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Insights into Programmed Cell Death through Structural Biology

2000; Cell Press; Volume: 103; Issue: 2 Linguagem: Inglês

10.1016/s0092-8674(00)00119-7

1097-4172

Stephen W. Fesik,

Mitochondrial Function and Pathology

Programmed cell death plays a critical role in controlling the number of cells in development and throughout an organism's life by the removal of cells at the appropriate time. It is an important biological process for the elimination of unwanted cells such as those with potentially harmful genomic mutations, autoreactive lymphocytes, or virally infected cells. Alterations of this normal process can result in the disruption of the delicate balance between cell proliferation and cell death and can lead to a variety of diseases (57Thompson C.B. Apoptosis in the pathogenesis and treatment of disease.Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6009) Google Scholar). For example, in many forms of cancer, key proapoptotic proteins are mutated or antiapoptotic proteins are upregulated, leading to the accumulation of cells and the inability to respond to harmful mutations, DNA damage, or chemotherapeutic agents. Since effective chemotherapy depends on the induction of programmed cell death, cancers with defects in the cell death signaling pathways are particularly difficult to treat. Programmed cell death is also important for eliminating autoreactive T cells after an immune response. When this normal process is disrupted through mutations of the proteins that trigger apoptosis (e.g., Fas ligand or the Fas receptor), an autoimmune lymphoproliferative syndrome (ALPS) can result, with complications such as hypersplenism, autoimmune hemolytic anemia, thrombocytopenia, and neutropenia (52Strauss S.E. Sneller M. Lenardo M.J. Puck J.M. Strober W. An inherited disorder of lymphocyte apoptosis the autoimmune lymphoproliferative syndrome.Ann. Intern. Med. 1999; 130: 591-601Crossref PubMed Scopus (248) Google Scholar). Inappropriate apoptosis also contributes to several neurological disorders. In Alzheimer's, Parkinson's, and Huntington's disease, specific neurons prematurely commit suicide, which can lead to irreversible memory loss, uncontrolled muscular movements, and depression. In the last few years, much has been learned about the signal transduction pathways of programmed cell death, providing us with insight into how programmed cell death works and how dysregulation of apoptosis contributes to disease. The overall process of programmed cell death occurs in several stages. In the first step, the apoptotic pathway is triggered, which can be accomplished by a wide variety of stimuli, including DNA damage, growth factor withdrawal, toxins, and radiation. Once activated, the signal is transduced by a series of protein–protein interactions that involve a conserved set of signaling modules. In the next stage, cell death is executed by the activation of specific proteases called caspases that cleave multiple substrates, leading to changes characteristic of apoptotic cells such as DNA fragmentation, chromatin condensation, cell shrinkage, and membrane blebbing. The signaling pathways of programmed cell death can be divided into two components, involving either the mitochondria or death receptors (Figure 1) (5Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Biochemical pathways of caspase activation during apoptosis.Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2185) Google Scholar). In the death receptor pathway, receptors such as TNFR1, Fas, DR-3, DR-4, or DR-5 interact with their cognate ligands. This binding event allows the recruitment of downstream signaling partners, which ultimately results in the activation of caspases and subsequent cell death. For example, in Fas-mediated apoptosis, the Fas ligand interacts with the Fas receptor, which leads to an interaction between the death domain of the cytoplasmic region of the Fas receptor and the death domain of the adaptor protein, FADD. FADD recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins. Once activated, caspase-8 activates downstream caspases such as caspase-3 (for a review, see 43Nagata S. Apoptosis by death factor.Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4455) Google Scholar). In the mitochondrial pathway, cytochrome c is released from the intermembrane space of mitochondria upon activation via a death signal. Although the details of this process at the molecular level are unknown, cytochrome c and ATP or dATP bind to the protein Apaf-1 to form a multimeric complex that recruits and activates procaspase-9. This is followed by the activation of caspase-3 and -7 (5Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Biochemical pathways of caspase activation during apoptosis.Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2185) Google Scholar). As one might imagine, apoptosis is highly regulated. One class of regulators is the Bcl-2 family of proteins. Antiapoptotic family members such as Bcl-2 and Bcl-xL inhibit the release of cytochrome c whereas the proapoptotic proteins Bax and tBID promote cytochrome c release from the mitochrondria. Pro- and antiapoptotic members of the Bcl-2 family interact with one another and modulate each others' activities (69Yin X.-M. Oltvai Z.N. Korsmeyer S.J. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax.Nature. 1994; 369: 321-323Crossref PubMed Scopus (1178) Google Scholar). Additional regulators of this pathway are the IAPs. One of their major functions is to bind to and inhibit the effectors of apoptosis—the caspase family of enzymes (15Deveraux Q.L. Reed J.C. IAP family proteins-suppressors of apoptosis.Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2217) Google Scholar). The three-dimensional structures for a number of proteins and protein domains involved in the signaling pathways of programmed cell death have been determined. These structures have helped us understand apoptosis at the molecular level by defining the interactions that stabilize complex formation, guiding site-directed mutagenesis experiments, elucidating enzyme mechanisms, and characterizing protein function. In this review, a compendium of recently determined three-dimensional structures of proteins involved in the cell death signaling pathways is described along with the information learned from these structures relevant to the chemistry and biology of apoptosis. Tumor necrosis factor (TNF) is the prototypic member of a family of cytokines that interact with a large number of receptors. Activation of these receptors by ligand binding leads to many diverse activities such as cell proliferation, differentiation, and apoptosis. The subset of receptors responsible for eliciting programmed cell death all contain an intracellular death domain (56Tartaglia L.A. Ayres T.M. Wong G.H.W. Goeddel D.V. A novel domain within the 55 kd TNF receptor signals cell death.Cell. 1993; 74: 845-853Abstract Full Text PDF PubMed Scopus (1137) Google Scholar). TNFα and TNFβ were found to crystallize as trimers (27Jones E.Y. Stuart D.I. Walker N.P. Structure of tumour necrosis factor.Nature. 1989; 338: 225-228Crossref PubMed Scopus (471) Google Scholar, 18Eck M.J. Sprang S.R. The structure of tumor necrosis factor alpha at 2.6 Å resolution implications for receptor binding.J. Biol. Chem. 1989; 264: 17595-17605Abstract Full Text PDF PubMed Google Scholar, 19Eck M.J. Ultsch M. Rinderknecht E. de Vos A.M. Sprang S.R. The structure of human lymphotoxin (tumor necrosis factor beta) at 1.9 Å resolution.J. Biol. Chem. 1992; 267: 2119-2122Abstract Full Text PDF PubMed Google Scholar). The structures of these proteins are similar and adopt the shape of a truncated pyramid. Each monomer consists of a β sandwich with a jellyroll topology. The structure of the complex between TNFβ and the soluble extracellular region of the TNF receptor (TNF-R1) provided the first view of the receptor and the interactions that stabilize complex formation between these two families of proteins (Figure 2A) (3Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H.-J. Broger C. Loetscher H. Lessiauer W. Crystal structure of the soluble human 55 kd TNF receptor-human TNFβ complex implications for TNF receptor activation.Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (950) Google Scholar). The extracellular portion of TNF-R1 consists of four pseudo repeats, called cysteine-rich domains (CRDs), which each contain three disulfides formed from six conserved cysteines. Three elongated receptor molecules bind to one TNF trimer at the interfaces formed between the TNF monomers (Figure 2A). There are two principal regions of contact in the complex that involve loops from the second (50s loop) and third (90s loop) cysteine-rich domains of the TNF receptor and two distinct regions of TNFβ. On the basis of the structure of the TNFβ/TNF-R1 complex, a model was proposed for TNF-mediated signaling in which the TNF trimer induces trimerization of the