1. BMOL 6106 - W20051 Cell death Part II: Regulation Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry

2. BMOL 6106 - W20052 Outline  Basic regulatory mechanisms:  Turnover;  Compartmentalization;  Alternative splicing;  Changes in protein conformation;  Post-translational modifications;  Protein-protein interactions

3. BMOL 6106 - W20053 Core apoptotic machinery Major control points:  Death receptor signalling;  Bax activation/translocation;  Initiator caspase activation;  Executioner caspase activation. Major regulatory mechanisms:  Cellular compartmentalization  Modulation of protein turnover ( transcription, translation, stability);  Changes in protein conformation;  Post-translational modifications (phosphorylation, nitrosylation, deamidation, ubiquitylation);  Alternative splicing;  Protein-protein interaction. Adapted from: Nature Struct. Biol. 2001. 8 [ 5] caspase-8 cFLIP

4. BMOL 6106 - W20054 Death receptor regulation Death receptors: multiple outcomes  TNF: can lead to cell survival or death (apoptosis, necrosis)  Fas: induction of cell death is dependent on the cell context (e.g. TCR stimulation)  TRAIL: preferentially induces death in tumor cells Major regulatory mechanisms:  Inhibition of DISC (Death Inducing Signalling Complex) formation;  Expression (transcription) of death receptors (p53 and Fas);  Decoy receptors;  Compartmentalization.

5. BMOL 6106 - W20055 Inhibition of DISC formation cFlip: Inactive homologs of Casp-8 and Casp-10;  cFlipL: Mutation of the active site Cys  cFlipS: Contains only two DED; Binds FADD and dimerizes with pro-Casp-8: initially thought to act as a dominant-negative inhibitor, leading to cell survival; However: recent data indicates that cFlip can also trigger cell death by promoting Casp-8/Casp-10 dimerization… Oncogene (2003) 22, 8634–8644 c-FlipS

6. BMOL 6106 - W20056 Inhibition of DISC formation Low c-FlipL levels:  Dimerizes with pro-Casp- 8 at the DISC;  Contributes to Casp-8 activation: c-Flip\Casp-8 dimers form more efficiently than Casp homodimers;  Cell death. High c-FlipL:  Incomplete processing of proCasp-8;  Casp-8 activated but remains at the DISC;  Cleave different substrates, leading to pro- survival function. Biochem. J. (2004) 382, e1–e3

7. BMOL 6106 - W20057 Decoy receptors - TRAIL receptors Cell Research (2004); 14(5):359-372

8. BMOL 6106 - W20058 Death receptor regulation Compartmentalization Complex 1:  Formed early after receptor activation;  Comprises: TNF-R, TRADD, TRAF2, RIP1  Leads to NF-  B activation by recruitment of the I-  B kinase signalsome (IKK1-IKK2-NEMO) Complex 2:  Found at later time points (>2 hrs), possibly after receptor internalization;  Dissociation from TNFR, and recruitment of FADD and proCasp-8; In situations where complex-1 formation trigger sufficient NF-kB signalling, c-FLIP and other anti-apoptotic proteins (e.g. IAPs) are synthesized, leading to inhibition Casp-8 activation in complex II; Also: localization in lipid rafts seems to promote the survival signalling function of TNFR, as cholesterol depletion favors complex II formation. Immunity, Vol. 21, 461–465, October, 2004

9. BMOL 6106 - W20059 Core apoptotic machinery Major control points:  Death receptor signalling;  Bax activation/translocation;  Initiator caspase activation;  Executioner caspase activation. Major regulatory mechanisms:  Cellular compartmentalization  Modulation of protein turnover ( transcription, translation, stability);  Changes in protein conformation;  Post-translational modifications (phosphorylation, nitrosylation, deamidation, ubiquitylation);  Alternative splicing;  Protein-protein interaction. Adapted from: Nature Struct. Biol. 2001. 8 [ 5] caspase-8 cFLIP

10. BMOL 6106 - W200510 Bax modulation Because of its central role in the intrinsic pathway, several mechanisms have evolved to control Bax activation:  Transcription (p53)  Alternative splicing  Subcellular localization  Protein-protein interaction  Inactivation of anti-apoptotic proteins

11. BMOL 6106 - W200511 Bax activation Bax exists as an inactive, cytosolic protein:  C-terminal TMD in the BH1/BH2/BH3 hydrophobic groove  Prevents Bax from anchoring into the mitochondrial outer membrane (MOM);  Prevents interaction of regulatory proteins with hydrophobic groove.  Prevents inappropriate aggregation in the cytosol;  N-terminal  1 helix not accessible.  Mitochondrial targeting sequence? Bax activation:  Requires the release of the TMD from the BH groove;  Results in the exposure of the NH 2 terminus (detectable by IP with 4G2 antibody); Biochimica et Biophysica Acta 1644 (2004) 83– 94 Helices:  2 (BH3) ;  4+5 (BH1);  7+8 (BH2)

