate oligomer, known as a prepore. The prepore model proposal suggests that the monomeric components assemble on the cell membrane surfacte into a prepore with prestem subunits packed inside. The formed prepore then goes through a conformational change prestem, forming the β-barrel pore. Several issues with the proposed pore formation mechanism have been identified including steric hindrance of the packed prestem structure.

[[Image:Ncomms5897-f5.jpg]]

This image shows the proposed mechanism of pore formation in the cell membrane. <ref name ="nat"/>

==Medical Implications==

===Role in infection===
Hemolysin lysis of red blood cells is a marker for many kinds of pathogenic infection characterized by death of red blood cells. <ref name="hem"/>

===Oncology===
Thermostable direct hemoslysin (TDH) is one type of hemolysin, secreted by ''Vibrio parahaemolyticus'', that may be linked to the down regulation of colon carcinoma cell proliferation. The presence of this hemolysin is responsible for the influx of calcium ions from the extracellular space, activating protein kinase C, an inhibitor of tyrosine kinase activity on a key growth factor of this cancer.<ref>http://www.sciencedirect.com/science/article/pii/S030441651200116X</ref>

===Hemolytic anemia===
Hemolytic anemia occurs when lysis of red blood cells occurs at rates faster than they can be replaced by bone marrow. Hypoxia due to this condition can result in dizziness, shortness of breath, poor maintenance of body temperature, and jaundice.<ref>https://www.nhlbi.nih.gov/health/health-topics/topics/ha</ref>

[[Image:Hemolysis3-147A9D970590BADE656.jpg]]

==References==
<references/>

Sandbox WWC6

2016-05-13T13:10:25Z

Dana Emmert:

[[Hemolysins]] are a lipid or protein toxins secreted by pathogens that lyse erythrocyte and some bacterial cell membranes. These toxins belong to a family of microbial exotoxins called cytolysins, which act on a broad number of cells<ref name ="cyt">http://www.uniprot.org/uniprot/P09616</ref>. The primary function of peptide hemolysins is pore formation at the cell membranes creating acytolytic effect, and is achieved by the release of cytosolic ions and small molecules through the hydrophilic, transmembrane portion of the beta-barrel pore <ref name ="bar>http://www.sciencedirect.com/science/article/pii/S0041010101001532</ref>.

<Structure load='7AHL' size='350' frame='true' align='right' caption='Stapholococcal alpha-hemolysin' scene='Insert optional scene name here' />

== Function ==

Hemolysins are most commonly proteins found in red blood cells that allow for the rapid release of small molecules and ions across the membrane. <ref name ="hem">https://en.wikipedia.org/wiki/Hemolysin#cite_note-pmid20692229-3</ref> or lipid biosurfactants that disrupt membrane composition resulting in cell lysis. Hemolysins act through disruption of the cell membrane. <ref>http://www.sciencedirect.com/science/article/pii/S0005273610002610</ref> Pore formation is the olgomerization of the pore sunbunits within the membrane. The pore is quickly filled with water, ions, and small molecules that rapidly exit the cell, dissipating ionic gradients and membrane potential. Osmotic pressure causes a rapid swelling of the cell, leading to total rupture of the membrane <ref name ="ion>http://www.ks.uiuc.edu/Research/hemolysin/</ref>. These proteins are important for some erythrocyte nutrient accession, but cause massive erythrocyte destruction in bacterial infection, specifically responsible forhemolytic anemia, which causes fatigue, pain, arrythmias, and even heart failure in affected individuals. <ref>http://www.nhlbi.nih.gov/health/health-topics/topics/ha/</ref>

==Structure==
Hemolysins have three structural variations: alpha, beta, and gamma. These hemolysin types are comprised of di-, hepta- or octomeric subunits.

*Alpha-hemolysin

<scene name='69/696302/Alpha-hemolysin/1'>Alpha-hemolysin</scene>

[http://proteopedia.org/wiki/index.php/Pore_forming_toxin,_%CE%B1-hemolysin Alpha hemolysin] causes a partial lysis of red blood cells. The heptameric pore assembles from water-soluble subunits. The alpha subunit, depicted right, consists seven identical monomeric units that exhibit rotational symmetry in oligomerized form. Each distinct subunit is differently colored for easy identification. The beta-barrel transmembrane domain is 50 Å in length. <ref>http://www.ks.uiuc.edu/Research/hemolysin/<ref>

<scene name='69/696302/Alpha-hemolysin_polar_residues/1'>Alpha-hemolysin residue polarity</scene>

{{Template:ColorKey_Hydrophobic}}

{{Template:ColorKey_Polar}}

*Beta-hemolysin

<scene name='69/696302/Beta-hemolysin/2'>Beta-hemolysin</scene>

Beta-hemolysins cause a total lysis of red blood cells.

*Gamma-hemolysin

<scene name='69/696302/Beta-hemolysin/1'>Gamma-hemolysin</scene>

[http://proteopedia.org/wiki/index.php/3b07 Gamma-hemolysin] is both hemolytic and leukotoxic.

<scene name='69/696302/Gamma-hemolysin_residue_polar/1'>Gamma-hemolysin residue polarity</scene>

{{Template:ColorKey_Hydrophobic}}

{{Template:ColorKey_Polar}}

==Pathogenic microorganisms==
Pore-forming toxins have been shown to closely relate to the pathogenicity of the toxin-producing organism. <ref>http://www.ncbi.nlm.nih.gov/pubmed/1930675<ref> Both gram positive and gram negative bacteria are producers of hemolysins, as well as some clinically relevant fungi. Toxin secretion facillitates the availability of water, ions, and small molecules like sugar for the secreting pathogen. Hemolysin producing pathogen are identified by their ability to lyse cells in vitro. <ref>https://en.wikipedia.org/wiki/Hemolysin<ref>

==Mechanism==

Four of each of the two subunits assemble in an alternating, circular pattern in the γ-HL pore, whereas seven distinct α-HL protomers assemble in a circular arrangement in the α-HL pore. These typically are comprised of three domains: the cap, rim and stem domains, named for the structural resemblance to a mushroom. The cap domain contains β-sandwiches from each protomer, while just below, the rim domain contains four looping β-strands. The stem domain takes on the antiparallel β-barrel, a portion of which becomes the transmembrane structure. <ref name ="nat">doi:10.1038/ncomms5897</ref>

===Pore formation===
Pore formation of hemolysins is believed to be a conserved process across subtypes. <ref name ="nat"/>
Studies suggest that pore formation is achieved through a nonlytic intermediate oligomer, known as a prepore. The prepore model proposal suggests that the monomeric components assemble on the cell membrane surfacte into a prepore with prestem subunits packed inside. The formed prepore then goes through a conformational change prestem, forming the β-barrel pore. Several issues with the proposed pore formation mechanism have been identified including steric hindrance of the packed prestem structure.

[[Image:Ncomms5897-f5.jpg]]

This image shows the proposed mechanism of pore formation in the cell membrane. <ref name ="nat"/>

==Medical Implications==

===Role in infection===
Hemolysin lysis of red blood cells is a marker for many kinds of pathogenic infection characterized by death of red blood cells. <ref name="hem"/>

===Oncology===
Thermostable direct hemoslysin (TDH) is one type of hemolysin, secreted by ''Vibrio parahaemolyticus'', that may be linked to the down regulation of colon carcinoma cell proliferation. The presence of this hemolysin is responsible for the influx of calcium ions from the extracellular space, activating protein kinase C, an inhibitor of tyrosine kinase activity on a key growth factor of this cancer.<ref>http://www.sciencedirect.com/science/article/pii/S030441651200116X<ref>

===Hemolytic anemia===
Hemolytic anemia occurs when lysis of red blood cells occurs at rates faster than they can be replaced by bone marrow. Hypoxia due to this condition can result in dizziness, shortness of breath, poor maintenance of body temperature, and jaundice.<ref>https://www.nhlbi.nih.gov/health/health-topics/topics/ha<ref>

[[Image:Hemolysis3-147A9D970590BADE656.jpg]]

==References==
<references/>

Sandbox WWC2

2016-05-13T12:07:43Z

Dana Emmert:

<Structure load='2Z5X' size='500' frame='top' align='right' caption='''Crystal structure of human monoamine oxidase A complex with Harmine''' scene='69/696303/Ntocrainbow/1' />

==Introduction==

Monoamine oxidase A (MAO-A) is an oxidoreductase flavoenzyme, encoded by the MAOA gene on the X chromosome; the enzyme is present throughout the brain, central nervous system, and stomach. Two isoforms of this enzyme (MAO-B being the other) are expressed on the outer surface of the mitochondrial membrane, and both are responsible for the oxidative deamination of various monoamine neurotransmitters and dietary amines [http://www.omim.org/entry/309850]. Each isoform is characterized by its unique substrate specificity and inhibitor sensitivity. For example, MAO-A (found primarily in catecholaminergic neurons) preferentially oxidizes 5-hydroxytryptamine (5-HT), epinephrine, and norepinephrine; while MAO-B (found primarily in serotonergic neurons) prefers phenylethylamine and benzylamine. Both of these enzymes oxidize dopamine, tyramine, and N,N-dimethyltryptamine as well as their respective unique substrates according to the following reaction:

RCH2NHR' + H2O + O2 --> RCHO + R'NH2 + H2O2

in which the H2O2 is enzymatically removed by glutathione reductase and glutathione peroxidase to produce a reduced glutathione since heightened levels of H2O2 promote apoptosis signalling within cells [https://books.google.com/books?id=6p_cBAAAQBAJ&pg=PA23&lpg=PA23&dq=h2o2+produced+by+mao+neutralized+by&source=bl&ots=xMja8jxpkk&sig=-o5KLxfPBe7m_selXCo9iVt8OuY&hl=en&sa=X&ved=0ahUKEwiDt_qYlJHMAhVIeD4KHcIDC3UQ6AEISzAF#v=onepage&q=h2o2%20produced%20by%20mao%20neutralized%20by&f=false]. Due to the important role MAOs play in controlling the prevalence of various neurotransmitters in the body as well as producing reactive oxygen species (ROS), an imbalance of these enzymes may be detrimental to human health.
http://www.proteopedia.org/wiki/skins/common/images/button_nowiki.png
The secondary structure, illustrated in the protein image to the right, in conjunction with the color key, below, indicates the directionality of the polypeptide chain.
{{Template:ColorKey_Amino2CarboxyRainbow}}

== Structure ==
Unlike its related dimers, human MAO-B and rat MAO-A, human MAO-A is a monomer. The enzyme shares 70% of its amino acid sequence with human MAO-B, and the amino acids interacting with the FAD binding site in both isoforms is conserved; therefore it is believed that the FAD binding sites are quite similar in both proteins <ref name="Edmondson et al"> PMID: 14697881</ref>. MAO-A composed of 527 amino acids, with a molecular weight of approximately 60.5 kDa. The first 497 amino acids in the polypeptide exist in the cytoplasm of the cell; while amino acids 498—518 are involved in an α-helix that anchors the enzyme to the mitochondrial membrane (this can clearly be seen in the provided crystal structure as the only α-helix that significantly sticks out away from the rest of the globular structure). Furthermore, amino acids 519—527 are present in the intermembrane space of the mitochondria [http://www.uniprot.org/uniprot/P2139].
The active site of MAO-A is a single hydrophobic pocket, lined with 11 aliphatic and 5 aromatic residues, with a volume of approximately 550 cubic Å [http://www.pnas.org/content/102/36/12684.full]. Moreover, the active site of human MAO-A is completely unique due to a loop-like conformation of residues 210-216, this is thought to be the result of interactions taking place across the monomer, which allow for the development of inhibitors specific to human MAO-A [http://www.ncbi.nlm.nih.gov/pubmed/16129825]. There are two crucial differences in the active sites of human MAO-A and -B, these include residues Phe-208 (Ile-199 in human MAO B) and Ile-335 (Tyr-326 in human hMAO B) <ref name="Colibus et al"> PMID: 16129825</ref>. Bach et al (1988) showed that the FAD is covalently bound to cysteine residues in both human MAO-A and -B, on Cys397 and Cys406, respectively [http://thirdworld.nl/the-fad-binding-sites-of-human-monoamine-oxidases-a-and-b].

== Function ==
As previously stated, the function of MAO-A is to deaminate 5-hydroxytryptamine (5-HT), epinephrine, norepinephrine, dopamine, tyramine, and N,N-dimethyltryptamine (DMT). When neurons release neurotransmitters into the synaptic cleft to bind to a receptor, some of the neurotransmitters do not bind to the receptor and are not reabsorbed. The monoamine oxidase enzymes are responsible for oxidizing some of these "free" neurotransmitters in an attempt to prevent their binding to unintended receptors, potentially causing deleterious side effects; they are also responsible for maintaining a steady state of certain neurotransmitters.
Although MAO-A is found concentrated in catecholaminergic neurons, it is primarily found in the digestive system, while MAO-B is found more abundantly in the brain.
There are two prevalent polymorphisms of the human MAOA gene, these include the low activity form (MAOA-L), which produces a smaller amount of the enzyme, and high activity form (MAOA-H), which results in the production of high levels of MAO-A [https://news.brown.edu/articles/2009/01/hotsauce]. Recent studies have found that individuals with the MAOA-L gene tend to display slightly higher levels of aggression than individuals with the MAOA-H gene (especially in the case of high-provocation situations). [http://scholar.harvard.edu/files/dtingley/files/pnas-2009.pdf]

Due to this association between aggression and MAO-A polymorphisms, the human MAOA gene has been dubbed the "warrior gene".

