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Proteases Jessica Bell Advanced Biochemistry Class University of Richmond March 12th, 2002

Proteases Jessica Bell Advanced Biochemistry Class University of Richmond March 12th, 2002. Adhesion P. gingivalis protease. Secretion signal peptidases. Immune Response T-cell protease. Development snake. Blood pressure regulation renin. Digestion trypsin. Coagulation thrombin.

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Proteases Jessica Bell Advanced Biochemistry Class University of Richmond March 12th, 2002

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  1. Proteases Jessica Bell Advanced Biochemistry Class University of Richmond March 12th, 2002

  2. Adhesion P. gingivalis protease Secretion signal peptidases Immune Response T-cell protease Development snake Blood pressure regulation renin Digestion trypsin Coagulation thrombin Complement Fixation CI protease Cell fusion hemaglutinase Tumor Invasion collagenase Reproduction and Fertilization acronase Pain Sensing kallikrein Fibrinolysis tissue plasminogen actvator Animal Virus Replication HIV protease Hormone Processing Kex 2 6 Broad Categories Function Protease Nutrition trypsin subtilisin, a-lytic protease Invasion matrix metallo proteases Evasion IgA protease Adhesion P. gingivalis protease Processing signal peptidase, viral proteases, proteosome Signaling caspases, granzymes

  3. H O H + H2O + +3HN C C N C COO- R1 H R2 H +3HN C COO- R1 H +3HN C COO- R2 Biology Differs, Chemistry is the Same endopeptidase exopeptidase

  4. Michaelis Complex 57 195 - O N: N 102 O H H O O HN P1

  5. Michaelis Complex 57 195 - O N: N 102 102 O O H H O O O HN P1 57 195 O d+ - N N H H d- O HN P1 Transition State to Acylation

  6. Michaelis Complex 57 195 - O N: N 102 102 102 O O O H H O O O O HN P1 57 195 O d+ - N N H H d- O HN P1 57 195 + - N H N O H O- HN P1 Transition State to Acylation Tetrahedral Intermediate I

  7. Michaelis Complex 57 195 - O N: N 102 102 102 102 O O O O H H O O O O O HN P1 57 195 O d+ - N N H H d- O HN P1 57 57 195 195 + - - N: N H N N O O H H O O- HN NH3+ P1 P1 Transition State to Acylation Acyl-Enzyme Tetrahedral Intermediate I

  8. Michaelis Complex 57 195 - O N: N 102 102 102 102 102 O O O O O H H O O O O O O HN P1 57 57 195 195 O d+ d+ - - N N N N H O d- H H H d- O O O HN H P1 P1 57 57 195 195 + - - N: N H N N O O H H O O- HN NH3+ P1 P1 Transition State to Deacylation Transition State to Acylation Acyl-Enzyme Tetrahedral Intermediate I

  9. Michaelis Complex Tetrahedral Intermediate II 57 57 195 195 + - - H N N O O N: N 102 102 102 102 102 102 H O- O O O O O O H H O O O O O O O O H HN P1 P1 57 57 195 195 O d+ d+ - - N N N N H O d- H H H d- O O O HN H P1 P1 57 57 195 195 + - - N: N H N N O O H H O O- HN NH3+ P1 P1 Transition State to Acylation Transition State to Deacylation Tetrahedral Intermediate I Acyl-Enzyme

  10. Mechanistic Sets of Proteases set feature inhibitor examples function serine protease active site serine fluorophosphates trypsin digestion H57, D102, S195 thrombin blood coagulation plasmin lysis of blood clots coccoonase mechanical subtilisin digestion acrosin sperm penetration metalloproteases Zn2+, E270 o-phenanthroline carboxypeptidase digestion Zn2+, Ca2+ o-phenanthroline thermolysin digestion E143, H231 sulfhydryl protease active site cysteine iodoacetate papain digestion C25, H159, N175 strept. proteinase digestion cathepsin B intracell. digestion Acid protease acidic pH optimum diazoketones pepsin digestion D32, D215 chymosin milk coagulation

