Proteopedia - User contributions [en]

Sandbox Reserved 508

2012-09-27T15:15:27Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain.<ref name="Berardi">PMID:21785437</ref> Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification.<ref name="Krauss">PMID:15738989</ref> There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above .<ref name="Teshima">PMID: 12855674</ref> shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production

Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS.

Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein. DCF fluorescence is barely visible, representing little to no ROS presence.

B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy. The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb]]
 
 
 
 
 
'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane'''
 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
 
 
 
 

[[Image:BCSMART_11-12_Figure_2.JPG|left|375px|thumb]]

 
 
 
 
 
 
 
 
 
 

'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
 
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb]]
 
 
 
 
 
 
 
 
 
 

'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
 
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|left|375px|thumb]]
 
 
 
 
 
 
 
 
 
 

'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/12' size='750' side='middle' caption='Uncoupling Protein 2, 2lck <ref name="Berardi">PMID:21785437</ref>Structure of UCP 2 displaying key features. In this model of UCP2, the light sea green parts represent the alpha helices which are thought to be involved in the transport of protons. All negatively-charged amino acids, glutamic and aspartic, are colored crimson. These may be involved in the direct transport of protons through UCP2. All positively charged amino acids, except for Lys141 and Arg185, are colored navy. These may be involved in the transport of deprotonated fatty acid molecules through UCP2, as well. Lys141 and Arg185, which are speculated to bind to the UCP2 inhibitor, GDP, are colored light sky blue. Helix sequences, conserved between different UCP proteins, which may also have a role in fatty acid transport, are colored orange. Helices found in the matrix or the inter-membrane space are colored light aquamarine. Loops, which are not involved in the active site, are colored white. '>

==References==
<references />

Sandbox Reserved 508

2012-09-17T21:12:59Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production

Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS.

Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein. DCF fluorescence is barely visible, representing little to no ROS presence.

B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy. The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb]]
 
 
 
 
 
'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane'''
 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
 
 
 
 

[[Image:BCSMART_11-12_Figure_2.JPG|left|375px|thumb]]

 
 
 
 
 
 
 
 
 
 

'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
 
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb]]
 
 
 
 
 
 
 
 
 
 

'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
 
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|left|375px|thumb]]
 
 
 
 
 
 
 
 
 
 

'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/12' size='750' side='middle' caption='Structure of UCP 2 displaying key features. In this model of UCP2, the light sea green parts represent the alpha helices which are thought to be involved in the transport of protons. All negatively-charged amino acids, glutamic and aspartic, are colored crimson. These may be involved in the direct transport of protons through UCP2. All positively charged amino acids, except for Lys141 and Arg185, are colored navy. These may be involved in the transport of deprotonated fatty acid molecules through UCP2, as well. Lys141 and Arg185, which are speculated to bind to the UCP2 inhibitor, GDP, are colored light sky blue. Helix sequences, conserved between different UCP proteins, which may also have a role in fatty acid transport, are colored orange. Helices found in the matrix or the inter-membrane space are colored light aquamarine. Loops, which are not involved in the active site, are colored white. '>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 508

2012-09-17T20:43:33Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production

Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS.

Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein. DCF fluorescence is barely visible, representing little to no ROS presence.

B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy. The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
 
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/12' size='750' side='middle' caption='Structure of UCP 2 displaying key features. In this model of UCP2, the light sea green parts represent the alpha helices which are thought to be involved in the transport of protons. All negatively-charged amino acids, glutamic and aspartic, are colored crimson. These may be involved in the direct transport of protons through UCP2. All positively charged amino acids, except for Lys141 and Arg185, are colored navy. These may be involved in the transport of deprotonated fatty acid molecules through UCP2, as well. Lys141 and Arg185, which are speculated to bind to the UCP2 inhibitor, GDP, are colored light sky blue. Helix sequences, conserved between different UCP proteins, which may also have a role in fatty acid transport, are colored orange. Helices found in the matrix or the inter-membrane space are colored light aquamarine. Loops, which are not involved in the active site, are colored white. '>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 509

2012-08-03T00:03:31Z

Nikil Prasad:

