Apoptosis

Sherwin Wilk, Ph.D.

Mount Sinai School of Medicine Department of Pharmacology and Biological Chemistry Cell Signaling Systems Course Spring 2005

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

C. elegans Death Genes



Pro-apoptotic

 ced-3  ced-4 

Anti-apoptotic

 ced-9

Pro-IL-1β (31 – 33 kDa) IL-1-β converting enzyme IL-1-β (17.5 kDa) Single cleavage at Asp 116 -Ala 117

Substrate specificity of ICE

P 4 -P 3 -P 2 -P 1 -P 1 ´-P 2 ´-P 3 ´ Asp is required in P 1 Synthetic Substrate acetyl-Tyr-Val-Ala-Asp-amc ac-YVAD-amc

ICE is a cysteine proteinase

   It is inactivated by –SH blocking reagents 14 C-iodoacetate is incorporated into the 20 kDa subunit The enzyme can be potently inhibited by a peptide aldehyde H RCHO + E-SH ES C R OH

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 modifier)

 38 kDa cowpox virus proteinase inhibitor  Member of the serpin family  Inhibits ICE  Inhibits apoptosis

DNA endonuclease oligonucleotides (-) inhibited by poly ADP ribosylation

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

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

Pro DN P17 ^ QACRG DS P12

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 oncogene), a Ced-9 homolog

 Large protein family  Many family members reside in the cytoplasmic face of the mitochondrial membrane  Transmits a survival signal when transfected into cells  Prevents cytochrome C release from mitochondria

Extrinsic Apoptotic Pathway

TNF-related apoptosis-inducing ligand (TRAIL) pathway

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.

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

Fig. 2. Proapoptotic and antiapoptotic signaling by TNFR1 and DR3. Ashkenazi and Dixit, Science. 1998 Aug 28;281(5381):1305-1308.

Comparison of signaling for NF-

κ

B or for apoptosis

NF κ B   TNFR1 TRADD  TRAF RIP Apoptosis   TNFR1 TRADD    FADD caspase 8 downstream ICE signaling

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

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

Intrinsic (mitochondrial) apoptotic pathway

Cell-free system for the activation of CPP32 (caspase 3)

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

Activation requires:

dATP Apaf-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 mitochondrial apoptosis

 IAP (inhibitors of apoptosis) – directly binds to active caspases  Smac [Diablo] (second mitochondrial activator of caspase) – directly binds IAP  AIF (apoptosis – inducing factor) and endonuclease G – involved in DNA fragmentation  Omi/HtrA2 (a serine proteinase) – interacts with IAP

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

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