ApoptosisApoptosis

Sherwin Wilk, Ph.D.Sherwin Wilk, Ph.D.Mount Sinai School of MedicineMount Sinai School of Medicine

Department of Pharmacology and Biological Department of Pharmacology and Biological ChemistryChemistry

Cell Signaling Systems CourseCell Signaling Systems CourseSpring 2005Spring 2005

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

C. elegansC. elegans Death Genes Death Genes

Pro-apoptoticPro-apoptoticcedced-3-3cedced-4-4

Anti-Anti-apoptoticapoptoticcedced-9-9

Pro-IL-1β (31 – 33 kDa)

IL-1-β converting enzyme

IL-1-β (17.5 kDa)

Single cleavage at Asp116-Ala117

Substrate specificity of Substrate specificity of ICEICE

P4-P3-P2-P1-P1´-P2´-P3´

Asp is required in P1

Synthetic Substrate

acetyl-Tyr-Val-Ala-Asp-amc

ac-YVAD-amc

ICE is a cysteine ICE is a cysteine proteinaseproteinase

It is inactivated by –SH blocking reagentsIt is inactivated by –SH blocking reagents 1414C-iodoacetate is incorporated into the 20 C-iodoacetate is incorporated into the 20

kDa subunitkDa subunit The enzyme can be potently inhibited by a The enzyme can be potently inhibited by a

peptide aldehydepeptide aldehydeH

OH

ES C RRCHO + E-SH

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

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

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

38 kDa cowpox virus proteinase 38 kDa cowpox virus proteinase inhibitorinhibitor

Member of the serpin familyMember of the serpin family Inhibits ICEInhibits ICE Inhibits apoptosisInhibits apoptosis

DNA endonuclease

oligonucleotides (-)

inhibited by poly ADP ribosylation

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

PARP cleava

ge produ

ct

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

Pro P17 P12

^

QACRGDN DS

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

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

Bcl-2 (B cell lymphoma Bcl-2 (B cell lymphoma oncogene), oncogene),

a Ced-9 homologa Ced-9 homolog Large protein familyLarge protein family Many family members reside in the Many family members reside in the

cytoplasmic face of the cytoplasmic face of the mitochondrial membranemitochondrial membrane

Transmits a survival signal when Transmits a survival signal when transfected into cellstransfected into cells

Prevents cytochrome C release from Prevents cytochrome C release from mitochondriamitochondria

Extrinsic Extrinsic Apoptotic Apoptotic PathwayPathway

TNF-related apoptosis-TNF-related apoptosis-inducing ligand (TRAIL) inducing ligand (TRAIL)

pathwaypathway

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

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

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

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

Figure 1. Apoptosis signaling by CD95. DD, death domain; DED, death effector domain.

Fig. 2. Proapoptotic and antiapoptotic signaling by TNFR1 and DR3.

Comparison of signaling for Comparison of signaling for NF-NF-κκB or for apoptosisB or for apoptosis

NF-NF-κκBB TNFR1TNFR1 TRADDTRADD TRAF TRAF RIPRIP

ApoptosisApoptosis TNFR1TNFR1 TRADDTRADD FADDFADD caspase 8caspase 8 downstream ICE downstream ICE

signalingsignaling

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

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

Intrinsic Intrinsic (mitochondrial) (mitochondrial)

apoptotic pathwayapoptotic pathway

Cell-free system for Cell-free system for the activation of the activation of

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

Activation requires:dATPApaf-1

Apaf-2 (cytochrome C)

Liu et al, Cell. 1996 Jul 12;86(1):147-157.

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

Proteins modulating Proteins modulating mitochondrial apoptosismitochondrial apoptosis

IAP (inhibitors of apoptosis) – directly IAP (inhibitors of apoptosis) – directly binds to active caspasesbinds to active caspases

Smac [Diablo] (second mitochondrial Smac [Diablo] (second mitochondrial activator of caspase) – directly binds IAPactivator of caspase) – directly binds IAP

AIF (apoptosis – inducing factor) and AIF (apoptosis – inducing factor) and endonuclease G – involved in DNA endonuclease G – involved in DNA fragmentationfragmentation

Omi/HtrA2 (a serine proteinase) – Omi/HtrA2 (a serine proteinase) – interacts with IAPinteracts with IAP

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 members of the BCL-2 family, such as BAX and BAK, to either open new pores or modify existing channels in the mitochondrial membrane, releasing cytochrome c and other proteins that lead to caspase activation and cell death. BCL-2 itself, which is antiapoptotic, somehow blocks the pore or channel opening.ILLUSTRATION: C. SLAYDEN

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

Figure 1. Pathways to cell death in C. elegans and mammals. The CED-9/Bcl-2 family integrates positive and negative signals and arbitrates whether apoptosis should occur; activation of CED-4/Apaf-1 commits to apoptosis, and CED-3/caspases mediate the death process. In mammalian cells, the Bcl-2 family rules on signals 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" CD95 proceeds primarily through the adaptor FADD, which directly activates caspase-8 and largely bypasses the Bcl-2 family (see text).