Proteolysis

• Proteolytic structure & biochemistry– Amino-acid catalyzed– Metalloproteases

• Ubiquitin-proteasome system

• Caspase cascade & apoptosis

Non-metal proteases

• Amino-acid centers– {Cysteine, serine, theonine} + histidine– Aspartate, glutamic acid

Hedstrom 2002

Serine proteases

• Chymotrypsin, subtilisin, assemblin

• Digestion, immune response, coagulation

• Coagulation– Protease cascade– Thrombin-mediated cleavage of fibrinogen– Spontaneous polymerization

Biochemistry Ch 10.5

Fibrinogen

Metalloproteases

• Zinc center

• Histidine

Kester & Matthews 1977

Tetrahedral intermediate

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

Autophagy

• Lysosomal degradation of cellular components

• ATG-mediated engulfment• Starvation--|mTOR--|ULK1/2• IP3--|beclinAATG14

Kroemer et al 2010

Proteasome

• Predominant pathway for protein degradation

• Anti-ribosome

• Ubiquitin

• Poly-Ub

• Proteasome

Ubiquitination

• E1 Ubiquitin activating enzyme

• E2 Ubiquitin conjugating enzyme

• E3 Ubiquitin ligase– E3– APC– HECT– SCF

Ubiquitin Ligase

Coordinate E2-ub with specific substrate

Cell cycle controlMisfolded proteins

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

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)• ROCK1MLC phosphorylation

Apoptosis

• Cellular condensation– Contraction– Membrane blebbing

• Nuclear condensation– DNA degradation– 200 bp “Ladder”– TUNEL

Apoptosis: extrinsic pathway

• TNFRDISCCasp8Casp3apoptosis

• eg: ligand mediatedFas aggregation– Death domain (dd)– Death effector domain (ded)– Death-inducing signaling

complex (DISC)• FADD• Casp8

Apoptosis: intrinsic/mitochondrial pathway

• CytochromeCApoptosomeCasp9casp3• 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)

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

Wullaert et al., 2006