Apoptotic Pathways in Mammals Douglas R. Green 1 Apoptosis A form of cell death that is defined morphologically, and features a number of biochemical events Programmed cell death Cell death that occurs at a predictable stage of development; It can be by apoptosis, but these terms are not synonymous 2 a b a Digits form during the development of the mammali an limb by programmed cell death; This cell death has the characteristics of apoptosis b From Choutan, M., et al., Current Biology 1999, 9:967 970 3 The screen versions of these slides have full details of copyright and acknowledgements 1
Blebbing Nuclear fragmentati on Chromatin condensati on Control Apoptosis 4 Joshua C. Goldstein and Douglas R. Green 5 Apoptosis Development Homeostasis Aging Loss of adhesion Oncogenesis (tumor suppressors) 6 The screen versions of these slides have full details of copyright and acknowledgements 2
Caspases (Cytokine processing) 3 6 7 3 6 7 8 10 9 2 8 10 9 2 1 4 5 1 4 5 Inflammatory Inflammatory 7 Inactive executioner caspase Active caspase 8 Destroying the DNA Chromatin Activated executioner caspase icad-cad complex 9 The screen versions of these slides have full details of copyright and acknowledgements 3
Substrate cleavage Gelsolin M E KK- 1 P AK- 2 G el- DQ TD 352 G - solin Blebbing P A- EV D 212 G - K- 2 ICAD- L (- S) FAK F- VSW D 704 S - AK F- DQ TD 772 S- AK M E- DTV D 874 G - KK- 1 Loss of adhesion? D N A- P K I C- DE PD 117 - AD - L I C- DAV D 224 - AD - S DNA fragmentation PKC- δ PA RP D N A - D E V D 27 12 N - P K P A - D E V D 213 G - RP Disable DNA repair? Ret inoblast oma PKC- DM Q D 330 N- δ Retino- DEAD 886 G - blastoma Disruption of cell cycle control? 10 Caspases 3 6 7 8 10 9 2 1 4 5 Inflammatory Cleavage site 11 caspase activation Adapter caspase (activated) Cleavage and activation of executioner caspase caspase 12 The screen versions of these slides have full details of copyright and acknowledgements 4
Caspases Prodomains Death effector domain (DED) Caspase recruitment domain (CARD) Protease subunits 3 6 7 8 10 9 2 1 4 5 Inflammatory Cleavage site 13 Death folds Death effector domain (DED) Caspase recruitment domain (CARD) Death domain (DD) Like-like interactions: DD-DD DED-DED CARD-CARD 14 Inhibitor of apoptosis proteins XIAP XIAP Caspase-3 Caspase-7 Caspase-9 Caspase-3 XIAP 15 Image from Reidl, et al., Cell Volume 104, Issue 5, 9 March 2001, Pages 791-800 The screen versions of these slides have full details of copyright and acknowledgements 5
Apoptotic pathways in mammals Death receptor ( extrinsic ) pathway Death ligands Death receptors Mitochondrial ( intrinsic ) pathway Bcl-2 family Mitochondria Caspase 8 APAF-1 Caspase 9 16 Death receptors TNFR1 Fas Apo-1 CD95 Trail-R1 DR4 Apo-2 Trail-R2 DR5 Killer DR3 Apo-3 WSL TRAMP DR6 Subset of the TNFR family 17 Death receptors Death ligands DD DED DD FADD DED DED Death inducing signaling complex (DISC) Active caspase-8 procaspase-8 F AS T R AIL-receptors Cleavage and activation of executioner pro and apoptosis 18 The screen versions of these slides have full details of copyright and acknowledgements 6
TNF receptor TNF DD Other proteins TRAFs TRADD Complex I FADD Activation of NF-κB and antiapoptotic effects procaspase-8 Cleavage and activation of executioner pro and apoptosis Complex II Active caspase-8 19 Death receptor Death ligand DED DD FADD DED DED c-flip procaspase-8 blocked pro apoptosis 20 Apoptosisinducing signals Mitochondrial pathway of apoptosis Bcl-2 family proteins BH3-only protein Pro-apoptotic multidomain protein Anti-apoptotic Bcl-2 protein pro Cytochrome c Apoptosis protease activating factor (APAF-1) Procaspase-9 Active executioner Apoptosis Apoptosome 21 The screen versions of these slides have full details of copyright and acknowledgements 7