== Disease ==
Due to the important role MAO-A plays in maintaining a balance of various neurotransmitters in the digestive and central nervous system, as well as its production of ROS, an imbalance of MAO-A may result in any of several diseases. For example, postpartum depression is a moderate to severe form of depression that occurs as the result of an excess of MAO-A in the brain that some women experience after childbirth [http://www.mpg.de]. In research conducted by Julia Sacher of the Max Planck Institute for Human Cognitive and Brain Sciences, it was concluded that the levels of MAO-A in women with postpartum depression are about 40% greater than those of average women. [https://www.mpg.de/8331073/postpartum-depression_monoamine-oxidase-a]
[[Image:Postpardam_depression_maoa.jpg|thumb|right|”Positron-Emission-Tomography (PET) of a depressive patient with elevated monoamine-oxidase-A-levels (green, yellow, red) without medication (left) and after a six-week-treatment with the monoamine-oxidase-A-inhibitor moclobemid (right)” [https://www.mpg.de/8331073/postpartum-depression_monoamine-oxidase-a]]].

Brunner's syndrome (BRUNS) is a disease associated with a mutation in the MAOA gene that results in a deficiency of MAO-A and as a result causes mild intellectual disability and aggressive behavior in males. Female heterzygotes for this mutation exhibit no mental retardation or aggressive behavior [https://ghr.nlm.nih.gov/gene/MAOA#conditions].
Many other diseases and disorders are associated with mutations in the MAOA gene, such as: Alzheimer's disease, bipolar affective disorder, antisocial behavior, major depressive disorder, and ADHD.

== Inhibition and Research ==
Monoamine oxidase inhibitors (MAOIs) are among the most effective anti-depressants available, however, many of these inhibit MAO-A alongside the target MAO-B, which results in the need to be on a very strict diet while on MAOIs. This is because MAO-A is responsible for oxidizing tyramine, a monoamine found in many different foods and drinks, has been found to have a strong effect on blood pressure. Due to MAO-As effect on blood pressure and digestion those whom are prescribed MAOIs often have to follow a strict diet to prevent side effects such as dizziness, nausea, drowsiness, low blood pressure, and insomnia from occuring. The diet requires that the patient avoid: alcohol, aged cheeses, cured meats, sauerkraut, tofu, fava beans, dried fruit, fermented soy products, and fish or meat refrigerated more than 24 hours [http://www.healthline.com/health/depression/monoamine-oxidase-inhibitors-maois]. Due to the affect MAOIs have on neurotransmitters and digestive enzymes they are rarely used as an antidepressant today, even though they are very effective when prescribed to individuals in which other anti-depressants have not worked.
A newer form of MAOI that has just recently come on the market, Selegiline, works by inhibiting primarily MAO-B as opposed to MAO-A; highly limiting the likeliness of side effects that are influenced by the excessive breakdown of tyramine. Isoform specific inhibitors are certainly a target for future research into treating MAO influenced disorders; particularly in child and adlolescent psychiatry [https://books.google.com/books?id=MEso7oyWMywC&pg=PA294&lpg=PA294&dq=future+research+in+monoamine+oxidase&source=bl&ots=FDX0pxkW6V&sig=xSSZ9HVigEdOTxEOTtyOti8_-oY&hl=en&sa=X&ved=0ahUKEwic4sCt5MPMAhULaT4KHWsmAOIQ6AEIVDAH#v=onepage&q=future%20research%20in%20monoamine%20oxidase&f=false].

== References ==
<references/>

Sandbox WWC2

2016-05-13T12:05:45Z

Dana Emmert:

<Structure load='2Z5X' size='500' frame='top' align='right' caption='''Crystal structure of human monoamine oxidase A complex with Harmine''' scene='69/696303/Ntocrainbow/1' />

==Introduction==

Monoamine oxidase A (MAO-A) is an oxidoreductase flavoenzyme, encoded by the MAOA gene on the X chromosome; the enzyme is present throughout the brain, central nervous system, and stomach. Two isoforms of this enzyme (MAO-B being the other) are expressed on the outer surface of the mitochondrial membrane, and both are responsible for the oxidative deamination of various monoamine neurotransmitters and dietary amines [http://www.omim.org/entry/309850]. Each isoform is characterized by its unique substrate specificity and inhibitor sensitivity. For example, MAO-A (found primarily in catecholaminergic neurons) preferentially oxidizes 5-hydroxytryptamine (5-HT), epinephrine, and norepinephrine; while MAO-B (found primarily in serotonergic neurons) prefers phenylethylamine and benzylamine. Both of these enzymes oxidize dopamine, tyramine, and N,N-dimethyltryptamine as well as their respective unique substrates according to the following reaction:

RCH2NHR' + H2O + O2 --> RCHO + R'NH2 + H2O2

in which the H2O2 is enzymatically removed by glutathione reductase and glutathione peroxidase to produce a reduced glutathione since heightened levels of H2O2 promote apoptosis signalling within cells [https://books.google.com/books?id=6p_cBAAAQBAJ&pg=PA23&lpg=PA23&dq=h2o2+produced+by+mao+neutralized+by&source=bl&ots=xMja8jxpkk&sig=-o5KLxfPBe7m_selXCo9iVt8OuY&hl=en&sa=X&ved=0ahUKEwiDt_qYlJHMAhVIeD4KHcIDC3UQ6AEISzAF#v=onepage&q=h2o2%20produced%20by%20mao%20neutralized%20by&f=false]. Due to the important role MAOs play in controlling the prevalence of various neurotransmitters in the body as well as producing reactive oxygen species (ROS), an imbalance of these enzymes may be detrimental to human health.
http://www.proteopedia.org/wiki/skins/common/images/button_nowiki.png
The secondary structure, illustrated in the protein image to the right, in conjunction with the color key, below, indicates the directionality of the polypeptide chain.
{{Template:ColorKey_Amino2CarboxyRainbow}}

== Structure ==
Unlike its related dimers, human MAO-B and rat MAO-A, human MAO-A is a monomer. The enzyme shares 70% of its amino acid sequence with human MAO-B, and the amino acids interacting with the FAD binding site in both isoforms is conserved; therefore it is believed that the FAD binding sites are quite similar in both proteins <ref name="Edmondson et al"> PMID: 14697881</ref>. MAO-A composed of 527 amino acids, with a molecular weight of approximately 60.5 kDa. The first 497 amino acids in the polypeptide exist in the cytoplasm of the cell; while amino acids 498—518 are involved in an α-helix that anchors the enzyme to the mitochondrial membrane (this can clearly be seen in the provided crystal structure as the only α-helix that significantly sticks out away from the rest of the globular structure). Furthermore, amino acids 519—527 are present in the intermembrane space of the mitochondria [http://www.uniprot.org/uniprot/P2139].
The active site of MAO-A is a single hydrophobic pocket, lined with 11 aliphatic and 5 aromatic residues, with a volume of approximately 550 cubic Å [http://www.pnas.org/content/102/36/12684.full]. Moreover, the active site of human MAO-A is completely unique due to a loop-like conformation of residues 210-216, this is thought to be the result of interactions taking place across the monomer, which allow for the development of inhibitors specific to human MAO-A [http://www.ncbi.nlm.nih.gov/pubmed/16129825]. There are two crucial differences in the active sites of human MAO-A and -B, these include residues Phe-208 (Ile-199 in human MAO B) and Ile-335 (Tyr-326 in human hMAO B) <ref name="Colibus et al"> PMID: 16129825</ref>. Bach et al (1988) showed that the FAD is covalently bound to cysteine residues in both human MAO-A and -B, on Cys397 and Cys406, respectively [http://thirdworld.nl/the-fad-binding-sites-of-human-monoamine-oxidases-a-and-b].

== Function ==
As previously stated, the function of MAO-A is to deaminate 5-hydroxytryptamine (5-HT), epinephrine, norepinephrine, dopamine, tyramine, and N,N-dimethyltryptamine (DMT). When neurons release neurotransmitters into the synaptic cleft to bind to a receptor, some of the neurotransmitters do not bind to the receptor and are not reabsorbed. The monoamine oxidase enzymes are responsible for oxidizing some of these "free" neurotransmitters in an attempt to prevent their binding to unintended receptors, potentially causing deleterious side effects; they are also responsible for maintaining a steady state of certain neurotransmitters.
Although MAO-A is found concentrated in catecholaminergic neurons, it is primarily found in the digestive system, while MAO-B is found more abundantly in the brain.
There are two prevalent polymorphisms of the human MAOA gene, these include the low activity form (MAOA-L), which produces a smaller amount of the enzyme, and high activity form (MAOA-H), which results in the production of high levels of MAO-A [https://news.brown.edu/articles/2009/01/hotsauce]. Recent studies have found that individuals with the MAOA-L gene tend to display slightly higher levels of aggression than individuals with the MAOA-H gene (especially in the case of high-provocation situations). [http://scholar.harvard.edu/files/dtingley/files/pnas-2009.pdf]

Due to this association between aggression and MAO-A polymorphisms, the human MAOA gene has been dubbed the "warrior gene".

== Disease ==
Due to the important role MAO-A plays in maintaining a balance of various neurotransmitters in the digestive and central nervous system, as well as its production of ROS, an imbalance of MAO-A may result in any of several diseases. For example, postpartum depression is a moderate to severe form of depression that occurs as the result of an excess of MAO-A in the brain that some women experience after childbirth [http://www.mpg.de]. In research conducted by Julia Sacher of the Max Planck Institute for Human Cognitive and Brain Sciences, it was concluded that the levels of MAO-A in women with postpartum depression are about 40% greater than those of average women. [https://www.mpg.de/8331073/postpartum-depression_monoamine-oxidase-a]
[[Image:Postpardam_depression_maoa.jpg|thumb|right|”Positron-Emission-Tomography (PET) of a depressive patient without medication (left) with elevated monoamine-oxidase-A-levels (green, yellow, red) and after a six-week-treatment with the monoamine-oxidase-A-inhibitor moclobemid (right)” [https://www.mpg.de/8331073/postpartum-depression_monoamine-oxidase-a]]].

Brunner's syndrome (BRUNS) is a disease associated with a mutation in the MAOA gene that results in a deficiency of MAO-A and as a result causes mild intellectual disability and aggressive behavior in males. Female heterzygotes for this mutation exhibit no mental retardation or aggressive behavior [https://ghr.nlm.nih.gov/gene/MAOA#conditions].
Many other diseases and disorders are associated with mutations in the MAOA gene, such as: Alzheimer's disease, bipolar affective disorder, antisocial behavior, major depressive disorder, and ADHD.

== Inhibition and Research ==
Monoamine oxidase inhibitors (MAOIs) are among the most effective anti-depressants available, however, many of these inhibit MAO-A alongside the target MAO-B, which results in the need to be on a very strict diet while on MAOIs. This is because MAO-A is responsible for oxidizing tyramine, a monoamine found in many different foods and drinks, has been found to have a strong effect on blood pressure. Due to MAO-As effect on blood pressure and digestion those whom are prescribed MAOIs often have to follow a strict diet to prevent side effects such as dizziness, nausea, drowsiness, low blood pressure, and insomnia from occuring. The diet requires that the patient avoid: alcohol, aged cheeses, cured meats, sauerkraut, tofu, fava beans, dried fruit, fermented soy products, and fish or meat refrigerated more than 24 hours [http://www.healthline.com/health/depression/monoamine-oxidase-inhibitors-maois]. Due to the affect MAOIs have on neurotransmitters and digestive enzymes they are rarely used as an antidepressant today, even though they are very effective when prescribed to individuals in which other anti-depressants have not worked.
A newer form of MAOI that has just recently come on the market, Selegiline, works by inhibiting primarily MAO-B as opposed to MAO-A; highly limiting the likeliness of side effects that are influenced by the excessive breakdown of tyramine. Isoform specific inhibitors are certainly a target for future research into treating MAO influenced disorders; particularly in child and adlolescent psychiatry [https://books.google.com/books?id=MEso7oyWMywC&pg=PA294&lpg=PA294&dq=future+research+in+monoamine+oxidase&source=bl&ots=FDX0pxkW6V&sig=xSSZ9HVigEdOTxEOTtyOti8_-oY&hl=en&sa=X&ved=0ahUKEwic4sCt5MPMAhULaT4KHWsmAOIQ6AEIVDAH#v=onepage&q=future%20research%20in%20monoamine%20oxidase&f=false].