receptor, which causes the cytoplasmic regions of the receptor to form a cluster. The cytoplasmic region of TNF-R1 and other death receptors contain death domains that oligomerize and recruit death domain–containing adaptor proteins, leading to the downstream signaling of programmed cell death (Figure 1). Recently, the crystal structure was determined of another death receptor, DR5, complexed to the ligand TRAIL (also called Apo2L) (Figure 2B) (25Hymowitz S.G. Christinger H.W. Fuh G. Ultsch M. O'Connell M. Kelley R.F. Ashkenazi A. de Vos A.M. Triggering cell death the crystal structure of Apo2L/TRAIL in a complex with death receptor 5.Mol. Cell. 1999; 4: 563-571Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 40Mongkolsapaya J. Grimes J.M. Chen N. Xu X.-N. Stuart D.I. Jones E.Y. Screaton G.R. Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation.Nat. Struct. Biol. 1999; 6: 1048-1053Crossref PubMed Scopus (217) Google Scholar). The overall structure of the TRAIL/DR5 complex is similar to TNFβ/TNF-R1. However, there are some important differences that define the binding specificities observed within these families of ligands and receptors. Unlike the TNF receptor that contains four extracellular cysteine-rich domains, DR5 contains only two CRDs, which correspond to domains 2 and 3 of TNF-R1. CRD2 of DR5 interacts with TRAIL in a similar fashion as observed for the corresponding residues in the TNF-β/TNF-R1 complex (Figure 2). However, CRD3 of DR5, when bound to TRAIL, adopts a different relative orientation compared to CRD3 of TNF-R1 in the TNFβ/TNF-R1 complex and forms a different set of interactions. The binding specificity observed in the TNF receptor superfamily could be important for developing selective therapeutic agents. Unlike TNF and Fas ligand, which are highly toxic, TRAIL selectively triggers apoptosis in tumor cells versus normal tissues and, when given to mice with human tumors, causes tumors to shrink without any apparent toxicity (64Walczak H. Miller R.E. Arial K. Gliniak HB Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. et al.Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2139) Google Scholar). A common theme in the signaling pathways of programmed cell death is the association of proteins that contain similar domains. Four protein modules have been identified that participate in cell death signaling through protein–protein interactions. These include the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and the N-terminal domains of DFF45, DFF40, and cell death-inducing DFF45-like effector (CIDE) proteins. The death domain is found in the intracellular portion of death receptors whereas the death effector and caspase recruitment domains are found in adaptor proteins such as FADD and procaspases. The three-dimensional structure of the Fas death domain was the first structure reported for one of these signaling modules (24Huang B. Eberstadt M. Olejniczak E.T. Meadows R.P. Fesik S.W. NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain.Nature. 1996; 384: 638-641Crossref PubMed Scopus (306) Google Scholar). The structure contains six antiparallel, amphipathic α helices with an unusual topology (Figure 3A). The surface of the Fas death domain mainly consists of charged residues, suggesting that charge–charge interactions mediate complex formation between the death domains. This was supported by site-directed mutagenesis in which the mutation of charged residues in the second and third α helices of the Fas DD reduced binding to FADD. The overall fold of other death domains were found to be similar to the Fas death domain with only minor differences in the length and orientation for some of the α helices (31Liepinsh E. Ilag L.L. Otting G. Ibáñez C.F. NMR structure of the death domain of the p75 neurotrophin receptor.EMBO J. 1997; 16: 4999-5005Crossref PubMed Scopus (249) Google Scholar, 26Jeong E.-J. Bang S. Lee T.H. Park Y.I. Sim W.-S. Kim K.