  11. Divergent vs. Convergent Evolution Catalytic Triad Conserved Trypsin Elastase Subtilisin Same Fold

  12. Thermolysin Carboxypeptidase A

  13. Pepsin HIV Protease

  14. OH CH3 O O HN HN NH NH NH O O OH NH3+ P2 P1 P1’ P2’ Peptide Scissile Bond Subsite of Protease S2 S1 S1’ S2’

  15. Substrate Selection within the Same Tertiary Fold

  16. Ecotin Serine Protease Inhibitor Unknown function Dimeric 1° and 2° binding sites Cleaved

  17. Cystatin Superfamily Cysteine protease inhibitors Non-canonical binding

  18. Serpins Serine protease inhibitors Irreversible Disruption of 3º structure

  19. A Zn2+ H H O H O H O H2N NH C C N C C N C C C NH Arg R2 H R1 H R1 O H2N A Zn2+ H H O H O H2N S CH2 C C N C C C NH Arg CH3 O H2N H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-His-Leu-Co2H Proangiotensin H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-Co2H Angiotensin - + carboxy-di-peptidase active site + - Captopril

  20. Ways to Determine Specificity Synthesis of short peptides [15 to 20a.a.], check for cleavage with PAGE Phage display of short peptides • Positional scanning synthetic combinatorial libraries [PS-SCL]

  21. X O O X HN HN NH NH X X O O HN O 7-amino-4-methyl coumarin R N D E Q G H I L A Ac-XXXO-AMC K F P S T W Y V m R N D E Q G H I L A Ac-XXOX-AMC K F P S T W Y V m R N D E Q G H I L A Ac-XOXX-AMC K F P S T W Y V m R N D E Q G H I L A Ac-OXXX-AMC K F P S T W Y V m

  22. P2 O O P4 HN HN NH NH P1 P3 O O

  23. Cytotoxic Lymphocytes Molecular Biology of the Cell, Garland

  24. Cytotoxic T Lymphocyte Apoptotic Pathway Cytotoxic T lymphocyte Granzymes Perforin Ca2+ Ca2+ Ca2+ 3 Fas Ca2+ GrnA GrnB MPR? DD FADD cleave pro-caspases serpins Nuclease? DED Mito. apoptosis Single stranded breaks in DNA Bcl-2 aggregrates pro-caspase 8, intermolecular cleavage to caspase 8, activation of effector caspases [3, 6, 7], apoptosis nucleus

  25. Granzymes: Lymphocyte Serine Proteases Name Activity Predicted P1 MW cleavage site A Trypsin-like R/K 60 (Dimer) BAsp-ase D/E 35 C Unknown N/S 27 D Unknown F/L 35-50 E Unknown F/L 35-45 F Unknown F/L 35-40 G Unknown F/L H Chymase F I Unknown J Unknown K Trypsin-like 30 M Met-ase M/L/nor-L 30

  26. Granzyme Structure Waugh et al. (2000) Nat. Struct. Biol. 7:762-765

  27. Granzyme A, Proposed Dimeric Structure

  28. Substrate Sequence P4 P3 P2 P1 FLUOROGENIC LIBRARIES V/I G/A/S N R PIL-1b D A P V R S L N C T THROMBIN RECEPTOR T L D P R S F L L R HISTONE H1 K L G L K S L V S K HISTONE H2b A P A P K K G S K K SET Q T Q N K A S R K R LAMIN B V T V S R A S S S R Granzyme A: Substrate Specificity and Macromolecule Substrates

  29. Research Aims Quaternary Structure and Substrate Selectivity Crystallographic Study Dimer vs. Monomer Cell Lysate Assays

  30. mOD/min @ 405nm 0 0.05 5 50 [Inhibitor], mM Macromolecular Inhibition of Granzyme A Control mM84R Eco dM84R Eco Tryp. Inh.

