Transcript Proteolysis - Georgia Institute of Technology

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
Fibrinogen
Biochemistry Ch 10.5
Metalloproteases
• Zinc center
• Histidine
Tetrahedral intermediate
Kester & Matthews 1977
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--|beclinAATG14
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
– E3a
– APC
– HECT
– SCF
Ubiquitin Ligase
Coordinate E2-ub
with specific
substrate
Cell cycle control
Misfolded 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)
• ROCK1MLC phosphorylation
Apoptosis
• Cellular condensation
– Contraction
– Membrane blebbing
• Nuclear condensation
– DNA degradation
– 200 bp “Ladder”
– TUNEL
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
Apoptosis: intrinsic/mitochondrial
pathway
• CytochromeCApoptosomeCasp9casp3
• Mitochondrial release of CyC
– Lysis – calcium/osmotic
– Bax-mediated pore formation
• Apoptotic proteaseactivating 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