== References ==
<references/>

Sandbox WWC2

2016-05-13T12:03:19Z

Dana Emmert:

<Structure load='2Z5X' size='500' frame='top' align='right' caption='''Crystal structure of human monoamine oxidase A complex with Harmine''' scene='69/696303/Ntocrainbow/1' />

==Introduction==

Monoamine oxidase A (MAO-A) is an oxidoreductase flavoenzyme, encoded by the MAOA gene on the X chromosome; the enzyme is present throughout the brain, central nervous system, and stomach. Two isoforms of this enzyme (MAO-B being the other) are expressed on the outer surface of the mitochondrial membrane, and both are responsible for the oxidative deamination of various monoamine neurotransmitters and dietary amines [http://www.omim.org/entry/309850]. Each isoform is characterized by its unique substrate specificity and inhibitor sensitivity. For example, MAO-A (found primarily in catecholaminergic neurons) preferentially oxidizes 5-hydroxytryptamine (5-HT), epinephrine, and norepinephrine; while MAO-B (found primarily in serotonergic neurons) prefers phenylethylamine and benzylamine. Both of these enzymes oxidize dopamine, tyramine, and N,N-dimethyltryptamine as well as their respective unique substrates according to the following reaction:

RCH2NHR' + H2O + O2 --> RCHO + R'NH2 + H2O2

in which the H2O2 is enzymatically removed by glutathione reductase and glutathione peroxidase to produce a reduced glutathione since heightened levels of H2O2 promote apoptosis signalling within cells [https://books.google.com/books?id=6p_cBAAAQBAJ&pg=PA23&lpg=PA23&dq=h2o2+produced+by+mao+neutralized+by&source=bl&ots=xMja8jxpkk&sig=-o5KLxfPBe7m_selXCo9iVt8OuY&hl=en&sa=X&ved=0ahUKEwiDt_qYlJHMAhVIeD4KHcIDC3UQ6AEISzAF#v=onepage&q=h2o2%20produced%20by%20mao%20neutralized%20by&f=false]. Due to the important role MAOs play in controlling the prevalence of various neurotransmitters in the body as well as producing reactive oxygen species (ROS), an imbalance of these enzymes may be detrimental to human health.
http://www.proteopedia.org/wiki/skins/common/images/button_nowiki.png
The secondary structure, illustrated in the protein image to the right, in conjunction with the color key, below, indicates the directionality of the polypeptide chain.
{{Template:ColorKey_Amino2CarboxyRainbow}}

== Structure ==
Unlike its related dimers, human MAO-B and rat MAO-A, human MAO-A is a monomer. The enzyme shares 70% of its amino acid sequence with human MAO-B, and the amino acids interacting with the FAD binding site in both isoforms is conserved; therefore it is believed that the FAD binding sites are quite similar in both proteins <ref name="Edmondson et al"> PMID: 14697881</ref>. MAO-A composed of 527 amino acids, with a molecular weight of approximately 60.5 kDa. The first 497 amino acids in the polypeptide exist in the cytoplasm of the cell; while amino acids 498—518 are involved in an α-helix that anchors the enzyme to the mitochondrial membrane (this can clearly be seen in the provided crystal structure as the only α-helix that significantly sticks out away from the rest of the globular structure). Furthermore, amino acids 519—527 are present in the intermembrane space of the mitochondria [http://www.uniprot.org/uniprot/P2139].
The active site of MAO-A is a single hydrophobic pocket, lined with 11 aliphatic and 5 aromatic residues, with a volume of approximately 550 cubic Å [http://www.pnas.org/content/102/36/12684.full]. Moreover, the active site of human MAO-A is completely unique due to a loop-like conformation of residues 210-216, this is thought to be the result of interactions taking place across the monomer, which allow for the development of inhibitors specific to human MAO-A [http://www.ncbi.nlm.nih.gov/pubmed/16129825]. There are two crucial differences in the active sites of human MAO-A and -B, these include residues Phe-208 (Ile-199 in human MAO B) and Ile-335 (Tyr-326 in human hMAO B) <ref name="Colibus et al"> PMID: 16129825</ref>. Bach et al (1988) showed that the FAD is covalently bound to cysteine residues in both human MAO-A and -B, on Cys397 and Cys406, respectively [http://thirdworld.nl/the-fad-binding-sites-of-human-monoamine-oxidases-a-and-b].

== Function ==
As previously stated, the function of MAO-A is to deaminate 5-hydroxytryptamine (5-HT), epinephrine, norepinephrine, dopamine, tyramine, and N,N-dimethyltryptamine (DMT). When neurons release neurotransmitters into the synaptic cleft to bind to a receptor, some of the neurotransmitters do not bind to the receptor and are not reabsorbed. The monoamine oxidase enzymes are responsible for oxidizing some of these "free" neurotransmitters in an attempt to prevent their binding to unintended receptors, potentially causing deleterious side effects; they are also responsible for maintaining a steady state of certain neurotransmitters.
Although MAO-A is found concentrated in catecholaminergic neurons, it is primarily found in the digestive system, while MAO-B is found more abundantly in the brain.
There are two prevalent polymorphisms of the human MAOA gene, these include the low activity form (MAOA-L), which produces a smaller amount of the enzyme, and high activity form (MAOA-H), which results in the production of high levels of MAO-A [https://news.brown.edu/articles/2009/01/hotsauce]. Recent studies have found that individuals with the MAOA-L gene tend to display slightly higher levels of aggression than individuals with the MAOA-H gene (especially in the case of high-provocation situations). [http://scholar.harvard.edu/files/dtingley/files/pnas-2009.pdf]

Due to this association between aggression and MAO-A polymorphisms, the human MAOA gene has been dubbed the "warrior gene".

== Disease ==
Due to the important role MAO-A plays in maintaining a balance of various neurotransmitters in the digestive and central nervous system, as well as its production of ROS, an imbalance of MAO-A may result in any of several diseases. For example, postpartum depression is a moderate to severe form of depression that occurs as the result of an excess of MAO-A in the brain that some women experience after childbirth [http://www.mpg.de]. In research conducted by Julia Sacher of the Max Planck Institute for Human Cognitive and Brain Sciences, it was concluded that the levels of MAO-A in women with postpartum depression are about 40% greater than those of average women. [[Image:Postpardam_depression_maoa.jpg|thumb|right|”Positron-Emission-Tomography (PET) of a depressive patient without medication (left) with elevated monoamine-oxidase-A-levels (green, yellow, red) and after a six-week-treatment with the monoamine-oxidase-A-inhibitor moclobemid (right)” [https://www.mpg.de/8331073/postpartum-depression_monoamine-oxidase-a]]].

Brunner's syndrome (BRUNS) is a disease associated with a mutation in the MAOA gene that results in a deficiency of MAO-A and as a result causes mild intellectual disability and aggressive behavior in males. Female heterzygotes for this mutation exhibit no mental retardation or aggressive behavior [https://ghr.nlm.nih.gov/gene/MAOA#conditions].
Many other diseases and disorders are associated with mutations in the MAOA gene, such as: Alzheimer's disease, bipolar affective disorder, antisocial behavior, major depressive disorder, and ADHD.

== Inhibition and Research ==
Monoamine oxidase inhibitors (MAOIs) are among the most effective anti-depressants available, however, many of these inhibit MAO-A alongside the target MAO-B, which results in the need to be on a very strict diet while on MAOIs. This is because MAO-A is responsible for oxidizing tyramine, a monoamine found in many different foods and drinks, has been found to have a strong effect on blood pressure. Due to MAO-As effect on blood pressure and digestion those whom are prescribed MAOIs often have to follow a strict diet to prevent side effects such as dizziness, nausea, drowsiness, low blood pressure, and insomnia from occuring. The diet requires that the patient avoid: alcohol, aged cheeses, cured meats, sauerkraut, tofu, fava beans, dried fruit, fermented soy products, and fish or meat refrigerated more than 24 hours [http://www.healthline.com/health/depression/monoamine-oxidase-inhibitors-maois]. Due to the affect MAOIs have on neurotransmitters and digestive enzymes they are rarely used as an antidepressant today, even though they are very effective when prescribed to individuals in which other anti-depressants have not worked.
A newer form of MAOI that has just recently come on the market, Selegiline, works by inhibiting primarily MAO-B as opposed to MAO-A; highly limiting the likeliness of side effects that are influenced by the excessive breakdown of tyramine. Isoform specific inhibitors are certainly a target for future research into treating MAO influenced disorders; particularly in child and adlolescent psychiatry [https://books.google.com/books?id=MEso7oyWMywC&pg=PA294&lpg=PA294&dq=future+research+in+monoamine+oxidase&source=bl&ots=FDX0pxkW6V&sig=xSSZ9HVigEdOTxEOTtyOti8_-oY&hl=en&sa=X&ved=0ahUKEwic4sCt5MPMAhULaT4KHWsmAOIQ6AEIVDAH#v=onepage&q=future%20research%20in%20monoamine%20oxidase&f=false].

== References ==
<references/>

Sandbox WWC11

2016-05-13T11:59:25Z

Dana Emmert:

{{STRUCTURE_1myp| PDB=1myp | SIZE=400| SCENE= |right|CAPTION=Dog glycosylated myeloperoxidase dimer: heavy chain (pink and yellow), light chain (grey and green) containing heme complex with Ca+2 ions (green), [[1D5L]] }}

----
'''Introduction'''

Myeloperoxidase (MPO) is a protein found in neutrophil, the most common white blood cell. This enzyme generates hypochlorous acid (HOCl) from hydrogen peroxide in addition to other hypohalous acids depending on what is available in the cell. The HOCl is then used, in combination with other molecules, in an oxidative burst that kills bacteria and other potentially harmful invaders once taken up by the cell. This protein falls into a group of heme peroxidase enzymes. The role of these enzymes is to oxidize molecules using heme.<ref name="Hampton">PMID:9787133</ref>

This protein is composed of two identical monomers linked by a cystine bridge. The total molecular weight of this protein is 146 kDa. These two monomers can work independently of each other and consist of a light and heavy amino acid chain. The active site on the protein consists of a deep crevice where a heme molecule is covalently attached. The deep pocket is believedto limit access to the heme and increase specificity for smaller molecules to be oxidized. This pocket also possesses a hydrophobic region for substrate stabilization.<ref name="Active Site Structure">PMID:16288920</ref>

A lack of or mutation in this enzyme can cause immune deficiency, although most individuals with this mutation seem to be able to compensate with other, still unknown mechanisms.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> Over activation of myloperoxidase has been linked to increased inflammation and is being studied in relation to inceased risk of cardiovascular disease.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> There are still many unknowns surrounding the complete function and role that myeloperoxidase plays in the body's immune response. However, it has become clear that this protein is an integral piece in the immune response and warrants further research.

'''Structure'''

[[Image:Heme_site.JPG]]

''Figure 1. Depicts key amino acids in stabilization of heme in myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:26884610</ref>

Myeloperoxidase is synthesized in bone marrow along with most other white blood cell components. The myloperoxidase enzyme is composed of two identical subunits. After translation of these subunits, they are cleaved into two parts: the heavy chain and the light chain. The heavy chain is a glycosylated domain that weighs approximately 58.5 kDa. This portion of the enzyme has the deep pocket where the heme is inserted by chaperones (calreticulin and calnexin). <ref name="Neutrophil">PMID:26904693</ref> Click on the green link to see the <scene name='69/696303/Heme_pocket_mpo/1'>MPO heme pocket</scene> in the structure above. The amino acid make up of heme pocket can be seen in Figure 1 above. The pink in this picture is the light chain, the blue is the heavy chain. The light chain (about 13.5 kDa) is attached to the heavy chain through disufide bonds. There have been discoveries of slight variation in the primary sequence of myeloperoxidase. However, for the most part these slight variations do not affect the enzymatic activity. Currently three isoforms have been isolated.<ref name="Enzymatic Activity">PMID:120019</ref>

'''Function'''

[[Image:Enzymatic_processes_of_myeloperoxidase.JPG]]

''Figure 2. The Enzymatic Activity of Myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:21297906</ref>

As stated above, the role of the myeloperoxidase in the immune response is to generate oxidative molecules that are used to damage invaders. Seen above in Figure 2 is a concise and basic diagram of the process. Compounds I, II and III represent myeloperoxidase with different oxidation states of the iron in the heme molecule. One of the processes facilitated by myeloperoxidase is to oxidize hydrogen peroxide (H2O2) and cause the iron to change oxidation states. In this state, the iron is then able to catalyze the production of the hypohalous acids. These molecules are able to wreak havoc on lipids, proteins and DNA. <ref name="Neutrophil">PMID:26904693</ref> These modifications are achieved by the oxidation (usually by the addition of the halogen) of the unlucky recipient. The heme iron can also be oxidized by radicals to form compounds II and III. The functions of these are less well known, but the production of these radicals is also helpful in the antibacterial response.<ref name="Myeloperoxidase">PMID:26884610</ref>

Now that the enzymatic properties of myeloperoxidase have been explored, it is time to turn to the bigger picture. These enzymes are stored in the subtype of leukocytes (white blood cells) called neutrophils. These are thought to be one of the first cells types to arrive at the site of infection.<ref name="Neutrophil">PMID:26904693</ref> An outline of the immune response of neutrophil, specifically highlighting MPO's role, can be seen in Figure 3 below. There are two paths seen in this figure. In the first path bacteria is taken up by neutrophil extracellular traps or NETs and then destroyed by the hypohalogous acids. The second path shows the MPO being released, signaled by the dendrite cells(DCs), and helping to destroy the pathogen.<ref name="Neutrophil">PMID:26904693</ref> ANCA stands for anti-neutrophil cytoplasmic antibody. The MPOs have a range of function that are steered by the neutophil cells. This intense oxidative power is very helpful when targeted to foreign bodies. However, as discussed in the next section, MPO can very destructive if released on our own tissue.

