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).