Upload
annick
View
54
Download
0
Tags:
Embed Size (px)
DESCRIPTION
Protein Families. sequence homology ― gene & protein Swiss Prot blastp . similar protein structure – includes S-S, 2 ndary structure patterns, 3D conformation . chymotrypsin trypsin. 3. related function. Reasons to Cleave Proteins. Destroy a) Digest food protein - PowerPoint PPT Presentation
Citation preview
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