[[Image:Neutrophil_Process.JPG]]

''Figure 3. The immune response of neutrophils with specific emphasis on myeloperoxidase.''<ref name="Neutrophil">PMID:26904693</ref>

'''Implication of Potential Defects or Deficiencies'''

Individuals with myeloperoxidase deficiency have been found to have a slight increased risk of chronic or severe infection. However, this condition is not fatal and the body seems to be able to compensate with other immune system mechanisms. <ref name="ANCA">PMID:11111115</ref> The most common source of MPO deficiency is ANCA (antineutrophil cytoplasmic antibodies)-associated vasculitis (AAV). This is an autoimmune disease that causes the production of antibodies for enzymes like MPO. <ref name="ANCA">PMID:26986103</ref> However, these diseases have also been linked to a decrease in risk of cardiovascular disease.<ref name="ANCA">PMID:11111115</ref>

'''Correlation to Cardiovascular Disease'''

Although MPO plays a key role in the fight against unwanted intruders, it has also been shown to turn those destructive powers against the body. Elevated levels of MPO in the blood has been found in patients with cardiovascular disease. <ref name="CAD">PMID:11694155</ref> It is speculated that over activation of the immune response causes inflammation, or damage to the surrounding tissue which increases risk of cardiovascular disease. More specifically, when MPO generate the oxidative agents such as HOCl, it not only chloronate invaders, but can also chloronate lipoproteins (both LDL and HDL). This changes the lipoprotein's affinity for the cell wall which can lead to plaque buildup. It can also effects the endothelial cells lining the vein or artery.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref>

Another of the identified affects of MPO on endothelial cells is in the production of nitric oxide (NO). This is a vasodilator that is inhibited when oxidized by MPO. Myloperoxidase can also oxidize NO2 - to its radical form which has been identified as a major contributor to inflammation.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref> Because of this clear correlation between MPO activity and the manifestation of cardiovascular disease, it has the potential to be used as an indicator of a cardiovascular problem.<ref name="Use in Medicine">PMID:12865732</ref>
==3D structures of myeloperoxidase==

Updated on {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

[[1myp]] – MPO – dog 
[[1mhl]], [[1cxp]] – hMPO C – human 
[[3f9p]] - hMPO 
[[1qo4]] - MPO – ''Arabidopsis thaliana'' 
[[1d2v]] – hMPO C + Br 
[[1d5l]] – hMPO + CN 
[[1d7w]] - hMPO + CN + Br 
[[1dnu]] - hMPO + SCN 
[[1dnw]] - hMPO + CN + SCN 
[[3zs0]], [[3zs1]], [[4c1m]] - hMPO + inhibitor 
[[4dl1]] - hMPO + thioxanthine 
[[4ejx]] - hMPO + ceruplasmin 
[[Category:Topic Page]]

== References ==
<references/>

Sandbox WWC2

2016-05-13T11:57:05Z

Dana Emmert:

<Structure load='2Z5X' size='500' frame='top' align='right' caption='''Crystal structure of human monoamine oxidase A complex with Harmine''' scene='69/696303/Ntocrainbow/1' />

==Introduction==

Monoamine oxidase A (MAO-A) is an oxidoreductase flavoenzyme, encoded by the MAOA gene on the X chromosome; the enzyme is present throughout the brain, central nervous system, and stomach. Two isoforms of this enzyme (MAO-B being the other) are expressed on the outer surface of the mitochondrial membrane, and both are responsible for the oxidative deamination of various monoamine neurotransmitters and dietary amines [http://www.omim.org/entry/309850]. Each isoform is characterized by its unique substrate specificity and inhibitor sensitivity. For example, MAO-A (found primarily in catecholaminergic neurons) preferentially oxidizes 5-hydroxytryptamine (5-HT), epinephrine, and norepinephrine; while MAO-B (found primarily in serotonergic neurons) prefers phenylethylamine and benzylamine. Both of these enzymes oxidize dopamine, tyramine, and N,N-dimethyltryptamine as well as their respective unique substrates according to the following reaction:

RCH2NHR' + H2O + O2 --> RCHO + R'NH2 + H2O2

in which the H2O2 is enzymatically removed by glutathione reductase and glutathione peroxidase to produce a reduced glutathione since heightened levels of H2O2 promote apoptosis signalling within cells [https://books.google.com/books?id=6p_cBAAAQBAJ&pg=PA23&lpg=PA23&dq=h2o2+produced+by+mao+neutralized+by&source=bl&ots=xMja8jxpkk&sig=-o5KLxfPBe7m_selXCo9iVt8OuY&hl=en&sa=X&ved=0ahUKEwiDt_qYlJHMAhVIeD4KHcIDC3UQ6AEISzAF#v=onepage&q=h2o2%20produced%20by%20mao%20neutralized%20by&f=false]. Due to the important role MAOs play in controlling the prevalence of various neurotransmitters in the body as well as producing reactive oxygen species (ROS), an imbalance of these enzymes may be detrimental to human health.
http://www.proteopedia.org/wiki/skins/common/images/button_nowiki.png
The secondary structure, illustrated in the protein image to the right, in conjunction with the color key, below, indicates the directionality of the polypeptide chain.
{{Template:ColorKey_Amino2CarboxyRainbow}}

== Structure ==
Unlike its related dimers, human MAO-B and rat MAO-A, human MAO-A is a monomer. The enzyme shares 70% of its amino acid sequence with human MAO-B, and the amino acids interacting with the FAD binding site in both isoforms is conserved; therefore it is believed that the FAD binding sites are quite similar in both proteins <ref name="Edmondson et al"> PMID: 14697881</ref>. MAO-A composed of 527 amino acids, with a molecular weight of approximately 60.5 kDa. The first 497 amino acids in the polypeptide exist in the cytoplasm of the cell; while amino acids 498—518 are involved in an α-helix that anchors the enzyme to the mitochondrial membrane (this can clearly be seen in the provided crystal structure as the only α-helix that significantly sticks out away from the rest of the globular structure). Furthermore, amino acids 519—527 are present in the intermembrane space of the mitochondria [http://www.uniprot.org/uniprot/P2139].
The active site of MAO-A is a single hydrophobic pocket, lined with 11 aliphatic and 5 aromatic residues, with a volume of approximately 550 cubic Å [http://www.pnas.org/content/102/36/12684.full]. Moreover, the active site of human MAO-A is completely unique due to a loop-like conformation of residues 210-216, this is thought to be the result of interactions taking place across the monomer, which allow for the development of inhibitors specific to human MAO-A [http://www.ncbi.nlm.nih.gov/pubmed/16129825]. There are two crucial differences in the active sites of human MAO-A and -B, these include residues Phe-208 (Ile-199 in human MAO B) and Ile-335 (Tyr-326 in human hMAO B) <ref name="Colibus et al"> PMID: 16129825</ref>. Bach et al (1988) showed that the FAD is covalently bound to cysteine residues in both human MAO-A and -B, on Cys397 and Cys406, respectively [http://thirdworld.nl/the-fad-binding-sites-of-human-monoamine-oxidases-a-and-b].

== Function ==
As previously stated, the function of MAO-A is to deaminate 5-hydroxytryptamine (5-HT), epinephrine, norepinephrine, dopamine, tyramine, and N,N-dimethyltryptamine (DMT). When neurons release neurotransmitters into the synaptic cleft to bind to a receptor, some of the neurotransmitters do not bind to the receptor and are not reabsorbed. The monoamine oxidase enzymes are responsible for oxidizing some of these "free" neurotransmitters in an attempt to prevent their binding to unintended receptors, potentially causing deleterious side effects; they are also responsible for maintaining a steady state of certain neurotransmitters.
Although MAO-A is found concentrated in catecholaminergic neurons, it is primarily found in the digestive system, while MAO-B is found more abundantly in the brain.
There are two prevalent polymorphisms of the human MAOA gene, these include the low activity form (MAOA-L), which produces a smaller amount of the enzyme, and high activity form (MAOA-H), which results in the production of high levels of MAO-A [https://news.brown.edu/articles/2009/01/hotsauce]. Recent studies have found that individuals with the MAOA-L gene tend to display slightly higher levels of aggression than individuals with the MAOA-H gene (especially in the case of high-provocation situations). It has also been shown that up to two thirds of the individuals in populations with a high rate of warfare in their history possess the MAOA-L gene. Due to this association between aggression and MAO-A polymorphisms, the human MAOA gene has been dubbed the "warrior gene".

== Disease ==
Due to the important role MAO-A plays in maintaining a balance of various neurotransmitters in the digestive and central nervous system, as well as its production of ROS, an imbalance of MAO-A may result in any of several diseases. For example, postpartum depression is a moderate to severe form of depression that occurs as the result of an excess of MAO-A in the brain that some women experience after childbirth [http://www.mpg.de]. In research conducted by Julia Sacher of the Max Planck Institute for Human Cognitive and Brain Sciences, it was concluded that the levels of MAO-A in women with postpartum depression are about 40% greater than those of average women. [[Image:Postpardam_depression_maoa.jpg|thumb|right|”Positron-Emission-Tomography (PET) of a depressive patient without medication (left) with elevated monoamine-oxidase-A-levels (green, yellow, red) and after a six-week-treatment with the monoamine-oxidase-A-inhibitor moclobemid (right)” [https://www.mpg.de/8331073/postpartum-depression_monoamine-oxidase-a]]].

Brunner's syndrome (BRUNS) is a disease associated with a mutation in the MAOA gene that results in a deficiency of MAO-A and as a result causes mild intellectual disability and aggressive behavior in males. Female heterzygotes for this mutation exhibit no mental retardation or aggressive behavior [https://ghr.nlm.nih.gov/gene/MAOA#conditions].
Many other diseases and disorders are associated with mutations in the MAOA gene, such as: Alzheimer's disease, bipolar affective disorder, antisocial behavior, major depressive disorder, and ADHD.

== Inhibition and Research ==
Monoamine oxidase inhibitors (MAOIs) are among the most effective anti-depressants available, however, many of these inhibit MAO-A alongside the target MAO-B, which results in the need to be on a very strict diet while on MAOIs. This is because MAO-A is responsible for oxidizing tyramine, a monoamine found in many different foods and drinks, has been found to have a strong effect on blood pressure. Due to MAO-As effect on blood pressure and digestion those whom are prescribed MAOIs often have to follow a strict diet to prevent side effects such as dizziness, nausea, drowsiness, low blood pressure, and insomnia from occuring. The diet requires that the patient avoid: alcohol, aged cheeses, cured meats, sauerkraut, tofu, fava beans, dried fruit, fermented soy products, and fish or meat refrigerated more than 24 hours [http://www.healthline.com/health/depression/monoamine-oxidase-inhibitors-maois]. Due to the affect MAOIs have on neurotransmitters and digestive enzymes they are rarely used as an antidepressant today, even though they are very effective when prescribed to individuals in which other anti-depressants have not worked.
A newer form of MAOI that has just recently come on the market, Selegiline, works by inhibiting primarily MAO-B as opposed to MAO-A; highly limiting the likeliness of side effects that are influenced by the excessive breakdown of tyramine. Isoform specific inhibitors are certainly a target for future research into treating MAO influenced disorders; particularly in child and adlolescent psychiatry [https://books.google.com/books?id=MEso7oyWMywC&pg=PA294&lpg=PA294&dq=future+research+in+monoamine+oxidase&source=bl&ots=FDX0pxkW6V&sig=xSSZ9HVigEdOTxEOTtyOti8_-oY&hl=en&sa=X&ved=0ahUKEwic4sCt5MPMAhULaT4KHWsmAOIQ6AEIVDAH#v=onepage&q=future%20research%20in%20monoamine%20oxidase&f=false].