'''Protein Kinase R'''

== Abstract ==
One mechanism against viral infection preventing viral propagation by inhibiting cellular protein synthesis. The protein kinase R (PKR) is an several enzymes involved in cellular immunity against viral infection. PKR is composed of two domains: two double stranded RNA (dsRNA) binding domains and a kinase domain (KD). The crystal structure of the PKR KD is bound to its substrate, initiation factor eIF2α revealing that each KD is composed of N-terminal (N-lobe) and C-terminal lobes (C-lobe). The active site lies between these two lobes, which also contains an ATP. The two N-lobes of each PKR KD interact to form a dimer whereas the C-lobe is bound to eIF2α composed of an S1 domain and a helical domain. Upon viral infection, PKR senses the dsRNA inserted by the virus, and is dimerized and activated. The active PKR molecule then binds to eIF2α, causing a conformational change in eIF2α, bringing the helix insert containing Ser51 (phospho-acceptor residue) closer to the ATP. The γ phosphate of ATP is then transferred to the Ser51, and the phosphorylated eIF2α inhibits protein synthesis in infected cells. Such fundamental insights into the mechanisms of substrate recognition and phosphorylation by PKR will help design a small molecule that will activate PKR, leading to improved immunity against multiple viral infections. In addition, the mechanism of PKR function may be applied to cancer therapy due to its role in controlling cell differentiation.

==Viral Defense against PKR Function==
Viruses spread throughout the body by invading cells and hijacking the protein production machinery of the cell in order to replicate. However, there are several defense mechanisms used to counter viral infection. One is protein kinase R (PKR). In an uninfected cell, PKR exists as an inactive molecule. However, the presence of viral RNA triggers the PKR to form an active dimer. The active PKR then binds to an initiation factor eIF2α and transfers a phosphate from an ATP to the eIF2α, thereby inhibiting cellular and viral protein synthesis.
[[Image:Figure 1-PKR and Viral Protein Conundrum.jpg|center|375px|thumb|'''Figure 1''']]
Viruses have evolved several mechanisms to subvert the PKR function (Figure 1). The VAI (Viral Associated I) RNA of adenovirus serves to prevent the dimerization and activation of PKR and inhibit the anti-viral response. Influenza virus p58IPK inhibits the activation of PKR. On the other hand, the smallpox virus K3L protein is a molecular mimic of eIF2α, acting as a competitive inhibitor and inhibits the phosphorylation reaction. Therefore fundamental understanding of this specific interaction between eIF2α and PKR and how viruses interfere with the reaction between the two is of paramount importance in developing anti-viral drugs.

==Data Pieces==
[[Image:Data Piece.jpg|center|375px|thumb|'''Mutation of remote residue T487 eliminates eIF2α phosphorylation, but not autophosphorylation''']]
Lane 1: Wild type PKR, wild type eIF2α and 33Pγ-ATP were mixed and were resolved in a gel (lower panel stain). The gel was dried and autoradiographed (upper panel 33P). PKR transfers 33Pγ of ATP to itself (autophosphorylation indicated as PKR~P) as well as to the eIF2α indicated here as eIF2α~P). Lane 2: When wild type PKR, a mutated form of eIF2α-S51A (phosphorylation site is mutated to an alanine) and 33Pγ-ATP were mixed. PKR transfers 33Pγ of ATP to itself, but not to the eIF2α-S51A. These data confirm that the Ser51 residue is the phosphorylation site of eIF2α. Lanes 3 and 4: when PKR-T487A (PKR-T487D for the lane 4), wild type eIF2α and 33Pγ-ATP were mixed, PKR transfers 33Pγ of ATP to itself, but not to the eIF2α suggesting that the remote residue T487 is important for Ser51 phosphorylation. The residue T487 is located on the contact surface of PKR with eIF2α (Fig. 2), thus providing an experimental evidence of PKR-eIF2α structure and interaction at the remote region.
[[Image:Data Piece 2.jpg|left|331px|thumb|'''Disruption of hydrophobic cluster leads to Ser51 phosphorylation of the T487D mutant, bypassing helix aG interaction with eIF2a.''']]
[[Image:Picture for Data Piece 2.jpg|right|350px|thumb|'''The movement of Ser51 is restricted by a cluster of hydrophobic residues''']]

Figure on Left: Mutation of residue L50 that disrupts hydrophobic interaction causes a conformation change of Ser51 position. As a result PKR-T487D protein is able to transfer 33Pγ of ATP to the eIF2α-L50S, but not to the wild type eIF2α (as seen in the comparison of eIF2α~P lanes 5 and 7).

