User:Cody Couperus

2014-05-18T05:17:58Z

Cody Couperus:

* Full Real Name: Cody Couperus

* Position: Undergraduate

* Institution (NO ABBREVIATIONS): University of Vermont

* City, State/Province, Country: Burlington, Vermont, USA

* Field of Expertise or Study: Undergraduate biochemistry student active in research related to the role of platelets and clotting factors in blood coagulation.

*[[User:Cody Couperus/Sandbox 1]]

*[[User:Cody Couperus/Sandbox 2]]

*[[User:Cody Couperus/Sandbox 3]]

User:Cody Couperus/Sandbox 2

2014-05-13T00:56:07Z

Cody Couperus: New page: ==Cathepsin Proteins== Cathepsins are proteases located in the lysosome, endosome, or extracellular space. They catalyze the hydrolysis of numerous peptide stubstrates utilizing serine, c...

==Cathepsin Proteins==

Cathepsins are proteases located in the lysosome, endosome, or extracellular space. They catalyze the hydrolysis of numerous peptide stubstrates utilizing serine, cystine, and aspartic protease mechanism.

==Cysteine Protease Cathepsins==
Are members of the C1 family of papain-like proteases

Cathepsin B

Cathepsin C

Cathepsin F

Cathepsin H

Cathepsin K

Cathepsin L

Cathepsin O

Cathepsin S

Cathepsin V

Cathepsin X

Cathepsin W

==Serine Protease Cathepsins==
Cathepsin A

Cathepsin G

==Aspartic Acid Protease Cathepsin==
Cathepsin D is the only aspartic protease of the cathepsin classification

== References ==
<references/>

Cody Couperus:

<StructureSection load='1PPB' size='350' side='right' caption='Thrombin (1PPB) viewed in cartoon representation colored by secondary structure. Active site residues Ser195, Asp102, and His57 are viewed in ball and stick form.' scene='58/583418/Thombin_main_secondary/2'>
==Introduction==
<scene name='58/583418/Thombin_main_secondary/2'>Thrombin</scene> is the serine protease that catalyzes the penultimate step in blood coagulation. It is activated from its [http://en.wikipedia.org/wiki/Zymogen zymogen], prothrombin, at the site of tissue injury by [[Factor_Xa | FXa]] and its cofactor [http://en.wikipedia.org/wiki/Factor_V FVa] in the presence of phospholipid membrane and calcium. Thrombin is then able to catalyze the cleavage of [[Fibrinogen | fibrinogen]] to insoluable fibrin which spontaneously polymerizes to form a stable clot.<ref name="zero">PMID: 7023326</ref><ref name="one">PMID: 11001069</ref> Thrombin also acts as a procoagulant by:

* Activating platelets through their [http://en.wikipedia.org/wiki/Protease-activated_receptor protease activated receptors (PARs)]<ref name="one"/>
* Preventing [http://en.wikipedia.org/wiki/Von_Willebrand_factor Von Willebrand factor (VWF)] processing by cleaving [http://en.wikipedia.org/wiki/ADAMTS13 ADAMTS13] <ref name="two">PMID: 15388580</ref><ref name="three">PMID: 15994286</ref>
* Enhancing its own production through [[Factor_IX | FIX]] activation <ref name="three"/>
* Activating the fibrin crosslinking transglutaminase [[Factor_XIII | FXIII]] <ref>PMID: 4811064</ref>
* Activation of [http://www.ncbi.nlm.nih.gov/pubmed/17008302 thrombin activatable fibrinolysis inhibitor (TAFI)] <ref>PMID: 20229688</ref>

Activity of thrombin is regulated physiologically by the serpin inhibitors:
* [[Antithrombin]] <ref name="three"/><ref name='four'>PMID: 24477356</ref><ref name='five'>PMID: 23809129</ref>
* [http://en.wikipedia.org/wiki/Heparin_cofactor_II Heparin cofactor II]<ref name="three"/><ref name='four'/><ref name='five'/>
* [http://en.wikipedia.org/wiki/Protein_C_inhibitor Protein C inhibitor]<ref name="three"/><ref name='four'/><ref name='five'/>
* [http://www.sciencedirect.com/science/article/pii/0968000484900744 Protease nexin 1]<ref name="three"/><ref name='four'/><ref name='five'/>

Generation of thrombin is decreased when thrombin reaches endothelial lining and interacts with [[1fge |thrombomodulin]] which significantly increases activity of thrombin in activating [http://en.wikipedia.org/wiki/Protein_C protein C (APC)].<ref>PMID: 3029867</ref> This enzyme will go on to inactivate FVa<ref>PMID: 7989361</ref><ref name='six'>PMID: 8639840</ref> and [[Factor_VIII | FVIIIa]]<ref name='six'/> , cofactors for activation of prothrombin<ref>PMID: 15147718</ref> and [[Factor_Xa | FXa]]<ref>PMID: 10881749</ref>, respectively.