== References ==
<references/>

Sandbox WWC11

2016-05-13T11:56:00Z

Dana Emmert:

{{STRUCTURE_1myp| PDB=1myp | SIZE=400| SCENE= |right|CAPTION=Dog glycosylated myeloperoxidase dimer: heavy chain (pink and yellow), light chain (grey and green) containing heme complex with Ca+2 ions (green), [[1D5L]] }}

-->

----
'''Introduction'''

Myeloperoxidase (MPO) is a protein found in neutrophil, the most common white blood cell. This enzyme generates hypochlorous acid (HOCl) from hydrogen peroxide in addition to other hypohalous acids depending on what is available in the cell. The HOCl is then used, in combination with other molecules, in an oxidative burst that kills bacteria and other potentially harmful invaders once taken up by the cell. This protein falls into a group of heme peroxidase enzymes. The role of these enzymes is to oxidize molecules using heme.<ref name="Hampton">PMID:9787133</ref>

This protein is composed of two identical monomers linked by a cystine bridge. The total molecular weight of this protein is 146 kDa. These two monomers can work independently of each other and consist of a light and heavy amino acid chain. The active site on the protein consists of a deep crevice where a heme molecule is covalently attached. The deep pocket is believedto limit access to the heme and increase specificity for smaller molecules to be oxidized. This pocket also possesses a hydrophobic region for substrate stabilization.<ref name="Active Site Structure">PMID:16288920</ref>

A lack of or mutation in this enzyme can cause immune deficiency, although most individuals with this mutation seem to be able to compensate with other, still unknown mechanisms.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> Over activation of myloperoxidase has been linked to increased inflammation and is being studied in relation to inceased risk of cardiovascular disease.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> There are still many unknowns surrounding the complete function and role that myeloperoxidase plays in the body's immune response. However, it has become clear that this protein is an integral piece in the immune response and warrants further research.

'''Structure'''

[[Image:Heme_site.JPG]]

''Figure 1. Depicts key amino acids in stabilization of heme in myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:26884610</ref>

Myeloperoxidase is synthesized in bone marrow along with most other white blood cell components. The myloperoxidase enzyme is composed of two identical subunits. After translation of these subunits, they are cleaved into two parts: the heavy chain and the light chain. The heavy chain is a glycosylated domain that weighs approximately 58.5 kDa. This portion of the enzyme has the deep pocket where the heme is inserted by chaperones (calreticulin and calnexin). <ref name="Neutrophil">PMID:26904693</ref> Click on the green link to see the <scene name='69/696303/Heme_pocket_mpo/1'>TextToBeDisplayed</scene>. The amino acid make up of heme pocket can be seen in Figure 1 above. The pink in this picture is the light chain, the blue is the heavy chain. The light chain (about 13.5 kDa) is attached to the heavy chain through disufide bonds. There have been discoveries of slight variation in the primary sequence of myeloperoxidase. However, for the most part these slight variations do not affect the enzymatic activity. Currently three isoforms have been isolated.<ref name="Enzymatic Activity">PMID:120019</ref>

'''Function'''

[[Image:Enzymatic_processes_of_myeloperoxidase.JPG]]

''Figure 2. The Enzymatic Activity of Myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:21297906</ref>

As stated above, the role of the myeloperoxidase in the immune response is to generate oxidative molecules that are used to damage invaders. Seen above in Figure 2 is a concise and basic diagram of the process. Compounds I, II and III represent myeloperoxidase with different oxidation states of the iron in the heme molecule. One of the processes facilitated by myeloperoxidase is to oxidize hydrogen peroxide (H2O2) and cause the iron to change oxidation states. In this state, the iron is then able to catalyze the production of the hypohalous acids. These molecules are able to wreak havoc on lipids, proteins and DNA. <ref name="Neutrophil">PMID:26904693</ref> These modifications are achieved by the oxidation (usually by the addition of the halogen) of the unlucky recipient. The heme iron can also be oxidized by radicals to form compounds II and III. The functions of these are less well known, but the production of these radicals is also helpful in the antibacterial response.<ref name="Myeloperoxidase">PMID:26884610</ref>

Now that the enzymatic properties of myeloperoxidase have been explored, it is time to turn to the bigger picture. These enzymes are stored in the subtype of leukocytes (white blood cells) called neutrophils. These are thought to be one of the first cells types to arrive at the site of infection.<ref name="Neutrophil">PMID:26904693</ref> An outline of the immune response of neutrophil, specifically highlighting MPO's role, can be seen in Figure 3 below. There are two paths seen in this figure. In the first path bacteria is taken up by neutrophil extracellular traps or NETs and then destroyed by the hypohalogous acids. The second path shows the MPO being released, signaled by the dendrite cells(DCs), and helping to destroy the pathogen.<ref name="Neutrophil">PMID:26904693</ref> ANCA stands for anti-neutrophil cytoplasmic antibody. The MPOs have a range of function that are steered by the neutophil cells. This intense oxidative power is very helpful when targeted to foreign bodies. However, as discussed in the next section, MPO can very destructive if released on our own tissue.

[[Image:Neutrophil_Process.JPG]]

''Figure 3. The immune response of neutrophils with specific emphasis on myeloperoxidase.''<ref name="Neutrophil">PMID:26904693</ref>

'''Implication of Potential Defects or Deficiencies'''

Individuals with myeloperoxidase deficiency have been found to have a slight increased risk of chronic or severe infection. However, this condition is not fatal and the body seems to be able to compensate with other immune system mechanisms. <ref name="ANCA">PMID:11111115</ref> The most common source of MPO deficiency is ANCA (antineutrophil cytoplasmic antibodies)-associated vasculitis (AAV). This is an autoimmune disease that causes the production of antibodies for enzymes like MPO. <ref name="ANCA">PMID:26986103</ref> However, these diseases have also been linked to a decrease in risk of cardiovascular disease.<ref name="ANCA">PMID:11111115</ref>

'''Correlation to Cardiovascular Disease'''

Although MPO plays a key role in the fight against unwanted intruders, it has also been shown to turn those destructive powers against the body. Elevated levels of MPO in the blood has been found in patients with cardiovascular disease. <ref name="CAD">PMID:11694155</ref> It is speculated that over activation of the immune response causes inflammation, or damage to the surrounding tissue which increases risk of cardiovascular disease. More specifically, when MPO generate the oxidative agents such as HOCl, it not only chloronate invaders, but can also chloronate lipoproteins (both LDL and HDL). This changes the lipoprotein's affinity for the cell wall which can lead to plaque buildup. It can also effects the endothelial cells lining the vein or artery.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref>

Another of the identified affects of MPO on endothelial cells is in the production of nitric oxide (NO). This is a vasodilator that is inhibited when oxidized by MPO. Myloperoxidase can also oxidize NO2 - to its radical form which has been identified as a major contributor to inflammation.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref> Because of this clear correlation between MPO activity and the manifestation of cardiovascular disease, it has the potential to be used as an indicator of a cardiovascular problem.<ref name="Use in Medicine">PMID:12865732</ref>
==3D structures of myeloperoxidase==

Updated on {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

[[1myp]] – MPO – dog 
[[1mhl]], [[1cxp]] – hMPO C – human 
[[3f9p]] - hMPO 
[[1qo4]] - MPO – ''Arabidopsis thaliana'' 
[[1d2v]] – hMPO C + Br 
[[1d5l]] – hMPO + CN 
[[1d7w]] - hMPO + CN + Br 
[[1dnu]] - hMPO + SCN 
[[1dnw]] - hMPO + CN + SCN 
[[3zs0]], [[3zs1]], [[4c1m]] - hMPO + inhibitor 
[[4dl1]] - hMPO + thioxanthine 
[[4ejx]] - hMPO + ceruplasmin 
[[Category:Topic Page]]

== References ==
<references/>

Sandbox WWC1

2016-05-12T21:18:12Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria under anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the BTX protein, named A through G, that are structurally similar but elicit different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven polypeptides are produced as an inactive chain in the bacterium, but is activated when a protease cleaves the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nontoxic-nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut. <ref name="Simpson"> PMID: 14744243</ref>

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminal of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminal half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntaxin, depends on the serotype of BTX where each serotype cuts at a different place. The cleaved SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref> Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to ''Clostridium botulism'' in their gut and therefore produce antibodies against ''Clostridium botulism'' to prevent the bacteria population from growing to an extreme. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. <ref>Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476.</ref> By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis are reduced. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. <ref>Lewis GE Jr. Approaches to the prophylaxis, immunotherapy,and chemotherapy of botulism. In: Lewis GE Jr, ed. Biomedical Aspects of Botulism. New York, NY: Academic Press; 1981: 261–270</ref> BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T21:08:33Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria under anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the BTX protein, named A through G, that are structurally similar but elicit different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven polypeptides are produced as an inactive chain in the bacterium, but is activated when a protease cleaves the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nontoxic-nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut. <ref name="Simpson"> PMID: 14744243</ref>

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminal of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminal half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntaxin, depends on the serotype of BTX where each serotype cuts at a different place. The cleaved SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref> Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to ''Clostridium botulism'' in their gut and therefore produce antibodies against ''Clostridium botulism'' to prevent the bacteria population from growing to an extreme. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis are reduced. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. <ref>Lewis GE Jr. Approaches to the prophylaxis, immunotherapy,and chemotherapy of botulism. In: Lewis GE Jr, ed. Biomedical Aspects of Botulism. New York, NY: Academic Press; 1981: 261–270</ref> BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T21:03:12Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria under anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the BTX protein, named A through G, that are structurally similar but elicit different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven polypeptides are produced as an inactive chain in the bacterium, but is activated when a protease cleaves the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nontoxic-nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut. <ref name="Simpson"> PMID: 14744243</ref>

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminal of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminal half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntaxin, depends on the serotype of BTX where each serotype cuts at a different place. The cleaved SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref> Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to ''Clostridium botulism'' in their gut and therefore produce antibodies against ''Clostridium botulism'' to prevent the bacteria population from growing to an extreme. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis are reduced. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T20:57:28Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria under anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the BTX protein, named A through G, that are structurally similar but elicit different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven polypeptides are produced as an inactive chain in the bacterium, but is activated when a protease cleaves the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nontoxic-nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut. <ref name="Simpson"> PMID: 14744243</ref>

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminal of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminal half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntaxin, depends on the serotype of BTX where each serotype cuts at a different place. The cleaved SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref> Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to ''Clostridium botulism'' in their gut and therefore produce antibodies against ''Clostridium botulism'' to prevent the bacteria population from growing to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis are reduced. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T20:41:36Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria under anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the BTX protein, named A through G, that are structurally similar but elicit different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven polypeptides are produced as an inactive chain in the bacterium, but is activated when a protease cleaves the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nontoxic-nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminal of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminal half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntaxin, depends on the serotype of BTX where each serotype cuts at a different place. The cleaved SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref> Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to ''Clostridium botulism'' in their gut and therefore produce antibodies against ''Clostridium botulism'' to prevent the bacteria population from growing to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis are reduced. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T20:39:42Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria under anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the BTX protein, named A through G, that are structurally similar but elicit different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven polypeptides are produced as an inactive chain in the bacterium, but is activated when a protease cleaves the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nontoxic-nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminal of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminal half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntaxin, depends on the serotype of BTX where each serotype cuts at a different place. The cleaved SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" />The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to ''Clostridium botulism'' in their gut and therefore produce antibodies against ''Clostridium botulism'' to prevent the bacteria population from growing to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis are reduced. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T16:06:52Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref>

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T16:05:51Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein. <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref> ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals. <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses. <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref> All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references). <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />)

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis. <ref name="Montecucco"> PMID: 8771234</ref> The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis. <ref name="Dolly"> PMID: 6694738</ref> Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis. <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E. <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref> If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally. <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref>

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq. <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months. <ref name="Nayyar"> PMID: 25654058</ref> Some of these diseases include cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction. <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref> By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T16:01:26Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref name="Montecucco"> PMID: 8771234</ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref name="Dolly"> PMID: 6694738</ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref name="Herrero"> PMID: 4960839</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref name="Nayyar"> PMID: 25654058</ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC11

2016-05-12T15:59:53Z

Dana Emmert:

{{STRUCTURE_1myp| PDB=1myp | SIZE=400| SCENE= |right|CAPTION=Dog glycosylated myeloperoxidase dimer: heavy chain (pink and yellow), light chain (grey and green) containing heme complex with Ca+2 ions (green), [[1D5L]] }}

Please use the "3D" button above this box to insert a Jmol applet (molecule) on this page.
Or use the four-green-boxes-button to insert scrollable text adjacent
to a Jmol applet. Check out the other buttons as well!
-->
The <scene name='72/727903/Heme_pocket/1'>heme pocket</scene> skjdjkfhjf...

----
'''Introduction'''

Myeloperoxidase (MPO) is a protein found in neutrophil, the most common white blood cell. This enzyme generates hypochlorous acid (HOCl) from hydrogen peroxide in addition to other hypohalous acids depending on what is available in the cell. The HOCl is then used, in combination with other molecules, in an oxidative burst that kills bacteria and other potentially harmful invaders once taken up by the cell. This protein falls into a group of heme peroxidase enzymes. The role of these enzymes is to oxidize molecules using heme.<ref name="Hampton">PMID:9787133</ref>

This protein is composed of two identical monomers linked by a cystine bridge. The total molecular weight of this protein is 146 kDa. These two monomers can work independently of each other and consist of a light and heavy amino acid chain. The active site on the protein consists of a deep crevice where a heme molecule is covalently attached. The deep pocket is believedto limit access to the heme and increase specificity for smaller molecules to be oxidized. This pocket also possesses a hydrophobic region for substrate stabilization.<ref name="Active Site Structure">PMID:16288920</ref>

A lack of or mutation in this enzyme can cause immune deficiency, although most individuals with this mutation seem to be able to compensate with other, still unknown mechanisms.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> Over activation of myloperoxidase has been linked to increased inflammation and is being studied in relation to inceased risk of cardiovascular disease.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> There are still many unknowns surrounding the complete function and role that myeloperoxidase plays in the body's immune response. However, it has become clear that this protein is an integral piece in the immune response and warrants further research.