  31. Potential Effects of Oligomer on Macromolecular Inhibitors grnA

  32. Potential Effects of Oligomer on Macromolecular Inhibitors grnB:dEcotin

  33. Potential Effects of Oligomer on Macromolecular Inhibitors mEcotin

  34. O O N C C N C C N C C CH2Cl O Small Molecule Inhibitor of Granzyme A mOD/minute @405nm 0 50 100 150 200 [Inhibitor], nM

  35. Box1 WellB3 Box1 WellC3 Box1 WellC5 Box3 WellC5 Box4 WellA2 Box1 WellD2 Box2 WellC4 Box2 WellC4a Crystallization Trials I Citrate pH5.6/Acetate/PEG4K Cac. pH6.5/AmSO4/PEG8K Tris pH8.5/LiSO4/PEG4K Citrate pH6.5/t-Butanol Acetate pH4.5/AmSO4/PEG4K Citrate pH6.5/i-propanol/PEG4K MES pH6.5/AmSO4/PEG-me5K

  36. Crystallization Trials II Citrate pH5.6/Acetate/PEG4K Citrate pH6.5/i-prop/PEG4K 30% 20% 20% 10% 25% 15%

  37. Diffraction!!! Unit Cell a=72.85 b=175.9 c=152.55, Å b=99.24º <I/sI> 14.3 redundancy 90% of the data is measured ≥2X Resolution 25-2.55Å completeness 97.5% [89%] [2.64-2.55Å shell] chi2 1.2 Rmerge 0.049

  38. Research Aims Quaternary Structure and Substrate Selectivity Crystallographic Study Dimer vs. Monomer Cell Lysate Assays Substrate Selection amongst Isozymes Structural Comparison of Human and Mouse Mutagenesis & Substrate Libraries

  39. Granzyme A: Human and Mouse Human MRNSYRFLAS SLSVVVSLLL IPEDVCEKII GGNEVTPHSR PYMVLLSLDR Mouse MRNASGPRGP SLATLLFLLL IPEGGCERII GGDTVVPHSR PYMALLKLSS Human KTICAGALIA KDWVLTAAHC NLNKRSQVIL GAHSITREEP TKQIMLVKKE Mouse NTICAGALIE KNWVLTAAHC NVGKRSKFIL GAHSINK-EP EQQILTVKKA # Human FPYPCYDPAT REGDLKLLQL TEKAKINKYV TILHLPKKGD DVKPGTMCQV Mouse FPYPCYDETT REGDLQLVRL KKKATVNRNV AILHLPKKGD DVKPGTRCRV # Human AGWGRTHNSA SWSDTLREVN ITIIDRKVCN DRNHYNFNPV IGMNMVCAGS Mouse AGWGRFGNKS APSETLREVN ITVIDRKICN DEKHYNFHPV IGLNMICAGD Human LRGGRDSCNG DSGSPLLCEG VFRGVTSFGL ENKCGDPRGP GVYILLSKKH Mouse LRGGKDSCNG DSGSPLLCDG ILRGITSFG- GEKCGDRRWP GVYTFLSDKH # * * Human LNWIIMTIKG AV Mouse LNWIKKIMKG SV 68% Identical! P4 P3 P2 P1 Human V/I G/A/S N R Mouse G F/Y F R

  40. Substrate Specificity of Granzyme A Species H57 D102 R99 P2 P1 S195 P3 P4 D189

  41. Substrate Specificity of Granzyme A Species P4 W224

  42. Substrate Specificity of Granzyme A Species P4 W224

  43. Substrate Specificity of Granzyme A Species

  44. C. S. Craik Craik Lab Members Granzyme A Sandy Waugh Sami Mahrus Carly Klein MT-SP1 Jeonghoon Sun Ami Bhatt R. J. Fletterick Fletterick Lab Members Mary Jane Budny and all those helpful people who... ALS 8.3.1 James Holton NIH: The $$$ people

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