By balancing substrate specificity, activity, and inhibition thrombin plays a central role in the blood coagulation cascade. <ref name="three"/>

==The Thrombin Life Cycle==
Upon tissue damage [http://en.wikipedia.org/wiki/Tissue_factor tissue factor (TF)] is released by subendothelial cells. This interacts with circulating [Factor_VII| FVIIa], a zymogen-like serine protease, significantly increasing its activity. The FVIIa-TF complex activates FVII, FIX, and FX. The now active FXa cleaves prothrombin bound to membranes through its [http://en.wikipedia.org/wiki/Gla_domain Gla domain], activating it to thrombin.

Thrombin interacts with platelet membrane protein [http://www.ncbi.nlm.nih.gov/pubmed/14720584 GpIbα] and subsequently cleave protease activated receptor-1 (PAR1) causing a signaling cascade which leads to platelet [http://en.wikipedia.org/wiki/Platelet_alpha-granule α-granule] release and membrane flipping exposing the negatively charged [http://en.wikipedia.org/wiki/Phosphatidylserine phosphatidylserine]. The platelet alpha granules contain the physiologically relevant pool of FVa. <ref>Camire, R. M. (2010). Platelet factor V to the rescue. Blood, 115(4), 753-754. [http://dx.doi.org/10.1002/bmb.2005.494033022442 DOI: 10.1182/blood-2009-11-252619]</ref>

Thrombin also causes activation of FIX, through FXI cleavage, and FVIII which form the Xase complex to activate FX. FVa and FXa form the prothrombinase complex in the presence of calcium and phospholipid. It causes rapid activation of prothrombin to thrombin. This increase in thrombin allows sufficient fibrinogen to be cleaved to fibrin which is able to polymerize to form a stable blood clot.

Further supporting coagulation, thrombin activates FXIII, a transglutaminase that crosslinks fibrin at lysine residues.
The structure of thrombin facilitates its inactivation.

Once the endothelial lining is reached thrombin binds heparin glycosaminoglycans. This facilitates its inactivation by serpin inhibitors antithrombin and heparin cofactor II. In addition, thrombin will interact with thrombomodulin which significantly increases its catalytic efficiency activating protein C. Activated protein C inactivates FVIIa and FVa thus down regulating thrombin generation.

Thrombin plays a critical dual role in blood coagulation. It must be both promiscuous and specific so that it may complete both procoagulant and anticoagulant functions. The structure of thrombin facilitates its important physiologic functions.

==Prothrombin Activation==
[[Image:Prothrombin activation scheme_2.png|300px|right|thumb| Activation scheme of in vivo activation of prothrombin by the prothrombinase complex in presence of calcium and a phospholipid bilayer]]
[[Image:Prothrombin activation scheme_nofva.png|300px|right|thumb| Activation scheme of in vivo activation of prothrombin by FXa in the absence of FVa.]]
Prothrombin is the zymogen form of thrombin. From N-terminal to C-terminal it consists of a Gla domain, two kringle domains, and a catalytic domain.

Prothrombin is activated by prothrombinase which consists of FXa, FVa, calcium, and a phospholipid surface. In vivo the first cleavage occurs at the R320-I321 bond, corresponding to residues 15-16 in thrombin which is the N-terminus of the B chain, producing meizothrombin.<ref name='seven'>PMID: 22944689</ref> Subsequent cleavage at R271-T272 yields thrombin.<ref name='seven'/> The initial cleavage can also occur at R271 resulting in prothrombin-2 which will then be cleaved at R320 to produce thrombin.<ref>PMID: 1995649</ref>

After cleavage by prothrombinase the new B chain N-terminus (Ile16) folds into the core protease domain and forms a salt bridge with Asp194.<ref name='seven'/> This leads to stabilization of regions of the 180s-loop, Na+ binding loop, and γ-loop (zymogen activation domains). These changes provide the correct conformation for the S1 pocket and oxyanion hole for catalysis.<ref name='seven'/><ref>PMID: 15890651</ref>

==Thrombin Structure and Function==

[[Image:Electrostatic labeled.png|300px|right|thumb| Thrombin (1PPB) overlayed with electrostatic surface. Structural features 60-loop, γ-loop, exosite I, and exosite II labeled]]
Thrombin is a α/β heterodimer composed of a 36 amino acid A chain and 259 amino acid B chain connected by a <scene name='58/583418/Disulfides_nospin/1'>disufide</scene> bridge between Cys1 and Cys122, in addition to 3 other intrachain disulfide bonds.<ref name='eight'>PMID: 2583108</ref> Its overall fold is similar to trypsin and chymotrypsin and it belongs to the peptidase S1 protease family<ref>PMID: 18768474</ref>. It is an overall spherical protein with approximate dimensions of 45 X 45 X 50 Å^3.<ref name='eight'/>
Important structural features include:
* A prominent active site cleft flanked by the 60- and γ-loops
* A sodium binding loop
* Two surface patches referred to as exosite I and exosite II.

