Protein Families

1. Protein Families • sequence homology ― gene & protein • Swiss Prot blastp • similar protein structure – • includes S-S, 2ndary structure patterns, 3D conformation. chymotrypsin trypsin 3. related function

2. Reasons to Cleave Proteins • Destroy • a) Digest food protein • b) Eliminate function after usefulness • Activate ― Proproteins  Active Protein Digestive Serine Proteases Trypsin – cleaves after Lys/Arg Chymotrypsin –cleaves after aromatic made in pancreas & secreted to intestines activated by enterokinase

3. Degrading Serine Proteases Plasmin(digestion of fibrin) kidney  plasma Elastase(digestion of elastin, etc.) varied sources Myeloblastin(digests laminin, fibronectin, elastase, vitronectin, collagens) Granzymes(target cell lysis in immune response) Proteinase C(deactivates some Clotting factors) Activating Serine Proteases enteropeptidase & Trypsin +... Blood Clotting Factors ― thrombin, VII, IX, X, XI, XII, Kallikreins Tissue Plasminogen Activator Complement C1 +  activates C2 & C4 HGF Activator

4. Serine proteases form a covalent intermediate 1. E + S  ES 2. ES  E-P2 + P1 fast 3. E-P2 E + P2 slow Chymotrypsin cleavage of N-acetyl-phenylalanine p-nitrophenyl ester

5. specificity pocket R Serine Proteases O Gly H - N - CH - C N - CH - C - H H O .. O - CH2 H .. Ser Asp N O C O His HN

6. R Serine Protease O Gly H covalent (acyl) intermediate - N - CH - C N - CH - C - H H O O - CH2 H .. Ser Asp N O C O His HN

7. Trp ― X Tyr ― X Phe ― X Lys ― X Arg ― X Ala ― X small Large aromatic basic

8. Intrinsic Cascade Kallikrein + TF released Kallikreinreleased Extrinsic + Intrinsic Cascade Blood Clotting Cascade

9. Intrinsic (Kininogen & Kallikrein) Extrinsic XII XIIa Trauma XI XIa IX IXaVIIaVII VIIIatTF & Ca X XaX Vat (Vit. K)ProthrombinThrombin Fibrinogen Fibrin XIIIat Clot Blood Clotting Cascade

10. 3 globular segments A A B B 2 rod segments Fibrinogen: Soluble monomer unit (AB

11. Thrombin A A A A B B B B Fibrinogen Fibrin II

13. B B B B B B B B B B B B B B A A A A A A B B B B B B A A A A A A A A B B B B B B B B A A A A A A B B B B B B A A A A A A A A A A A A A A 46 nm 23 nm

14. O CH2-CH2-C-NH2 H H2N-(CH2)3-CH2 N-(CH2)3-CH2 GLN LYS Factor XIII Transglutaminase

15. B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B A A A A A A B B B B B B B B A A A A A A B B B B B B B B A A A A A A B B B B B B B B B B A A A A B B B B A A A A A A A A A A B B B B B B A A A A A A A A B B B B B B A A A A A A A A B B B B B B B B B B B B B B A A A A A A A A A A A A A A B B B B B B B B A A A A A A B B B B B B B B A A A A A A B B B B B B B B B B B B B B A A A A A A A A A A A A A A B B B B B B A A A A A A A A B B B B B B A A A A A A A A B B B B B B A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A Fibrin Stabilizing Factor (XIII) is a transglutaminase - forms GLN-LYS, covalent cross-links. HARDCLOT

16. g-carboxy Glu chelates Ca++ Prothrombin Sequence Binds membrane His Asp Ser Disulfide bond 336-482

17. 10 Glu residues, 49-75 converted to g-carboxy Glu by Vit K dependent carboxylase cleaved by Thrombin (in vitro only?) cleaved by Xa 363-364 cleaved by Xa - still held together by disulfide bond (336-482). Serine Protease Domain 328-362 N Activation Peptide #1 44-198 Activation Peptide #2 199-327 C Prothrombin

18. CH2-CH2-COO- CH2-CH Vit K dependent Carboxylase COO- + + Ca++ COO- + + Modified Glu residues anchor Prothrombin to platelets (Ca2+) While Kringle domains bind damaged tissue

19. Control of Clotting Antithrombin III (inhibits soluble thrombin, XII, XI, IX, & X) Heparin - released by damaged mast cells (enhances Antithrombin III) Protein C - activated by thrombin (destroys factor VIII & V)

20. Kallikrein & TF released Anti-Thrombin III + Heparin prevents off-site clotting Protein C: inhibits additional clot TPA & Plasmin: dissolves clot Blood Clot time after trauma

21. Clot  Dissolved clot B B B B B B B B B B B B B B A A A A A A B B B B B B A A A A A A A A B B B B B B B B A A A A A A B B B B B B A A A A A A A A A A A A A A Reversal of Clotting TPA (72K) Plasminogen(bound) Plasmin

22. Plasmin TPA Fibrin binding Growth Factor Domain Thrombin Kringle : binds tissue Serine Protease

23. Medical Reasons to prevent Clotting (heparin) Angioplasty(restenosis & rethrombosis 6-8%) Deep-Vein Thrombosis Unstable Angina Medical Reasons to dissolve Clots (cloned TPA) Myocardial infarction (MI) or stroke Hemophilia & Factor VIII 1. X linked - 1 in 10,000 males 2. 50% severe : < 1% F8 activity 3. 10% moderate : 2-5% F8 4. 40% mild : 5-30% F8

24. NSAIDs Membrane Lipids arachadonic acid prostaglandins (Cox-2) platelet activating factor (Cox-1) (+ gastric protection) leukotrienes Glucose Acetyl CoA cholesterol cortisone NSAIDs (aspirin)

25. Aspirin binds more tightly to COX-1 than COX-2. This means it will block platelet aggregation at much lower [ ] than is required to block inflammation. NSAIDS can be harmful to individuals at risk of internal bleeding. One ‘holy grail’ of pharmacology was to find a selective COX-2 inhibitor.

26. COOH O O O O S – CH3 | O S – NH2 | O O O N F3C N Celebrex Aspirin Vioxx COX-2 has an extension of the hydrophobic pocket binding site for aspirin relative to Cox-1.

27. O O S – CH3 | O O Vioxx recall FDA approved May 1999 Vioxx VIGOR submitted Feb 2001 designed to show gastric protection also illustrated ↑cardiovascular risk Withdrawn Sept 2004 Vioxx effects ― desired effect - ↓prostacyclin - ↓inflammation response Additional effect - ↑thromboxanes - ↑bp and ↑clotting