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 Biochemistry Ch 10.5 Fibrinogen

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

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 Kroemer et 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 –E3  –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

14. Apoptosis: extrinsic pathway TNFR  DISC  Casp8  Casp3  apoptosis eg: ligand mediated Fas aggregation –Death domain (dd) –Death effector domain (ded) –Death-inducing signaling complex (DISC) FADD Casp8

15. 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)

16. 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

17. Wullaert et al., 2006