The <scene name='58/583418/A_chain_nospin/1' target='0'>A chain</scene> is mostly helical and is wound around the B chain and shaped like a boomerang. It is bound to the B chain mostly through side chain interactions including a salt bridge and H-bond cluster at residues D14, E8, and E14c.<ref name='eight'/> Furthermore the C-terminus region forms a short amphipathic helix with hydrophobic side chains interacting with the B chain.<ref name='eight'/>

The <scene name='58/583418/B_chain/1' target='0'>B chain</scene> contains the active site of the protein and has numerous notable structural features. The active site is formed at the rims of two interacting 6 stranded <scene name='58/583418/Beta_barrel/2'>beta barrel domains</scene>(N-terminal barrel in red and C-terminal barrel in orange) which are surrounded by 4 helical regions and many turns.

The serine protease <scene name='58/583418/Catalytic_triad/1'>catalytic triad</scene>, based on chymotrypsin numbering, are Ser195, His57, and Asp102. As is common with serine proteases, an <scene name='58/583418/Oxyanion_hole/2'>oxyanion hole</scene> hole is formed by backbone amides of Ser195 and Gly193.<ref name='seven'/> This has the functional role of stabilizing the oxyanion intermediate involved in the serine protease mechanism by hydrogen bonding to the oxygen of the P1 residue (traditional substrate-protein nomeclature <ref>PMID: 22925665</ref>. In addition, since thrombin cleaves after Arg/Lys the <scene name='58/583418/S1/2'>S1 specificity site</scene>, formed by the 180s- and 220s- loops, has Asp189 at the base to form a salt bridge with the incoming substrate. Furthermore, the S4 binding pocket accommodates hydrophobic substrate residues.

The '''active site''' cleft rims are formed by the hydrophobic and rigid <scene name='58/583418/Loops/2'>60-loops</scene> (residues L60, Y60a, P60b, P60c, W60d, D60e, K60f, N60g, F60h, T60i, and N60g) and the <scene name='58/583418/Loops/1'>γ-loop</scene> (residues T147, W147a, T147b, A147c, N147d, and V147f) while the base is mostly hydrophilic negatively charged amino acids. The cleft is deep compared to more promiscuous serine proteases, consequently substrates must either have a large loop that is cleaved or have favorable interactions with the insertion loops <ref>PMID: 16102053</ref>.

Many other loops project out of the B chain but most are rigid due to proline and tryptophan residues.

'''Exosite I''' is located on the B chain and had both basic and hydrophobic character. It is important in binding fibrinogen, platelet activated receptors, and thrombomodulin.

'''Exosite II''' is also part of the B chain, and derived from numerous basic amino acids, this is the site of heparin binding through the sulfate groups on the glycosaminoglycan. It is also the site of GpIα on the platelet surface.

The <scene name='58/583418/Sodium_binding_loop/1'>sodium binding site</scene> is formed by the 180s- and 220s- loops. Na+ is bound by the backbone oxygens of Arg221a and Lys224 in addition to four water molecules in a classic octahedral geometry<ref>PMID: 9108691</ref>. Through the covelent disulfide linkage between Cys220 and Cys 191 the sodium binding site is linked to Ser195 and the oxyanion hole.

==Thrombin Allostery==

Binding of thrombin by sodium or at exosite I stabilizes a form of thrombin that improves substrate recognition.<ref name='seven'/> This occurs due to energetic linkage between these sites to the S1 binding pocket and oxyanion hole. Rapid kinetic analysis suggests that thrombin is in a dynamic equilibrium that consists of a fast, slow, and inactive state<ref name='seven'/>. There is question as to the physiologic relevance of the inactive state. Regardless, there will be a proportion of fast:slow thrombin and sodium binding to the fast form stabilizes that conformation. Indeed, mutation of the residues involved in sodium binding diminishes the activity of thrombin.<ref name='seven'/> It should be restated, that current data suggest that sodium binding does not induce a conformation change, rather, it stabilizes a conformation of thrombin that has greater activity.

==Interesting Secondary Structure==
Thrombin has a <scene name='58/583418/Cis_proline/1'>cis peptide bond</scene> in a loop containing proline 37. The dihedral (Cα-C'-N-Cα) angle omega is 142.7 degrees.

[[Image:Capping.png|450px|center|thumb| “Capping box” motif in alpha thrombin (PDB: 1PPB) represented by His230 (Ncap) side chain and main chain hydrogen bonded with the backbone nitrogen of Arg233 (N3). An additional feature is a weak hydrophobic interaction between Thr229 (N’) and Val234 (N4) termed the “hydrophobic staple.” This motif derives it’s name from the box shaped hydrogen bonding pattern.]]

[[Image:Cation pi.png|450px|center|thumb| A cation-π interaction between Trp128 and Arg 129 in alpha thrombin (PDB: 2BDY). The guanidinium carbon is 3.6 angstroms from the top edge of the Trp. It is expected that the epsilon nitrogen forms the primary cation-π interaction. The electrostatic and Van der Waals interaction energies were calculated to be -3.29 kcal/mol and -3.08 kcal/mol respectively by the CaPTURE program (http://capture.caltech.edu/result.cgi).]]
</StructureSection>

== References ==

<references/>