'''Structure'''

[[Image:Heme_site.JPG]]

''Figure 1. Depicts key amino acids in stabilization of heme in myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:26884610</ref>

Myeloperoxidase is synthesized in bone marrow along with most other white blood cell components. The myloperoxidase enzyme is composed of two identical subunits. After translation of these subunits, they are cleaved into two parts: the heavy chain and the light chain. The heavy chain is a glycosylated domain that weighs approximately 58.5 kDa. This portion of the enzyme has the deep pocket where the heme is inserted by chaperones (calreticulin and calnexin). <ref name="Neutrophil">PMID:26904693</ref> The amino acid make up of heme pocket can be seen in Figure 1 above. The pink in this picture is the light chain, the blue is the heavy chain. The light chain (about 13.5 kDa) is attached to the heavy chain through disufide bonds. There have been discoveries of slight variation in the primary sequence of myeloperoxidase. However, for the most part these slight variations do not affect the enzymatic activity. Currently three isoforms have been isolated.<ref name="Enzymatic Activity">PMID:120019</ref>

'''Function'''

[[Image:Enzymatic_processes_of_myeloperoxidase.JPG]]

''Figure 2. The Enzymatic Activity of Myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:21297906</ref>

As stated above, the role of the myeloperoxidase in the immune response is to generate oxidative molecules that are used to damage invaders. Seen above in Figure 2 is a concise and basic diagram of the process. Compounds I, II and III represent myeloperoxidase with different oxidation states of the iron in the heme molecule. One of the processes facilitated by myeloperoxidase is to oxidize hydrogen peroxide (H2O2) and cause the iron to change oxidation states. In this state, the iron is then able to catalyze the production of the hypohalous acids. These molecules are able to wreak havoc on lipids, proteins and DNA. <ref name="Neutrophil">PMID:26904693</ref> These modifications are achieved by the oxidation (usually by the addition of the halogen) of the unlucky recipient. The heme iron can also be oxidized by radicals to form compounds II and III. The functions of these are less well known, but the production of these radicals is also helpful in the antibacterial response.<ref name="Myeloperoxidase">PMID:26884610</ref>

Now that the enzymatic properties of myeloperoxidase have been explored, it is time to turn to the bigger picture. These enzymes are stored in the subtype of leukocytes (white blood cells) called neutrophils. These are thought to be one of the first cells types to arrive at the site of infection.<ref name="Neutrophil">PMID:26904693</ref> An outline of the immune response of neutrophil, specifically highlighting MPO's role, can be seen in Figure 3 below. There are two paths seen in this figure. In the first path bacteria is taken up by neutrophil extracellular traps or NETs and then destroyed by the hypohalogous acids. The second path shows the MPO being released, signaled by the dendrite cells(DCs), and helping to destroy the pathogen.<ref name="Neutrophil">PMID:26904693</ref> ANCA stands for anti-neutrophil cytoplasmic antibody. The MPOs have a range of function that are steered by the neutophil cells. This intense oxidative power is very helpful when targeted to foreign bodies. However, as discussed in the next section, MPO can very destructive if released on our own tissue.

[[Image:Neutrophil_Process.JPG]]

''Figure 3. The immune response of neutrophils with specific emphasis on myeloperoxidase.''<ref name="Neutrophil">PMID:26904693</ref>

'''Implication of Potential Defects or Deficiencies'''

Individuals with myeloperoxidase deficiency have been found to have a slight increased risk of chronic or severe infection. However, this condition is not fatal and the body seems to be able to compensate with other immune system mechanisms. <ref name="ANCA">PMID:11111115</ref> The most common source of MPO deficiency is ANCA (antineutrophil cytoplasmic antibodies)-associated vasculitis (AAV). This is an autoimmune disease that causes the production of antibodies for enzymes like MPO. <ref name="ANCA">PMID:26986103</ref> However, these diseases have also been linked to a decrease in risk of cardiovascular disease.<ref name="ANCA">PMID:11111115</ref>

'''Correlation to Cardiovascular Disease'''

Although MPO plays a key role in the fight against unwanted intruders, it has also been shown to turn those destructive powers against the body. Elevated levels of MPO in the blood has been found in patients with cardiovascular disease. <ref name="CAD">PMID:11694155</ref> It is speculated that over activation of the immune response causes inflammation, or damage to the surrounding tissue which increases risk of cardiovascular disease. More specifically, when MPO generate the oxidative agents such as HOCl, it not only chloronate invaders, but can also chloronate lipoproteins (both LDL and HDL). This changes the lipoprotein's affinity for the cell wall which can lead to plaque buildup. It can also effects the endothelial cells lining the vein or artery.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref>

Another of the identified affects of MPO on endothelial cells is in the production of nitric oxide (NO). This is a vasodilator that is inhibited when oxidized by MPO. Myloperoxidase can also oxidize NO2 - to its radical form which has been identified as a major contributor to inflammation.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref> Because of this clear correlation between MPO activity and the manifestation of cardiovascular disease, it has the potential to be used as an indicator of a cardiovascular problem.<ref name="Use in Medicine">PMID:12865732</ref>
==3D structures of myeloperoxidase==

Updated on {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

[[1myp]] – MPO – dog 
[[1mhl]], [[1cxp]] – hMPO C – human 
[[3f9p]] - hMPO 
[[1qo4]] - MPO – ''Arabidopsis thaliana'' 
[[1d2v]] – hMPO C + Br 
[[1d5l]] – hMPO + CN 
[[1d7w]] - hMPO + CN + Br 
[[1dnu]] - hMPO + SCN 
[[1dnw]] - hMPO + CN + SCN 
[[3zs0]], [[3zs1]], [[4c1m]] - hMPO + inhibitor 
[[4dl1]] - hMPO + thioxanthine 
[[4ejx]] - hMPO + ceruplasmin 
[[Category:Topic Page]]

== References ==
<references/>

Sandbox WWC1

2016-05-12T15:58:43Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref name="Montecucco"> PMID: 8771234</ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref name="Dolly"> PMID: 6694738</ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref name="Arnon" /> Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T15:14:02Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

Leah, the problem with the references was that you were missing a ">" and instead had a period behind the name of one of your references.

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref name="Montecucco"> PMID: 8771234</ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref name="Dolly"> PMID: 6694738</ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T15:13:04Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon"> PMID: 11209178</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref name="Montecucco"> PMID: 8771234</ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref name="Dolly"> PMID: 6694738</ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:44:13Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon". PMID: 11209178</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref name="Montecucco"> PMID: 8771234</ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref name="Dolly"> PMID: 6694738</ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />

== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:42:06Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref name="Arnon". PMID: 11209178</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref name="Minton"> PMID: 8542753</ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref name="Montecucco"> PMID: 8771234</ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref name="Dolly"> PMID: 6694738</ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:38:06Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref name="Popoff"> PMID: 8542750</ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref name="Popoff" />).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:35:57Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref name="Ward"> PMID: 5340653</ref> <ref name="Smith"> PMID: 355208</ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref>Popoff MR. Ecology of neurotoxigenic strains of clostridia. In: Montecucco C, ed. Current Topics in Microbiology: Clostridial Neurotoxins. The Molecular Pathogenesis of Tetanus and Botulism. Vol 195. Berlin, Germany: Springer-Verlag; 1995: 1–29. </ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref> Popoff MR, Marvaud J-C. 1999. Structural and genomic features of clostridial neurotoxins. See Ref. 132, pp. 174–
201 </ref>).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:21:59Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref name="Hall"> PMID: 3988908</ref> <ref name="Aureli"> PMID: 3722863</ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref>Ward BQ, Carroll BJ, Garrett ES, GB Reese. Survey of the U.S. Gulf Coast for the presence of Clostridium botulinum. Appl Microbiol. 1967;15:629–636. 26.</ref> <ref> Smith LDS. The occurrence of Clostridium botulinum and Clostridium tetani in the soil of the United States. Health Lab Sci. 1978;15:74–80. 27. </ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref>Popoff MR. Ecology of neurotoxigenic strains of clostridia. In: Montecucco C, ed. Current Topics in Microbiology: Clostridial Neurotoxins. The Molecular Pathogenesis of Tetanus and Botulism. Vol 195. Berlin, Germany: Springer-Verlag; 1995: 1–29. </ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref> Popoff MR, Marvaud J-C. 1999. Structural and genomic features of clostridial neurotoxins. See Ref. 132, pp. 174–
201 </ref>).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:19:57Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref>Hall JD, McCroskey LM, Pincomb BJ, Hatheway CL. Isolation of an organism resembling Clostridium baratii which produces type F botulinal toxin from an infant with botulism. J Clin Microbiol. 1985;21:654-655. 36. </ref> <ref name="Aureli"> PMID: 3722863</ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref>Ward BQ, Carroll BJ, Garrett ES, GB Reese. Survey of the U.S. Gulf Coast for the presence of Clostridium botulinum. Appl Microbiol. 1967;15:629–636. 26.</ref> <ref> Smith LDS. The occurrence of Clostridium botulinum and Clostridium tetani in the soil of the United States. Health Lab Sci. 1978;15:74–80. 27. </ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref>Popoff MR. Ecology of neurotoxigenic strains of clostridia. In: Montecucco C, ed. Current Topics in Microbiology: Clostridial Neurotoxins. The Molecular Pathogenesis of Tetanus and Botulism. Vol 195. Berlin, Germany: Springer-Verlag; 1995: 1–29. </ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref> Popoff MR, Marvaud J-C. 1999. Structural and genomic features of clostridial neurotoxins. See Ref. 132, pp. 174–
201 </ref>).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:16:04Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref>Hall JD, McCroskey LM, Pincomb BJ, Hatheway CL. Isolation of an organism resembling Clostridium baratii which produces type F botulinal toxin from an infant with botulism. J Clin Microbiol. 1985;21:654-655. 36. </ref> <ref>Aureli P, Fenicia L, Pasolini B, Gianfranceschi M, McCroskey LM, Hatheway CL. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J Infect Dis. 1986;154: 207-211. 37. </ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref>Ward BQ, Carroll BJ, Garrett ES, GB Reese. Survey of the U.S. Gulf Coast for the presence of Clostridium botulinum. Appl Microbiol. 1967;15:629–636. 26.</ref> <ref> Smith LDS. The occurrence of Clostridium botulinum and Clostridium tetani in the soil of the United States. Health Lab Sci. 1978;15:74–80. 27. </ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref>Popoff MR. Ecology of neurotoxigenic strains of clostridia. In: Montecucco C, ed. Current Topics in Microbiology: Clostridial Neurotoxins. The Molecular Pathogenesis of Tetanus and Botulism. Vol 195. Berlin, Germany: Springer-Verlag; 1995: 1–29. </ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 10518945</ref> <ref> Popoff MR, Marvaud J-C. 1999. Structural and genomic features of clostridial neurotoxins. See Ref. 132, pp. 174–
201 </ref>).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:14:24Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref>Hall JD, McCroskey LM, Pincomb BJ, Hatheway CL. Isolation of an organism resembling Clostridium baratii which produces type F botulinal toxin from an infant with botulism. J Clin Microbiol. 1985;21:654-655. 36. </ref> <ref>Aureli P, Fenicia L, Pasolini B, Gianfranceschi M, McCroskey LM, Hatheway CL. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J Infect Dis. 1986;154: 207-211. 37. </ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref>Ward BQ, Carroll BJ, Garrett ES, GB Reese. Survey of the U.S. Gulf Coast for the presence of Clostridium botulinum. Appl Microbiol. 1967;15:629–636. 26.</ref> <ref> Smith LDS. The occurrence of Clostridium botulinum and Clostridium tetani in the soil of the United States. Health Lab Sci. 1978;15:74–80. 27. </ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref>Popoff MR. Ecology of neurotoxigenic strains of clostridia. In: Montecucco C, ed. Current Topics in Microbiology: Clostridial Neurotoxins. The Molecular Pathogenesis of Tetanus and Botulism. Vol 195. Berlin, Germany: Springer-Verlag; 1995: 1–29. </ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 3722863</ref> <ref> Popoff MR, Marvaud J-C. 1999. Structural and genomic features of clostridial neurotoxins. See Ref. 132, pp. 174–
201 </ref>).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref name="Lacy" />
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC1

2016-05-12T14:10:30Z

Dana Emmert:

'''Looking great! You need to add something interactive with your structure. Perhaps feature the consensus sequence, or the heavy and light chains?

