13346 wilktr16 slides

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Apoptosis ApoptosisSherwin Wilk, Ph.D.Sherwin Wilk, Ph.D.

Mount Sinai School of MedicineMount Sinai School of MedicineDepartment of Pharmacology and Biological Chemistry Department of Pharmacology and Biological Chemistry

Cell Signaling Systems CourseCell Signaling Systems CourseSpring 2005Spring 2005


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W ojcik, C. et al, Apoptosis. 1997;2(5):455-462.


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C. elegans C. elegans Death GenesDeath Genes

ProPro--apoptoticapoptoticc ed c ed --33c ed c ed --44

Anti Anti--apoptoticapoptoticc ed c ed --99


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Pro-IL-1 (31 33 kDa)

IL-1- converting enzyme

IL-1- (17.5 kDa)

Single cleavage at Asp116-Ala117


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Substrate specificity of ICESubstrate specificity of ICE

P4-P3-P2-P1-P1-P2-P3

Asp is required in P 1

Synthetic Substrate

acetyl-Tyr-Val-Ala-Asp-amc

ac-YVAD-amc


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ICE is a cysteine proteinaseICE is a cysteine proteinase

It is inactivated by It is inactivated by SH blocking reagentsSH blocking reagents1414CC--iodoacetate is incorporated into the 20 kDa subunitiodoacetate is incorporated into the 20 kDa subunit

The enzyme can be potently inhibited by a peptide The enzyme can be potently inhibited by a peptidealdehydealdehyde


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Thornberry et al, Nature. 1992 Apr 30;356(6372):768-774.


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Thornberry et al, Nature. 1992 Apr 30;356(6372):768-774.


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crmA (cytokine response modifier)crmA (cytokine response modifier)

38 kDa cowpox virus proteinase inhibitor38 kDa cowpox virus proteinase inhibitorMember of the serpin family Member of the serpin family

Inhibits ICEInhibits ICEInhibits apoptosisInhibits apoptosis


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DNA endonuclease

oligonucleotides (-)

inhibited by poly ADP ribosylation


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Nicholson et al, Nature. 1995 Jul 6;376(6535):37-43.

PARPcleavageproduct


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Nicholson et al, Nature. 1995 Jul 6;376(6535):37-43.


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Pro P17 P12^

QACRGDN DS


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Alnemri et al, Cell. 1996 Oct 18;87(2):171


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Stennicke and Salvesen, Biochim Biophys Acta. 1998 Sep 8;1387(1-2):17-31.


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B clB cl--2 ( B cell lymphoma oncogene),2 (B cell lymphoma oncogene),

a Ceda Ced--9 homolog9 homologLarge protein family Large protein family Many family members reside in the cytoplasmicMany family members reside in the cytoplasmic

face of the mitochondrial membraneface of the mitochondrial membrane Transmits a survival signal when transfected into Transmits a survival signal when transfected intocellscells

Prevents cytochrome C release fromPrevents cytochrome C release frommitochondriamitochondria


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Extrinsic ApoptoticExtrinsic Apoptotic

PathwayPathway TNF TNF--related apoptosisrelated apoptosis--inducing ligandinducing ligand

(TRAIL) pathway (TRAIL) pathway


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Schulze-Osthoff, Trends Cell Biol. 1994 Dec;4(12):421-426.


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Schulze-Osthoff, Trends Cell Biol. 1994 Dec;4(12):421-426.


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Tartaglia et al, Cell. 1993 Sep 10;74(5):845-853.


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Ashkenazi and Dixit, Science. 1998 Aug 28;281(5381):1305-1308.

F igure 1. Apoptosis signaling by CD95. DD, death domain; DED, deatheffector domain.

F ig. 2. Proapoptotic and antiapoptotic signaling by TNFR1 and DR3.


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Comparison of signaling for N FComparison of signaling for N F -- B B or for apoptosisor for apoptosis

NFNF-- BB TNFR1 TNFR1

TRADD TRADD TRAF TRAF RIPRIP

Apoptosis Apoptosis TNFR1 TNFR1

TRADD TRADDFADDFADDcaspase 8caspase 8downstream ICEdownstream ICEsignaling signaling


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Nagata, Cell. 1997 Feb 7;88(3):355-365.


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Enari et al, Nature. 1998 Jan 1;391(6662):43-50.


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Intrinsic (mitochondrial)Intrinsic (mitochondrial)apoptotic pathwayapoptotic pathway


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CellCell--free system for thefree system for the

activation of CPP32activation of CPP32(caspase 3)(caspase 3)Liu et al, Cell. 1996 Jul 12;86(1):147-157.

Activation requires:dATP

Apaf-1 Apaf-2 (cytochrome C)


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Z ou et al, Cell. 1997 Aug 8;90(3):405-413.


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Proteins modulatingProteins modulating

mitochondrial apoptosismitochondrial apoptosisIAP (inhibitors of apoptosis)IAP (inhibitors of apoptosis) directly binds todirectly binds toactive caspasesactive caspases

Smac [Diablo] (second mitochondrial activator of Smac [Diablo] (second mitochondrial activator of caspase)caspase) directly binds IAPdirectly binds IAP AIF (apoptosis AIF (apoptosis inducing factor) andinducing factor) andendonuclease Gendonuclease G involved in DNA fragmentationinvolved in DNA fragmentationOmi/HtrA2 (a serine proteinase)Omi/HtrA2 (a serine proteinase) interacts withinteracts withIAPIAP


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Finkel, Science. 2001 Apr 27;292(5517):624-626.

Center stage in apoptosis In this view, numerous cell-death stimuli work through the mitochondrion. They cause pro-apoptotic membersof the BCL-2 family, such as BAX and BAK, to either open new poresor modify existing channels in themitochondrial membrane, releasing cytochrome c and other proteinsthat lead to caspase activation andcell death. BCL-2 itself, which isantiapoptotic, somehow blocks thepore or channel opening.ILLUSTRATION: C. SLAYDEN


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Adams and Cory, Science. 1998 Aug 28;281(5381):1322-1326.

F igure 1. Pathways to cell death in C. elegans and mammals. The CED-9/Bcl-2 family integrates positive andnegative signals and arbitrates whether apoptosis should occur; activation of CED-4/Apaf-1 commits toapoptosis, and CED-3/caspases mediate the death process. In mammalian cells, the Bcl-2 family rules onsignals from diverse cytotoxic stimuli (for example, cytokine deprivation and exposure to glucocorticoids,DNA damage, or staurosporine). However, the signal induced by engagement of the "death receptor" CD95proceeds primarily through the adaptor FADD, which directly activates caspase-8 and largely bypasses theBcl-2 family (see text).

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