protein families

Protein Families1. sequence homology ― gene & protein

Swiss Prot blastp

2. similar protein structure – includes S-S, 2ndary structure patterns, 3D conformation.

3. related function

chymotrypsin trypsin

Reasons to Cleave Proteins1. Destroy a) Digest food protein

b) Eliminate function after usefulness

2. Activate ― Proproteins Active Protein

Digestive Serine ProteasesTrypsin – cleaves after Lys/ArgChymotrypsin – cleaves after aromatic

made in pancreas & secreted to intestinesactivated by enterokinase

Degrading Serine Proteases

Elastase (digestion of elastin, etc.) varied sourcesPlasmin (digestion of fibrin) kidney plasma

Myeloblastin (digests laminin, fibronectin, elastase, vitronectin, collagens)

Granzymes (target cell lysis in immune response)

Proteinase C (deactivates some Clotting factors)

Activating Serine Proteasesenteropeptidase & Trypsin +...Blood Clotting Factors ― thrombin, VII, IX, X, XI, XII, KallikreinsTissue Plasminogen Activator Complement C1 + activates C2 & C4

HGF Activator

1. E + S ES

2. ES E-P2 + P1 fast

3. E-P2 E + P2 slow

Serine proteases form a covalent intermediate

Chymotrypsin cleavage of N-acetyl-phenylalanine p-nitrophenyl ester

Serine Proteases

N

HN

..His

O - CH2H

..

SerAsp OC O

specificity pocket

H- N - CH - C N - CH - C -

R

OH

OH

Gly

O - CH2Ser

N

HN

..His

HAsp OC O

H

Serine Protease

- N - CH - C N - CH - C -

R

OH

OH

Glycovalent (acyl) intermediate

Trp ― XTyr ― XPhe ― X

Lys ― XArg ― X Ala ― X

Large aromatic basic small

Blood Clotting CascadeIntrinsic Cascade

Kallikrein released

Extrinsic + Intrinsic Cascade

Kallikrein + TF released

Blood Clotting Cascade

XII XIIa Trauma XI XIa

IX IXa VIIa VII VIIIa

t TF & Ca

X Xa X Va

t

(Vit. K) Prothrombin Thrombin

Fibrinogen Fibrin XIIIa

t Clot

Intrinsic (Kininogen & Kallikrein) Extrinsic

(AB

A A

B B

2 rod segments

3 globular segments Fibrinogen: Soluble monomer unit

Fibrinogen

Fibrin II

A A

B B

Thrombin

A A

B B

AA

BB AA

BB AA

BB

AA

BB

AA

BB AA

BB

AA

BB AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

23 nm46 nm

O

CH2-CH2-C-NH2

H2N-(CH2)3-CH2

N-(CH2)3-CH2

H

GLN

LYSFactor XIIITransglutaminase

AA

BB AA

BB AA

BB

AA

BB

AA

BB AA

BB

AA

BB AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BBAA

BB AA

BB AA

BB

AA

BB

AA

BB AA

BB

AA

BB AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BBAA

BB AA

BB AA

BB

AA

BB

AA

BB AA

BB

AA

BB AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BBAA

BB AA

BB AA

BB

AA

BB

AA

BB AA

BB

AA

BB AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

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

HARD CLOT

FT SIGNAL 1 ?FT PROPEP ? 43FT CHAIN 44 622 PROTHROMBIN.FT PEPTIDE 44 198 ACTIVATION PEPTIDE (FRAGMENT 1).FT PEPTIDE 199 327 ACTIVATION PEPTIDE (FRAGMENT 2).FT CHAIN 328 363 THROMBIN LIGHT CHAIN (A).FT CHAIN 364 622 THROMBIN HEAVY CHAIN (B).FT DOMAIN 108 186 KRINGLE 1.FT DOMAIN 213 291 KRINGLE 2.FT DOMAIN 364 622 CATALYTIC.FT SITE 198 199 CLEAVAGE (BY THROMBIN).FT SITE 327 328 CLEAVAGE (BY FACTOR XA).FT SITE 363 364 CLEAVAGE (BY FACTOR XA).FT ACT_SITE 406 406 CHARGE RELAY SYSTEM.FT ACT_SITE 462 462 CHARGE RELAY SYSTEM.FT ACT_SITE 568 568 CHARGE RELAY SYSTEM.FT MOD_RES 49, 50, 57, 59, 62, 63, 68, 69, 72, 75FT CARBOHYD 121, 143, 416

Binds membrane

-carboxy Glu chelates Ca++

HisAspSer

Prothrombin Sequence

Disulfide bond 336-482

N

C

Activation Peptide #1 44-198

Activation Peptide #2 199-327cleaved by Thrombin (in vitro only?)

cleaved by Xa363-364 cleaved by Xa - still heldtogether by disulfide bond (336-482).

10 Glu residues, 49-75 converted to -carboxy Glu by Vit K dependent carboxylase

Serine Protease Domain 328-362

Prothrombin

CH2-CH2-COO-

Vit K dependent Carboxylase

Ca++ +

+

+

+

Modified Glu residues anchor Prothrombin to platelets (Ca2+) While Kringle domains bind damaged tissue

CH2-CHCOO-

COO-

Control of Clotting

Protein C - activated by thrombin (destroys factor VIII & V)

Antithrombin III (inhibits soluble thrombin, XII, XI, IX, & X)

Heparin - released by damaged mast cells (enhances Antithrombin III)

Blood Clot

time after trauma

Anti-Thrombin III + Heparinprevents off-site clotting

Kallikrein & TF released

Protein C: inhibits additional clot TPA & Plasmin: dissolves clot

Reversal of Clotting

TPA (72K) Plasminogen (bound) Plasmin

AA

BB AA

BB AA

BB

AA

BB

AA

BB AA

BB

AA

BB AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

AA

BB

Clot Dissolved clot

TPA

SerineProtease

Kringle :binds tissue

Fibrin bindingGrowth Factor DomainThrombin

Plasmin

Medical Reasons to prevent Clotting (heparin)Angioplasty (restenosis & rethrombosis 6-8%)

Deep-Vein ThrombosisUnstable Angina

Medical Reasons to dissolve Clots (cloned TPA)Myocardial infarction (MI) or stroke

Hemophilia & Factor VIII1. X linked - 1 in 10,000 males2. 50% severe : < 1% F8 activity3. 10% moderate : 2-5% F84. 40% mild : 5-30% F8

NSAIDs

Glucose

Acetyl CoA

cholesterol

cortisone

Membrane Lipids

arachadonic acid

prostaglandins (Cox-2)platelet activating factor(Cox-1) (+ gastric protection)

leukotrienes

NSAIDs (aspirin)

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.

Aspirin

S – CH3

|O

O

O

O Vioxx

S – NH2

|O

O

NN

F3C

Celebrex

COOH

O

O

COX-2 has an extension of the hydrophobic pocket binding site for aspirin relative to Cox-1.

S – CH3

|O

O

O

OVioxx

Vioxx recall

FDA approved May 1999

VIGOR submitted Feb 2001 designed to show gastric protection also illustrated ↑cardiovascular risk

Withdrawn Sept 2004

Vioxx effects ― desired effect - ↓prostacyclin - ↓inflammation responseAdditional effect - ↑thromboxanes - ↑bp and ↑clotting