Figure on Right: A cluster of hydrophobic residues (L47, L50, I58 and I62) restricts the movement of Ser51 residue of eIF2α. These hydrophobic residues pull Ser51 in a hydrophobic pocket.

== Conclusion ==
The mechanism of the PKR response to viral infection involves PKR activation (dimerization and autophosphorylation) and phosphorylation of eIF2α. To counteract PKR function, viruses have evolved specific mechanisms. Therefore understanding the structure and mechanism of PKR-eIF2α recognition will help design anti-viral drugs through preventing viruses from binding to PKR and through mimicking eIF2α’s function.

==References==

Dey, M., Velyvis, A., Li, J., Chiu, E., Chiovitti, D., Kay, L., Sicheri, F., Dever, T. E. Binding to PKR induces a conformational change in eIF2α to expose Ser-51 for phosphorylation. (2011) Proc. Natl. Acad. Sci., USA (in press).
 

Dey, M., Cao, C., Dar, A., Tamura, T., Ozato, K., Sicheri, F., and Dever, T. E. Mechanistic link between protein kinase PKR catalytic domain dimerization, autophosphorylation and eIF2α phosphorylation. (2005) Cell, 122, 901-913.
 

Dar AC, Dever TE, & Sicheri F (2005) Higher-order substrate recognition of eIF2α by the RNA-dependent protein kinase PKR. Cell 122:887-900.
Dhaliwal, S., Hoffman, D.W. J.Mol.Biol. (2003) 334: 187-195

Sandbox Reserved 509

2012-08-03T00:01:11Z

Nikil Prasad:

File:Picture for Data Piece 2.jpg

2012-08-02T23:51:42Z

Nikil Prasad:

File:Data Piece 2.jpg

2012-08-02T23:48:54Z

Nikil Prasad:

File:Data Piece.jpg

2012-08-02T23:41:32Z

Nikil Prasad:

Sandbox Reserved 509

2012-08-02T23:38:48Z

Nikil Prasad:

File:Figure 1-PKR and Viral Protein Conundrum.jpg

2012-08-02T23:35:41Z

Nikil Prasad:

Sandbox Reserved 508

2012-08-02T23:22:00Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production

Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS.

Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein. DCF fluorescence is barely visible, representing little to no ROS presence.

B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy. The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
 
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/12' size='750' side='middle' caption='Structure of UCP 2 displaying key features. In this model of UCP2, the light sea green parts represent the alpha helices which are thought to be involved in the transport of protons. All negatively-charged amino acids, glutamic and aspartic, are colored crimson. These may be involved in the direct transport of protons through UCP2. All positively charged amino acids, except for Lys141 and Arg185, are colored navy. These may be involved in the transport of deprotonated fatty acid molecules through UCP2, as well. Lys141 and Arg185, which are speculated to bind to the UCP2 inhibitor, GDP, are colored light sky blue. Helix sequences, conserved between different UCP proteins, which may also have a role in fatty acid transport, are colored orange. Helices found in the matrix or the inter-membrane space are colored light aquamarine. Loops, which are not involved in the active site, are colored white. '>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 508

2012-08-02T21:39:49Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production
Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS. Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein.DCF fluorescence is barely visible, representing little to no ROS presence.
 