12. BMOL 6106 - W200512 Bax activation Biochimica et Biophysica Acta 1644 (2004) 83– 94 J Cell Biol. 2004. 164 ( 7): 1021–1032

13. BMOL 6106 - W200513 Bax Activation Bax activation can be induced by several triggers:  pH alkalinization  Direct tBid/Bim binding  Ser 184 dephosphorylation  Ser 184 PO 4 by AKT  Inactivation of Bcl-2 / Bcl-xL  PO 4 (Bcl-2: Ser 87, Thr 69 - JNK)  Ubiquitylation (Bcl-2)  Casp-mediated cleavage  BH3 protein binding (e.g. Bad, Noxa, Puma)  Bcl-xL deamidation (Asn 52/66)  Release of binding by KU70  Removal of the N-terminal 20 first amino acids (calpain-mediated) SCIENCE. 2002. 298: 1346-1347

14. BMOL 6106 - W200514 Bax modulation Inactivation of Bcl-xL Cell, Vol. 87, 619–628, November 15, 1996 Cell, Vol. 91, 231–241, October 17, 1997 Mol. Cell. 2004. 13: 627–638

15. BMOL 6106 - W200515 Core apoptotic machinery Major control points:  Death receptor signalling;  Bax activation/translocation;  Initiator caspase activation;  Executioner caspase activation. Major regulatory mechanisms:  Cellular compartmentalization  Modulation of protein turnover ( transcription, translation, stability);  Changes in protein conformation;  Post-translational modifications (phosphorylation, nitrosylation, deamidation, ubiquitylation);  Alternative splicing;  Protein-protein interaction. Adapted from: Nature Struct. Biol. 2001. 8 [ 5] caspase-8 cFLIP

16. BMOL 6106 - W200516 IAPs BIR: Baculovirus IAP repeat: RING: ubiquitin ligase (E3) domain UBC: ubiquitin E2 domain CARD: caspase recruitment domain NACHT: putative ATP-binding domain Nature Reviews Molecular Cell Biology 5, 897-907 (2004)

17. BMOL 6106 - W200517 IAPs Mammals:  BIR-3: inhibits active Casp-9  Linker segment between BIR-1/2: inhibits active Casp-3/7 Drosophila:  BIR1: inhibits Drice (Casp-3 homolog)  BIR2: inhibits Dronc (Casp-9 homolog) Nature Reviews Molecular Cell Biology 5, 897-907 (2004)

18. BMOL 6106 - W200518 XIAP and caspase inhibition Biochem. J. (2004) 384, 201–232

19. BMOL 6106 - W200519 XIAP and effector caspase inhibition TRENDS in Biochemical Sciences Vol.27 No.2 February 2002: 94-101

20. BMOL 6106 - W200520 XIAP and caspase-9 inhibition The BIR-3 domain of XIAP traps Casp-9 in a monomeric, inactive conformation:  BIR-3 binds Casp-9 through an interface which is required for Casp-9 homodimerization and its interaction wiht the apoptosome;  An N-terminal segment of the small subunit of Casp-9 (A 298 -T-P-F 301 ) anchors this interaction by binding a conserved groove on BIR-3 (next slide). Nat. Rev. Mol. Cell Biol. 5, 897-907 (2004)

21. BMOL 6106 - W200521 XIAP and caspase-9 inhibition Biochem. J. (2004) 384, 201–232

22. BMOL 6106 - W200522 DIAP and Dronc inhibition Recruitment of E2/E3 enzymes Cell, Vol. 109, 793–796, June 28, 2002: 793-796

23. BMOL 6106 - W200523 Inhibiting the inhibitors: SMAC/DIABLO Nature Reviews Molecular Cell Biology 5, 897-907 (2004)

24. BMOL 6106 - W200524 DIAP and Dronc inhibition Recruitment of E2/E3 enzymes Cell, Vol. 109, 793–796, June 28, 2002: 793-796

25. BMOL 6106 - W200525 Core apoptotic machinery Major control points:  Death receptor signalling;  Bax activation/translocation;  Initiator caspase activation;  Executioner caspase activation. Major regulatory mechanisms:  Cellular compartmentalization  Modulation of protein turnover ( transcription, translation, stability);  Changes in protein conformation;  Post-translational modifications (phosphorylation, nitrosylation, deamidation, ubiquitylation);  Alternative splicing;  Protein-protein interaction. Adapted from: Nature Struct. Biol. 2001. 8 [ 5] caspase-8 cFLIP

26. BMOL 6106 - W200526 DIAP and Dronc inhibition Involvement of the N-end rule pathway NATURE CELL BIOLOGY VOL 5 MAY 2003: 373-376

27. BMOL 6106 - W200527 Inhibiting the inhibitors: The case of Drosophila NATURE STRUCTURAL BIOLOGY. 2003. 10 (9): 892-898