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Cell death

Cell death. Part II: Regulation. Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry. Outline. Basic regulatory mechanisms: Turnover; Compartmentalization; Alternative splicing; Changes in protein conformation; Post-translational modifications; Protein-protein interactions.

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Cell death

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  1. Cell death Part II: Regulation Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry BMOL 6106 - W2005

  2. Outline • Basic regulatory mechanisms: • Turnover; • Compartmentalization; • Alternative splicing; • Changes in protein conformation; • Post-translational modifications; • Protein-protein interactions BMOL 6106 - W2005

  3. 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. Core apoptotic machinery caspase-8 cFLIP Adapted from: Nature Struct. Biol. 2001. 8 [ 5] BMOL 6106 - W2005

  4. 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. BMOL 6106 - W2005

  5. Oncogene (2003) 22, 8634–8644 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… c-FlipS BMOL 6106 - W2005

  6. 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 BMOL 6106 - W2005

  7. Decoy receptors - TRAIL receptors Cell Research (2004); 14(5):359-372 BMOL 6106 - W2005

  8. Death receptor regulationCompartmentalization • Complex 1: • Formed early after receptor activation; • Comprises: TNF-R, TRADD, TRAF2, RIP1 • Leads to NF-kB activation by recruitment of the I-kB 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 BMOL 6106 - W2005

  9. 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. Core apoptotic machinery caspase-8 cFLIP Adapted from: Nature Struct. Biol. 2001. 8 [ 5] BMOL 6106 - W2005

  10. 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 BMOL 6106 - W2005

  11. 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 a1 helix not accessible. • Mitochondrial targeting sequence? • Bax activation: • Requires the release of the TMD from the BH groove; • Results in the exposure of the NH2 terminus (detectable by IP with 4G2 antibody); Biochimica et Biophysica Acta 1644 (2004) 83– 94 Helices: a2 (BH3) ; a4+5 (BH1); a7+8 (BH2) BMOL 6106 - W2005

  12. Bax activation Biochimica et Biophysica Acta 1644 (2004) 83– 94 J Cell Biol. 2004. 164 ( 7): 1021–1032 BMOL 6106 - W2005

  13. Bax Activation • Bax activation can be induced by several triggers: • pH alkalinization • Direct tBid/Bim binding • Ser 184 dephosphorylation • Ser 184 PO4 by AKT • Inactivation of Bcl-2 / Bcl-xL • PO4 (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) BMOL 6106 - W2005 SCIENCE. 2002. 298: 1346-1347

  14. Bax modulationInactivation of Bcl-xL Cell, Vol. 87, 619–628, November 15, 1996 Mol. Cell. 2004. 13: 627–638 BMOL 6106 - W2005 Cell, Vol. 91, 231–241, October 17, 1997

  15. 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. Core apoptotic machinery caspase-8 cFLIP Adapted from: Nature Struct. Biol. 2001. 8 [ 5] BMOL 6106 - W2005

  16. IAPs • BIR: Baculovirus IAP repeat: • RING: ubiquitin ligase (E3) domain • UBC: ubiquitin E2 domain • CARD: caspase recruitment domain • NACHT: putative ATP-binding domain BMOL 6106 - W2005 Nature Reviews Molecular Cell Biology5, 897-907 (2004)

  17. 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) BMOL 6106 - W2005 Nature Reviews Molecular Cell Biology5, 897-907 (2004)

  18. XIAP and caspase inhibition Biochem. J. (2004) 384, 201–232 BMOL 6106 - W2005

  19. XIAP and effector caspase inhibition BMOL 6106 - W2005 TRENDS in Biochemical Sciences Vol.27 No.2 February 2002: 94-101

  20. Nat. Rev. Mol. Cell Biol.5, 897-907 (2004) 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 (A298-T-P-F301) anchors this interaction by binding a conserved groove on BIR-3 (next slide). BMOL 6106 - W2005

  21. XIAP and caspase-9 inhibition Biochem. J. (2004) 384, 201–232 BMOL 6106 - W2005

  22. DIAP and Dronc inhibitionRecruitment of E2/E3 enzymes Cell, Vol. 109, 793–796, June 28, 2002: 793-796 BMOL 6106 - W2005

  23. Inhibiting the inhibitors:SMAC/DIABLO Nature Reviews Molecular Cell Biology5, 897-907 (2004) BMOL 6106 - W2005

  24. DIAP and Dronc inhibitionRecruitment of E2/E3 enzymes Cell, Vol. 109, 793–796, June 28, 2002: 793-796 BMOL 6106 - W2005

  25. 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. Core apoptotic machinery caspase-8 cFLIP Adapted from: Nature Struct. Biol. 2001. 8 [ 5] BMOL 6106 - W2005

  26. DIAP and Dronc inhibitionInvolvement of the N-end rule pathway NATURE CELL BIOLOGY VOL 5 MAY 2003: 373-376 BMOL 6106 - W2005

  27. Inhibiting the inhibitors:The case of Drosophila NATURE STRUCTURAL BIOLOGY. 2003. 10 (9): 892-898 BMOL 6106 - W2005

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