'''You need to format your references a little differently. I prefer that you use the PMID (Pubmed ID) whenever possible. This allows a user to have the paper one click away. Your citations should be placed outside the sentence, like this.<ref name="Aureli"> PMID: 3722863</ref>
By naming the reference as I have, if you use it again, you only need to insert that name.<ref name="Aureli" />'''

'''I have additional comments that are easier to show on paper. I will scan the comments and send the to you, or give them to you during lab tomorrow.''''''

== Background ==
Botulinum Toxin (BTX) is produced by three species of obligate anaerobe bacterium, primarily ''Clostridium botulism'', but ''Clostridium baratii'' and ''Clostridium butyricum'' also produce the protein <ref>Hall JD, McCroskey LM, Pincomb BJ, Hatheway CL. Isolation of an organism resembling Clostridium baratii which produces type F botulinal toxin from an infant with botulism. J Clin Microbiol. 1985;21:654-655. 36. </ref> <ref>Aureli P, Fenicia L, Pasolini B, Gianfranceschi M, McCroskey LM, Hatheway CL. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J Infect Dis. 1986;154: 207-211. 37. </ref> <ref>Arnon SS. Botulism as an intestinal toxemia. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, eds. Infections of the Gastrointestinal Tract. New York, NY: Raven Press; 1995:257-271.</ref>. ''Clostridium botulism'' is commonly found in soil, marine sediments, and the gut of grazing animals <ref>Ward BQ, Carroll BJ, Garrett ES, GB Reese. Survey of the U.S. Gulf Coast for the presence of Clostridium botulinum. Appl Microbiol. 1967;15:629–636. 26.</ref> <ref> Smith LDS. The occurrence of Clostridium botulinum and Clostridium tetani in the soil of the United States. Health Lab Sci. 1978;15:74–80. 27. </ref> <ref>Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980;44:419–448. 28. Dodds KL. Clostridium botulinum in the environment. In: Hauschild AHW </ref> <ref>Dodds KL, eds. Clostridium botulinum—Ecology and Control in Foods. New York, NY: Marcel Dekker, Inc; 1992: 21–51. 29. </ref> <ref>Popoff MR. Ecology of neurotoxigenic strains of clostridia. In: Montecucco C, ed. Current Topics in Microbiology: Clostridial Neurotoxins. The Molecular Pathogenesis of Tetanus and Botulism. Vol 195. Berlin, Germany: Springer-Verlag; 1995: 1–29. </ref> .BTX is only produced by these bacteria when there are anaerobic conditions that induce spore germination. Once produced, the protein can have toxic effects on animals and humans by causing botulism, a potentially fatal illness. BTX is also the active component of Botox.
<Structure load='3BTA' size='350' frame='true' align='right' caption='Botulinum Toxin Neurotoxin Serotype A' scene='Insert optional scene name here' />

== Structure ==

There are seven forms of the protein, named A through G, that are structurally similar but create different immune responses <ref> Hatheway cL. Clostridium botulinum and other clostridia that produce botulinum neurotoxins. in: Hauschild aHW, Dodds kL, eds. Clostridium botulinum—Ecology and Control in Foods. new york, ny: marcel Dekker, inc; 1992: 3–10 </ref>. All seven structures are produced as a single chain protein in the bacterium, but becomes active when a protease cuts the protein into a heavy and light chain connected by a single disulfide bond. The heavy chain is approximately 100 kDa and the light chain is 50 kDa (for reviews about structure see references <ref>Sakaguchi G. 1983. Clostridium botulinum toxins. Pharmacol. Ther. 19:165–
94. </ref> <ref> Minton NP. 1995. Molecular genetics of
clostridial neurotoxins. Curr. Top. Microbiol.
Immunol. 195:161–94 </ref> <ref> Oguma K, Fujinaga Y, Inoue K. 1995.Structure and function of Clostridium botulinum toxins. Microbiol. Immunol. 39:161–68 </ref> <ref name="Lacy"> PMID: 3722863 </ref> <ref> Popoff MR, Marvaud J-C. 1999. Structural and genomic features of clostridial neurotoxins. See Ref. 132, pp. 174–
201 </ref>).

The light chain contains the <scene name='69/696299/Consensus_sequence_heilh/1'>consensus sequence HELIH</scene> that codes for the binding of zinc, which subsequently regulates the endopeptidase activity of the light chain.

BTX also has two auxiliary proteins that compromise a multimeric complex: hemagglutinins (HA) and nonhemagglutinin (NTNH). HA and NTNH do not directly play a role in the toxic effect of BTX, but have an indirect role during ingestion of the protein by making the BTX more resistant to low pH environments and proteolytic enzymes found in the gut.

== Function ==

The active form of BTX has three functional domains responsible for binding, translocation, and catalysis <ref>Montecucco and Schiavo 1995 C. Montecucco, G. Schiavo Structure and function of tetanus and botulinum neurotoxins Quart. Rev. Biophys., 28 (1995), pp. 423–472. </ref>. The C terminus of the heavy chain is responsible for binding a ganglioside and protein receptors on the nerve terminal while the N terminus half functions by translocating the protein across the membrane by signaling endocytosis <ref>Dolly et al 1984 J.O. Dolly, J. Black, R.S. Williams, J. Melling Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization Nature, 307 (1984), pp. 457–460 </ref>. Once in a vesicle in the cytosol of a nerve terminal, the light chain dissociates from the heavy chain and cleaves SNARE proteins involved in synaptic vesicle fusion. The cleavage of these SNARE proteins, VAMP, SNAP-25, and syntax, depends on the serotype of BTX where each serotype cuts at a different place. The cut SNAREs inhibit the binding of vesicles containing acetylcholine which effectively stops the signaling from nerves to muscle cells, resulting in paralysis <ref>Lacy, D. B.; Stevens, R. C. Sequence Homology and Structural Analysis of the Clostridial neurotoxins1. J. Mol. Biol. 1999, 291 (5), 1091–1104.
</ref>.
== Disease ==

Of the seven serotypes of BTX, the forms that most often cause botulism in humans are A, B, and E <ref> arnon SS, Schechter r, inglesby tV, et al. botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.</ref>. Humans can be exposed to the neurotoxin through inhalation, ingestion, or surface wounds. Within 12 to 72 hours of exposure to BTX, the inhibition of acetylcholine signaling at nerve synapses creates symptoms of vomiting, nausea, diarrhea, blurred vision, ptosis, dysarthria and dysphagia, and finally weakness of muscles beginning with the head and moving down to the lower extremities <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>. If not treated, BTX can cause death due to respiratory failure or due to complications when on prolonged medical ventilation.

The lethal doses for a human weighing 70 kg is 0.09-0.15 μg when administered intravenously or intramuscularly, 0.70 - 0.90 μg through inhalation, and 70 μg orally <ref>  Franz DR, Pitt LM, Clayton MA, Hanes MA, Rose KJ. Efficacy of prophylactic and therapeutic administration of antitoxin for inhalation botulism. In: DasGupta BR, ed. Botulinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects. New York, NY: Plenum Press; 1993:473-476. </ref> <ref>  Herrero BA, Ecklung AE, Streett CS, Ford DF, King JK. Experimental botulism in monkeys: a clinical pathological study. Exp Mol Pathol. 1967;6:84-95.</ref>.

The treatment for human exposure to BTX is an antitoxin developed from horse plasma. Horses are exposed naturally to "Clostridium botulism" in their gut and therefore produce antibodies against "Clostridium botulism" in case the bacteria population grows to an extreme. Antitoxins have been developed by isolating this antibody from horses, and in some cases, removing the Fc from the immunoglobulin and leaving the F(ab')2 fragment. By removing the Fc fragment, the incidence of hypersensitivity and anaphylaxis. There are divalent, trivalent, and heptavalent antitoxins available that treat botulinum serotypes A and B (divalent), serotypes A, B, and C (trivalent), and all seven serotypes (heptavalent). An antitoxin, botulism immune globulin (BabyBIG) has also been developed that can be used in infantile cases of botulism. BabyBIG is produced by using the human antibodies produced when a person is injected with pentavalent botulinum toxoid vaccine. By using human antibodies, there is less of a risk of anaphylactic shock and hypersensitivity to equine antigens <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

The antitoxins cannot stop BTX from inhibiting acetylcholine signaling once it has started in parts of the body, but it can stop the progression of paralysis. Thus, treatment can decrease fatalities to 5-10% if administered within 24 hours of exposure <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007.</ref>.

== Relevance ==

Due to the powerful toxicity of BTX, the protein could be used as a biological weapon. The countries that have developed BTX to be used in warfare include Japan, Germany, United States, Russia, and Iraq <ref> Dembek, Z. F.; Smith, L. A.; Rusnak, J. Botulinum Toxin. In Medical Aspects of Biological Warfare; 2007. </ref>.

BTX has also been used medically and cosmetically as Botox. Various disorders characterized by involuntary muscle contraction can be treated with a small injection of BTX to weaken the spastic muscles for three to four months <ref>Nayyar, P.; Kumar, P.; Nayyar, P. V.; Singh, A. BOTOX: Broadening the Horizon of Dentistry. J. Clin. Diagn. Res. JCDR 2014, 8 (12), ZE25–ZE29. </ref>. Some of these diseases include
cervical dystonia, blepharospasm, strabismus, chronic migraines, primary axillary hyperhidrosis, oesophageal achalasia, vaginismus, and detrusor overactivity among others.

Botulinum toxin is also used in small quantities to prevent the formation of wrinkles. Small muscles in the face create wrinkles after years of repeated contraction <ref>Botulinum Toxin Injection for Facial Wrinkles - American Family Physician http://www.aafp.org/afp/2014/0801/p168.html#sec-1 (accessed Apr 28, 2016).</ref>. By injecting botulinum toxin into specific muscles, contraction is inhibited and wrinkles are prevented. Continual injections are needed every few weeks because the SNAP-25 proteins regenerate allowing signaling for muscle contraction to begin again.

== References ==
<references/>

Sandbox WWC7

2016-05-12T03:16:21Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, most known PPR proteins are found in plants, and they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of [https://en.wikipedia.org/wiki/Maize ''Zea mays'']. PPR10 is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned before specific PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized in order to prevent off target binding. Additionally, no entirely new PPR proteins have been engineered, and design strategies must rely on modifying preexisting proteins.<ref name = "engineering">DOI:10.1111/tpj.12377</ref> Finally, the vast majority of PPR proteins exist in plants and operate only within organelles. The reasons or this fact, both evolutionary and mechanistic, are unknown, but it is likely to complicate the process of using PPR proteins in other organisms and in other locations.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T03:13:33Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, most known PPR proteins are found in plants, and they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of [https://en.wikipedia.org/wiki/Maize ''Zea mays''] which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned before specific PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized in order to prevent off target binding. Additionally, no entirely new PPR proteins have been engineered, and design strategies must rely on modifying preexisting proteins.<ref name = "engineering">DOI:10.1111/tpj.12377</ref> Finally, the vast majority of PPR proteins exist in plants and operate only within organelles. The reasons or this fact, both evolutionary and mechanistic, are unknown, but it is likely to complicate the process of using PPR proteins in other organisms and in other locations.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T03:11:12Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, most known PPR proteins are found in plants, and they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned before specific PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized in order to prevent off target binding. Additionally, no entirely new PPR proteins have been engineered, and design strategies must rely on modifying preexisting proteins.<ref name = "engineering">DOI:10.1111/tpj.12377</ref> Finally, the vast majority of PPR proteins exist in plants and operate only within organelles. The reasons or this fact, both evolutionary and mechanistic, are unknown, but it is likely to complicate the process of using PPR proteins in other organisms and in other locations.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T03:09:23Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned before specific PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized in order to prevent off target binding. Additionally, no entirely new PPR proteins have been engineered, and design strategies must rely on modifying preexisting proteins.<ref name = "engineering">DOI:10.1111/tpj.12377</ref> Finally, the vast majority of PPR proteins exist in plants and operate only within organelles. The reasons or this fact, both evolutionary and mechanistic, are unknown, but it is likely to complicate the process of using PPR proteins in other organisms and in other locations.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T02:50:51Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. P-class PPR proteins generally bind irreversibly to non-coding regions of RNA, whereas PLS-class PPR proteins bind reversibly to coding regions. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these neighboring RNA regions are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned about them before PPR design becomes a viable technology. Most importantly, the mechanism of specificity must be completely characterized so that manufactured PPR proteins will be entirely specific and not cause off-target effects.

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T02:24:00Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these mRNAs are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific applications.<ref>doi:10.1126/science.1231143</ref> As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned about them before this becomes a viable technology. . .

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T01:25:03Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31–36 amino acid repeats. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these mRNAs are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-[[Cas9]]) have shown incredible potential for medical, agricultural, and scientific application. As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned about them before this becomes a viable technology. . .

