1. Apoptosis Sherwin Wilk, Ph.D. Mount Sinai School of Medicine Department of Pharmacology and Biological Chemistry Cell Signaling Systems Course Spring 2005

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

3. C. elegans Death Genes Pro-apoptotic Pro-apoptotic ced-3 ced-3 ced-4 ced-4 Anti-apoptotic ced-9

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

5. 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

6. ICE is a cysteine proteinase It is inactivated by –SH blocking reagents It is inactivated by –SH blocking reagents 14 C-iodoacetate is incorporated into the 20 kDa subunit 14 C-iodoacetate is incorporated into the 20 kDa subunit The enzyme can be potently inhibited by a peptide aldehyde The enzyme can be potently inhibited by a peptide aldehyde

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

9. crmA (cytokine response modifier) 38 kDa cowpox virus proteinase inhibitor 38 kDa cowpox virus proteinase inhibitor Member of the serpin family Member of the serpin family Inhibits ICE Inhibits ICE Inhibits apoptosis Inhibits apoptosis

10. DNA endonuclease oligonucleotides (-) inhibited by poly ADP ribosylation

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

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

13. Pro P17P12 ^ QACRG DNDS

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

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

16. Bcl-2 (B cell lymphoma oncogene), a Ced-9 homolog Large protein family Large protein family Many family members reside in the cytoplasmic face of the mitochondrial membrane Many family members reside in the cytoplasmic face of the mitochondrial membrane Transmits a survival signal when transfected into cells Transmits a survival signal when transfected into cells Prevents cytochrome C release from mitochondria Prevents cytochrome C release from mitochondria

17. Extrinsic Apoptotic Pathway TNF-related apoptosis-inducing ligand (TRAIL) pathway

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

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

21. 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.

22. Comparison of signaling for NF- κ B or for apoptosis NF- κ B TNFR1 TNFR1 TRADD TRADD TRAF RIP TRAF RIP Apoptosis TNFR1 TRADD FADD caspase 8 downstream ICE signaling

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

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

25. Intrinsic (mitochondrial) apoptotic pathway

26. 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)

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

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

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

30. 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

31. 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).