Green: cytochrome c Red (yellow): mitochondrial DY Blue: PS externalization Red: membrane integrity 22 Joshua C. Goldstein and Douglas R. Green Apoptosome Dome (caspase-9) Monomeric Apaf-1 WD WD cytochrome c Hub CARD Procaspase-9 23 Wild-type Knock-out APAF-1 Caspase-9 Cytochrome c K72A Kuida, et al., 1998 Cell 94, 325-337 24 The screen versions of these slides have full details of copyright and acknowledgements 8
The Bcl-2 family proteins A n ti-apoptotic Bcl-2 homology (BH) domains P ro -apoptotic M ulti-domain B H 3-3 only 25 *adapted from Kuwana et al., 2003, drawn by J. Chipuk Bcl-2 family proteins with different functions have similar structures Anti-apoptotic (Bcl-xL) Pro-apoptotic multidomain (BAX) Pro-apoptotic BH3-only (BID) 26 The pro-apoptotic multidomain proteins Bax and Bak oligomerize upon activation and cause mitochondrial outer membrane permeabilization (MOMP) Ol igomerization Bax or Bak oligomers Bax oligomers M O M P and apoptosis Bax Monomeric Bax or Bak Bax alone +Buffer +Control protein +Active BID 27 The screen versions of these slides have full details of copyright and acknowledgements 9
Anti-apoptotic Bcl-2 proteins sequester direct activator BH3-only proteins Anti-apoptotic Bcl-2 proteins sequester direct activator BH3-only proteins B c l-xl l Liberated activator Activation and repression B ad Activation and de-repression De-repressor BH3-only protein Bim B c l-xl l B im Displace B ad B c l-xl l B im Other BH3-only proteins free them Activator BH3-only proteins trigger Bax and Bak to cause MOMP A ctivation Monomeric Bax or Bak Ol igomerization Bim A p optosis Bax or Bak oligomers 28 MOMP also promotes apoptosis inhibiting XIAP Smac OMI XIAP Caspase-3 Caspase-7 Caspase-9 29 The BH3-only protein BID bridges the death receptor and mitochondrial pathways 30 The screen versions of these slides have full details of copyright and acknowledgements 10
The mitochondrial pathway also leads to caspase-independent cell death Apoptosisinducing signals Anti-apoptotic Bcl-2 protein Bcl-2 Family Proteins BH3-only protein Pro-apoptotic multidomain protein Caspasedependent death pro Cas pase- independent deat h Los s of mitochondrial f unction APAF-1 Release of other t oxic molecules Procaspase-9 Apoptosome Apoptosis Active executioner 31 Cells die in the absence of caspase activation Control Apoptosis Caspases inhibited 32 Caspase independent cell death in the developing interdigital web Viable cell Apoptotic cell Dying cell in the web of an Apaf-1 -/- mouse 33 The screen versions of these slides have full details of copyright and acknowledgements 11
Apoptotic cells are eaten and removed from the system Phagocyte Don t eat me CD31 Find me Lysophosphatidylcholine Apoptotic cell Eat me (Externalization of phosphatidylserine, Other signals) 34 Phospholipids scramble in the plasma membrane during apoptosis Outer leaflet Phosphatidylserine Inner leaflet Lipid scrambling during apoptosis Annexin V binds phosphatidylserine MFG-8, Annexin I bind Dying cell recognized by phagocytes 35 Apoptotic pathways in mammals 36 The screen versions of these slides have full details of copyright and acknowledgements 12
37 The screen versions of these slides have full details of copyright and acknowledgements 13