==References==

{{Reflist}}

Sandbox WWC7

2016-05-12T01:22:32Z

Dana Emmert:

Pentatricopeptide repeat (PPR) proteins are a family of sequence specific RNA-binding proteins which participate in organelle RNA metabolism. Although the mechanisms of RNA binding and the functions of PPR proteins are not fully understood, PPR proteins are thought to assist in RNA editing,<ref>PMID:17015439</ref> translation,<ref name = "translation">DOI:10.1073/pnas.1012076108</ref> and organelle biogenesis.<ref>PMID:15269332</ref> While PPR proteins are found in many eukaryotes, they make up the majority of RNA-binding factors in plant organelles. PPR proteins are characterized by a series of tandem-repeat amino acid consensus sequences which form α-helix <scene name='69/696301/Hairpins/1'>hairpins</scene>. These hairpin structures accumulate to form an <scene name='69/696301/Monomer/1'>α-solenoid tertiary structure</scene> (blue to red from N terminus to C terminus). PPR proteins belong to one of two classes: P-class and PLS-class, with the P-class containing 35 amino acid repeats and the PLS-class containing 31-36 amino acid repeats. PPR10 (shown to the right dimerized and bound to RNA) is a well-characterized P-class PPR protein found in the chloroplast of ''Zea mays'' which is often used as a model PPR protein.<ref name = "translation"/>
<Structure load='4OE1' size='350' frame='true' align='right' caption='PPR10 dimer bound to psaJ. pdb code: 4OE1' scene='Insert optional scene name here' />

==Function==
In the ''Zea mays'' plastid, PPR10 binds specifically to the ssRNA oligonucleotides atpH (17 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') and <scene name='69/696301/Atph/1'>spaJ</scene> (18 nucleotides: 5'-GUAUUCUUUAAUUAUUUC-3') where PPR10 has been shown to prevent degradation of sequences both upstream and downstream of its binding sites. In addition to stabilizing these RNA sequences, PPR10 increases the rate at which these mRNAs are translated.<ref name = "translation"/>

==Mechanism==
The primary factor in the ability of PPR10 to bind RNA bases in a modular fashion lies in the identities of the residue at position 6 on a repeat and the residue at position 1 on the next repeat (designated 1'). For example, in the structure to the right, <scene name='69/696301/G1binding/1'>T178 (blue) forms a hydrogen bond with G1 (green) of psaJ.</scene> Through Van der Waals interactions, V210 and R175 (both orange) also contribute to the specific binding of guanine in this example. These residues force G1 into a conformation where it forms a hydrogen bond with T178. The example of PPR10 binding G1 exemplifies the general rules by which PPR proteins bind specific nucleotides: firstly, a residue at the 6 position of one repeat (T178 in the previous example) forms a hydrogen bond with the base. The identity of this residue determines whether the repeat will bind a purine (adenine and guanine) or pyrimidine (cytosine and uracil). At position 6, serine and threonine are specific for purines, and asparagine at position 6 is specific for pyrimidines.<ref name = "barkan">doi:10.1371/journal.pgen.1002910</ref> Secondly, a residue at position 1' (Val210 in the previous example) completes the specificity of the interaction. Through Van der Waals interactions, this residue determines between A/G and C/U. Other amino acids further contribute to this mechanism, but the previously described rules always apply when PPR proteins bind RNA sequences with modularity.<ref name = "engineering">DOI:10.1111/tpj.12377</ref>

[[Image:PPR10binding.png]]

This image shows the general code by which PPR proteins recognize and bind RNA in a modular fashion.<ref name = "barkan"/> "A" and "B" refer to the first and second helices of each repeat on PPR10.

While crystallographic structures show PPR10 binding RNA in a dimerized configuration, further evidence by [http://www.sciencedirect.com/science/article/pii/S0021967309003057 EC-SY-SAX] has shown that this result is likely an artifact of the high concentrations necessary for crystallography. In a natural setting, PPR10 does not form a dimer.<ref name = "gully">DOI:10.1093/nar/gkv027</ref><ref name = "li">DOI:10.1074/jbc.M114.575472</ref>

===RNA Stabilization===
PPR10 stabilizes RNA upstream and downstream of its binding sites by blocking exonucleases from degrading RNA in either the 5' or 3' direction.<ref name = "translation"/>

===Translation Enhancement===
The mechanism by which PPR10 increases the rate of translation is still unknown. However, it is predicted that PPR10 binds to sequences near the ribosome binding site of the RNA transcript. By doing so, PPR10 prevents the ~20 nucleotides to which it is bound from base pairing to the ribosome binding site, a phenomenon which would impair translation. Considering the bacterial origins of the chloroplast, it is interesting to note that the RNA stabilizing and translation enhancing properties of PPR10 in the plastid mirror some of the functions of small RNAs (smRNA) in bacteria. In bacterial cells, smRNAs similarly bind regions near ribosome binding sites of mRNA, preventing degradation by nucleases and increasing translation by preventing base-pairing to ribosome binding sites.<ref name = "translation"/>

==Synthetic Applications==
Recent advances in programmable site-directed DNA-binding proteins (such as CRISPR-Cas9) have shown incredible potential for medical, agricultural, and scientific application. As a result, it is not surprising that researchers are attempting to develop similar protein-based tools for programmable RNA binding. Unfortunately, few RNA-binding proteins act in a manner which is predictable enough to facilitate convenient RNA manipulation. PPR proteins hold great promise for this application. As described previously, PPR proteins bind RNA nucleotides in a specific and predictable manner, so researchers are attempting to develop custom-made PPR proteins for use in manipulative gene expression experiments.

===Limitations===
As PPR proteins themselves were only discovered recently, there is still a great deal to be learned about them before this becomes a viable technology. . .

==References==

{{Reflist}}

Sandbox WWC11

2016-05-12T01:05:04Z

Dana Emmert:

{{STRUCTURE_1myp| PDB=1myp | SIZE=400| SCENE= |right|CAPTION=Dog glycosylated myeloperoxidase dimer: heavy chain (pink and yellow), light chain (grey and green) containing heme complex with Ca+2 ions (green), [[1D5L]] }}

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----
'''Introduction'''

Myeloperoxidase (MPO) is a protein found in neutrophil, the most common white blood cell. This enzyme generates hypochlorous acid (HOCl) from hydrogen peroxide in addition to other hypohalous acids depending on what is available in the cell. The HOCl is then used, in combination with other molecules, in an oxidative burst that kills bacteria and other potentially harmful invaders once taken up by the cell. This protein falls into a group of heme peroxidase enzymes. The role of these enzymes is to oxidize molecules using heme.<ref name="Hampton">PMID:9787133</ref>

This protein is composed of two identical monomers linked by a cystine bridge. The total molecular weight of this protein is 146 kDa. These two monomers can work independently of each other and consist of a light and heavy amino acid chain. The active site on the protein consists of a deep crevice where a heme molecule is covalently attached. The deep pocket is believedto limit access to the heme and increase specificity for smaller molecules to be oxidized. This pocket also possesses a hydrophobic region for substrate stabilization.<ref name="Active Site Structure">PMID:16288920</ref>

A lack of or mutation in this enzyme can cause immune deficiency, although most individuals with this mutation seem to be able to compensate with other, still unknown mechanisms.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> Over activation of myloperoxidase has been linked to increased inflammation and is being studied in relation to inceased risk of cardiovascular disease.<ref name="Association between myeloperoxidase levels and risk of coronary artery disease.">PMID:11694155</ref> There are still many unknowns surrounding the complete function and role that myeloperoxidase plays in the body's immune response. However, it has become clear that this protein is an integral piece in the immune response and warrants further research.

'''Structure'''

[[Image:Heme_site.JPG]]

''Figure 1. Depicts key amino acids in stabilization of heme in myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:26884610</ref>

Myeloperoxidase is synthesized in bone marrow along with most other white blood cell components. The myloperoxidase enzyme is composed of two identical subunits. After translation of these subunits, they are cleaved into two parts: the heavy chain and the light chain. The heavy chain is a glycosylated domain that weighs approximately 58.5 kDa. This portion of the enzyme has the deep pocket where the heme is inserted by chaperones (calreticulin and calnexin). <ref name="Neutrophil">PMID:26904693</ref> The amino acid make up of heme pocket can be seen in Figure 1 above. The pink in this picture is the light chain, the blue is the heavy chain. The light chain (about 13.5 kDa) is attached to the heavy chain through disufide bonds. There have been discoveries of slight variation in the primary sequence of myeloperoxidase. However, for the most part these slight variations do not affect the enzymatic activity. Currently three isoforms have been isolated.<ref name="Enzymatic Activity">PMID:120019</ref>

'''Function'''

[[Image:Enzymatic_processes_of_myeloperoxidase.JPG]]

''Figure 2. The Enzymatic Activity of Myeloperoxidase.'' <ref name="Myeloperoxidase">PMID:21297906</ref>

As stated above, the role of the myeloperoxidase in the immune response is to generate oxidative molecules that are used to damage invaders. Seen above in Figure 2 is a concise and basic diagram of the process. Compounds I, II and III represent myeloperoxidase with different oxidation states of the iron in the heme molecule. One of the processes facilitated by myeloperoxidase is to oxidize hydrogen peroxide (H2O2) and cause the iron to change oxidation states. In this state, the iron is then able to catalyze the production of the hypohalous acids. These molecules are able to wreak havoc on lipids, proteins and DNA. <ref name="Neutrophil">PMID:26904693</ref> These modifications are achieved by the oxidation (usually by the addition of the halogen) of the unlucky recipient. The heme iron can also be oxidized by radicals to form compounds II and III. The functions of these are less well known, but the production of these radicals is also helpful in the antibacterial response.<ref name="Myeloperoxidase">PMID:26884610</ref>

Now that the enzymatic properties of myeloperoxidase have been explored, it is time to turn to the bigger picture. These enzymes are stored in the subtype of leukocytes (white blood cells) called neutrophils. These are thought to be one of the first cells types to arrive at the site of infection.<ref name="Neutrophil">PMID:26904693</ref> An outline of the immune response of neutrophil, specifically highlighting MPO's role, can be seen in Figure 3 below. There are two paths seen in this figure. In the first path bacteria is taken up by neutrophil extracellular traps or NETs and then destroyed by the hypohalogous acids. The second path shows the MPO being released, signaled by the dendrite cells(DCs), and helping to destroy the pathogen.<ref name="Neutrophil">PMID:26904693</ref> ANCA stands for anti-neutrophil cytoplasmic antibody. The MPOs have a range of function that are steered by the neutophil cells. This intense oxidative power is very helpful when targeted to foreign bodies. However, as discussed in the next section, MPO can very destructive if released on our own tissue.

[[Image:Neutrophil_Process.JPG]]

''Figure 3. The immune response of neutrophils with specific emphasis on myeloperoxidase.''<ref name="Neutrophil">PMID:26904693</ref>

'''Implication of Potential Defects or Deficiencies'''

Individuals with myeloperoxidase deficiency have been found to have a slight increased risk of chronic or severe infection. However, this condition is not fatal and the body seems to be able to compensate with other immune system mechanisms. <ref name="ANCA">PMID:11111115</ref> The most common source of MPO deficiency is ANCA (antineutrophil cytoplasmic antibodies)-associated vasculitis (AAV). This is an autoimmune disease that causes the production of antibodies for enzymes like MPO. <ref name="ANCA">PMID:26986103</ref> However, these diseases have also been linked to a decrease in risk of cardiovascular disease.<ref name="ANCA">PMID:11111115</ref>

'''Correlation to Cardiovascular Disease'''

Although MPO plays a key role in the fight against unwanted intruders, it has also been shown to turn those destructive powers against the body. Elevated levels of MPO in the blood has been found in patients with cardiovascular disease. <ref name="CAD">PMID:11694155</ref> It is speculated that over activation of the immune response causes inflammation, or damage to the surrounding tissue which increases risk of cardiovascular disease. More specifically, when MPO generate the oxidative agents such as HOCl, it not only chloronate invaders, but can also chloronate lipoproteins (both LDL and HDL). This changes the lipoprotein's affinity for the cell wall which can lead to plaque buildup. It can also effects the endothelial cells lining the vein or artery.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref>

Another of the identified affects of MPO on endothelial cells is in the production of nitric oxide (NO). This is a vasodilator that is inhibited when oxidized by MPO. Myloperoxidase can also oxidize NO2 - to its radical form which has been identified as a major contributor to inflammation.<ref name="Specifics on Correlation to CVD">PMID:16476484</ref> Because of this clear correlation between MPO activity and the manifestation of cardiovascular disease, it has the potential to be used as an indicator of a cardiovascular problem.<ref name="Use in Medicine">PMID:12865732</ref>
==3D structures of myeloperoxidase==

Updated on {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

[[1myp]] – MPO – dog 
[[1mhl]], [[1cxp]] – hMPO C – human 
[[3f9p]] - hMPO 
[[1qo4]] - MPO – ''Arabidopsis thaliana'' 
[[1d2v]] – hMPO C + Br 
[[1d5l]] – hMPO + CN 
[[1d7w]] - hMPO + CN + Br 
[[1dnu]] - hMPO + SCN 
[[1dnw]] - hMPO + CN + SCN 
[[3zs0]], [[3zs1]], [[4c1m]] - hMPO + inhibitor 
[[4dl1]] - hMPO + thioxanthine 
[[4ejx]] - hMPO + ceruplasmin 
[[Category:Topic Page]]

== References ==
<references/>