B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy. The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
 
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/10' size='500' side='right' caption='Structure of UCP 2 displaying key features. In this model of UCP2, the light sea green parts represent the alpha helices which are thought to be involved in the transport of protons. All negatively charged amino acids, glutamic and aspartic, are colored crimson. These may be involved in the direct transport of protons through UCP2. All positively charged amino acids, except for Lys141 and Arg185, are colored navy. These may be involved in the transport of deprotonated fatty acid molecules through UCP2, as well. Lys141 and Arg185, which are speculated to bind to the UCP2 inhibitor, GDP, are colored light sky blue. Helix sequences, conserved between different UCP proteins, which may also have a role in fatty acid transport, are colored orange. Helices found in the matrix or the inter-membrane space are colored light aquamarine. Loops, which aren’t involved in the active site, are colored white. '>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 508

2012-08-02T19:36:15Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production
Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS. Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein.DCF fluorescence is barely visible, representing little to no ROS presence.
 
B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy.
 
The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/10' size='500' side='right' caption='Structure of Yeast Poly(A) Polymerase with ATP and oligo(A) (PDB entry [[2q66]])'>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 508

2012-08-02T19:35:17Z

Nikil Prasad:

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production
Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS. Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein.DCF fluorescence is barely visible, representing little to no ROS presence.
 
B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy.
 
The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<StructureSection load='2lck_pdb' scene='Sandbox_Reserved_508/2lck_pdb/4' size='500' side='right' caption='Structure of Yeast Poly(A) Polymerase with ATP and oligo(A) (PDB entry [[2q66]])'>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 508

2012-08-02T01:07:24Z

Nikil Prasad: Scene Authoring

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production
Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS. Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein.DCF fluorescence is barely visible, representing little to no ROS presence.
 
B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy.
 
The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

<scene name='Sandbox_Reserved_508/2lck_pdb/4'>TextToBeDisplayed</scene>

==References==
<references />
Berardi, M. J., Shih, W. M., Harrison, S. C., & Chou, J. J. (2011, August 4). Mitochondrial uncoupling protein 2 structure determined by
NMR molecular fragment searching. Nature, 476, 109-113. doi:10.1038/nature10257
 
Krauss, S., Zhang, C.-Y., & Lowell, B. B. (2005, March). The Mitochondrial Uncoupling-Protein Homologues. Nature, 6, 248-259.
Retrieved from http://www.nature.com/reviews/molcellbio
 
Teshima, Y., Akao, M., Jones, S. P., & Marbán, E. (2003, July 10). Uncoupling Protein-2 Over expression Inhibits Mitochondrial Death
Pathway in Cardiomyocytes. Circulation Research, 192-200. doi:10.1161/01.RES.0000085581.60197.4D

Sandbox Reserved 508

2012-08-02T00:52:49Z

Nikil Prasad: Scene Authoring

'''Uncoupling Protein 2'''

== Introduction ==
Ischemic Heart Disease, the underlying cause of myocardial infarctions (heart attacks), is the leading cause of death in the world. Annually 785,000 American alone experience their first heart attack and 470,000 have a second or third. Cardiomyocytes (contracting heart cells) need energy to function optimally. Mitochondria in the cardiomyocytes provide that energy in the form of ATP through the metabolic process of oxidative phosphorylation, specifically the electron transport chain coupled to the enzyme ATP synthase. However, these mitochondria are vulnerable to injury during recovery after heart attack when the cardiomyocytes again receive blood flow (reperfusion) due to the over-production of reactive oxygen species (ROS or oxygen radicals). An excess of oxygen radicals can be very damaging due to their high chemical reactivity, specifically their ability to break chemical bonds in DNA, RNA, and proteins within the mitochondria and in the cell cytoplasm.

