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Proteolysis
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Proteolysis

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  1. Proteolysis • Proteolytic structure & biochemistry • Amino-acid catalyzed • Metalloproteases • Ubiquitin-proteasome system • Caspase cascade & apoptosis

  2. Non-metal proteases • Amino-acid centers • {Cysteine, serine, theonine} + histidine • Aspartate, glutamic acid Hedstrom 2002

  3. Serine proteases • Chymotrypsin, subtilisin, assemblin • Digestion, immune response, coagulation • Coagulation • Protease cascade • Thrombin-mediated cleavage of fibrinogen • Spontaneous polymerization Fibrinogen Biochemistry Ch 10.5

  4. Metalloproteases • Zinc center • Histidine Tetrahedral intermediate Kester & Matthews 1977

  5. Metalloproteases • Matrix metalloproteinase (MMP) • Zinc center • Autoinhibitory domain • Secreted • ADAM • A Disintegrin And Metalloprotease • Snake venom • Membrane anchored • ECM remodeling • Cell adhesion • Cytokine maturation Protease Disintigrin Cysteine rich Cytoplasmic

  6. Autophagy • Lysosomal degradation of cellular components • ATG-mediated engulfment • Starvation--|mTOR--|ULK1/2 • IP3--|beclinAATG14 Kroemeret al 2010

  7. Proteasome • Predominant pathway for protein degradation • Anti-ribosome • Ubiquitin • Poly-Ub • Proteasome

  8. Ubiquitination • E1 Ubiquitin activating enzyme • E2 Ubiquitin conjugating enzyme • E3 Ubiquitin ligase • E3a • APC • HECT • SCF

  9. Ubiquitin Ligase Coordinate E2-ub with specific substrate Cell cycle control Misfolded proteins

  10. Caspase • Cleavage-activated protease cascade • Active domain • Large & small • Inhibitory domain • Targeting domain Caspase action removes pro-domain, separates large & small subunits Assembly into functional tetramer or octamer Jekeley, 1998

  11. Caspase classes • Initiator • Activation by aggregation • Death effector domain • Caspase activation and recruitment domain • Limited substrates - caspases • Effector • Limited aggregation • Limited autolysis • Non-caspase substrates • poly(ADP-ribose) polymerase (DNA repair) • DNA fragmentation factor (aka: CAD) • ROCK1MLC phosphorylation

  12. Apoptosis • Cellular condensation • Contraction • Membrane blebbing • Nuclear condensation • DNA degradation • 200 bp “Ladder” • TUNEL

  13. Apoptosis: extrinsic pathway • TNFRDISCCasp8Casp3apoptosis • eg: ligand mediatedFas aggregation • Death domain (dd) • Death effector domain (ded) • Death-inducing signaling complex (DISC) • FADD • Casp8

  14. Apoptosis: intrinsic/mitochondrial pathway • CytochromeCApoptosomeCasp9casp3 • Mitochondrial release of CyC • Lysis – calcium/osmotic • Bax-mediated pore formation • Apoptotic protease-activating factor-1 (Apaf-1) • CyC induced oligomerization • Casp9 scaffold – Caspase recruitment domain (card)

  15. Bax/Bcl • Mitochondrial pore forming proteins • Bax (Bax, Bad, Bak, Bid, Bik) • Pore dimers • Large molecule release from intermembrane space • Bcl2 (Bcl2, bcl-x, bcl-w, Mcl-1) • Bax/Bcl2 heterodimers • Non-pore forming • Competitive regulation of intrinsic pathway • Casp8-mediated activation of Bid

  16. Wullaert et al., 2006