== Abstract ==
Heart diseases are the leading cause of death for Americans today. Mitochondria play a crucial role in recovery following ischemia (blood flow restriction) and reperfusion (blood flow return) injury, when a surge of reactive oxygen species (radicals) originating from the mitochondrial electron transport chain causes damage to proteins, lipids and DNA. Uncoupling protein 2 (UCP2), an inner mitochondrial membrane transport protein, is speculated to participate in this protection. The presumed function of UCP2 is carrying protons (H+) into the mitochondrial matrix along a concentration gradient generated by the electron transport chain. Normally, this proton (H+) gradient is used by ATP synthase to phosphorylate ADP to ATP. Under certain conditions, protons (H+) may preferentially be transported through UCP2, creating a detour past ATP synthase (“uncoupling”). Such uncoupling reduces damaging reactive oxygen species whose presence may actually activate UCP2 by residue modification. There are two proposed mechanisms for the transport of protons (H+) into the matrix. One is the direct transport of protons (H+) through UCP2. Alternatively, a fatty acid anion is transported out of the matrix through UCP2, while the protonated fatty acid permeates through the membrane into the matrix. UCP2 must be tightly regulated so it is only active when required, enabling the mitochondria to produce ATP. Understanding transport mechanism and regulation of UCP2 could lead to effective prevention of tissue injury due to heart attack. The Brookfield Central High School SMART Team created a physical model of UCP2 using 3-D modeling printing technology in order to better understand the structure-function relationship of UCP2.
== Data Piece ==
[[Image:BCSMART_11-12_Figure_7.JPG|center|375px]]
'''The Effect of UCP2 on ROS Production'''
 
The data above shows the effect of UCP2 on ROS production. The ROS is made visible by DCF fluorescence(dichloro-fluorescein, which fluoresces in the presence of H2O2). ROS species are introduced into neonatal rat cardiac ventricular myocytes by injecting H2O2.
 
A. Confocal microscope images of cardiomyocytes’ ROS production
Top row: ROS in myoctes which have been exposed to adenovirus without the UCP2 protein. Increased DCF fluorescence can be seen over time, representing the presence of a significant level of ROS. Bottom row: ROS in a myocytes exposed to an adenovirus producing the UCP2 protein.DCF fluorescence is barely visible, representing little to no ROS presence.
 
B. Quantitative representation of the changes in ROS levels corresponding to the fluorescence images in A using time-lapse confocal microscopy.
 
The presence of UCP2 greatly reduces ROS levels, limiting damage to cardiac ventricular myocytes.
== Figures ==
[[Image:BCSMART_11-12_Figure_1.JPG|left|375px|thumb|'''FIGURE 1: Movement of Protons (H+) Across Inner Mitochondrial Membrane''' 
1. The electron transport chain pumps protons (H+) into the inter membrane space.
 
2. Protons (H+) in the matrix bond with oxygen (O2) to form water (H2O) in a reduction reaction.
 
3. Protons (H+) from the inter membrane space are used by ATP synthase, to provide energy for the conversion
of ADP to ATP.
]]
[[Image:BCSMART_11-12_Figure_2.JPG|right|375px|thumb|'''FIGURE 2: ROS Production'''
 
1. During reperfusion (return of blood flow), the electron transport chain sends more protons (H+) into the inter membrane space, producing a high concentration.
 
2. A high concentration gradient of protons (H+) “backs up” the electrons, allowing them to leave the ETC.
 
3. The excess electrons in the matrix can bind to oxygen, generating reactive oxygen species (ROS)
that carry a negative charge (O2
-)]]
[[Image:BCSMART_11-12_Figure_3.JPG|left|375px|thumb|'''FIGURE 3: UCP2 Relieving the High Concentration Gradient'''
 
One way the mitochondria can alleviate the proton buildup in the inter membrane space is to transport the protons (H+) back into the matrix. One possible protein involved in this transportation is uncoupling protein 2 (UCP2).
1.UCP2 brings protons (H+) into the matrix, relieving the high concentration gradient.
 
2.The “back up” of electrons and radical production is reduced, minimizing damage to cardiomyocytes.
]]
[[Image:BCSMART_11-12_Figure_4.JPG|right|375px|thumb|'''FIGURE 4: Proposed Mechanisms for UCP2 Transport of Protons (H+)'''
 
There are two proposed mechanisms for UCP2 transport of protons (H+) into the mitochondria.
 
1. Direct proton (H+) transport via negatively charged UCP2 residues through the membrane.
 
2. A neutral fatty acid is flipped through the mitochondrial membrane into the matrix, and because of the basic environment, it releases the proton (H+). The now negatively charged anion is then transported
through the membrane by UCP2.]]

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==References==
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