-S. The solution structure of FADD death domain.J. Biol. Chem. 1999; 274: 16337-16342Crossref PubMed Scopus (96) Google Scholar). However, the manner in which death domains or modules with similiar folds interact with one another was found to be different. This was recently illustrated in the X-ray structure of a complex between the interacting domains of the serine/threonine kinase Pelle and the adaptor protein Tube that recruits Pelle to the plasma membrane during Drosophila embryogenesis (68Xiao T. Towb P. Wasserman S.A. Sprang S.R. Three-dimensional structure of a complex between the death domains of Pelle and Tube.Cell. 1999; 99: 545-555Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Although not involved in apoptosis, the interacting domains of Pelle and Tube adopt the same overall fold as death domains but do not appear to interact in the same manner as the Fas and FADD death domains. Unlike the second and third α helices, which have been implicated in the Fas death domain for binding to FADD, residues in the fourth and fifth α helices in Pelle and the sixth α helix in Tube were found to play a critical role in complex formation for these proteins such that the binding interface involves a unique surface that is not even present in other death domains. The death effector domain of FADD (Figure 3B) adopts a fold that is similar to that of the death domains (17Eberstadt M. Huang B. Chen Z. Meadows R.P. Ng S.-C. Zheng L. Lenardo M.J. Fesik S.W. NMR structure and mutagenesis of the FADD (Mort1) death-effector domain.Nature. 1998; 392: 941-945Crossref PubMed Scopus (197) Google Scholar). However, mutations in the Fas death domain that inhibit the binding of Fas to FADD have no effect when introduced in the FADD DED. In contrast to the charged surface of the Fas DD, the FADD DED has two hydrophobic patches. One of these patches contains a conserved set of hydrophobic residues that is important for its apoptotic activity and binding to the DEDs of procaspase-8. The three-dimensional structures of RAIDD CARD (Figure 3C) (10Chou J.J. Matsuo H. Duan H. Wagner G. Solution structure of the RAIDD CARD and model for CARD/CARD interaction in caspase-2 and caspase-9 recruitment.Cell. 1998; 94: 171-180Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar) and Apaf-1 CARD (13Day C.L. Dupont C. Lackmann M. Vaux D.L. Hinds M.G. Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1.Cell Death and Diff. 1999; 6: 1125-1132Crossref PubMed Scopus (43) Google Scholar, 45Qin H. Srinivasula S.M. Wu G. Fernandes-Alnemri T. Alnemri E.S. Shi Y. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1.Nature. 1999; 399: 549-557Crossref PubMed Scopus (349) Google Scholar, 61Vaughn D.E. Rodriguez J. Lazebnik Y. Joshua-Tor L. Crystal structure of Apaf-1 caspase recruitment domain an α-helical greek key fold for apoptotic signaling.J. Mol. Biol. 1999; 293: 439-447Crossref PubMed Scopus (66) Google Scholar, 70Zhou P. Chou J. Olea R.S. Yuan J. Wagner G. Solution structure of Apaf-1 CARD and its interaction with caspase-9 CARD a structural basis for specific adaptor/caspase interaction.Proc. Natl. Acad. Sci. USA. 1999; 96: 11265-11270Crossref PubMed Scopus (130) Google Scholar) also adopt the same fold as the death domain and the death effector domain. Thus, all three signaling modules (DD, DED, and CARD) that participate in homophilic interactions to transduce the cell death signal adopt very similar structures. This is consistent with the presence of a conserved set of hydrophobic amino acids in all of these domains that compose the hydrophobic core of the proteins (23Hofmann K. Bucher P. Tschopp J. The CARD domain a new apoptotic signalling motif.Trends Biochem. Sci. 1997; 22: 155-156Abstract Full Text PDF PubMed Scopus (444) Google Scholar). The differences between the domains are the surface residues that stabilize complex formation. In the Apaf-1 CARD/procaspase-9 CARD complex (45Qin H. Srinivasula S.M. Wu G. Fernandes-Alnemri T. Alnemri E.S. Shi Y. Structural basis of procaspase-9 recruitment by the apoptotic protease-activating factor 1.Nature. 1999; 399: 549-557Crossref PubMed Scopus (349) Google Scholar), the positively charged surface formed by the basic residues Arg13, Arg52, and Arg56 of procaspase-9 CARD interacts with the negatively charged face of Apaf-1 CARD, which is composed of Asp27 and Glu40 (Figure 4). Another domain that participates in homophilic interactions in the programmed cell death cascade is found in the N terminus of DFF45, DFF40, and CIDE proteins. DFF45 (also called ICAD) forms a complex with DFF40 (also called CAD) through interactions involving their N-terminal domains and inhibits the DNA nuclease activity of DFF40, which is localized in the C-terminal portion of the protein (32Liu X. Zou H. Slaughter C. Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis.Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1593) Google Scholar, 20Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.Nature. 1998; 391: 43-50Crossref PubMed Scopus (2721) Google Scholar). Caspase-3 cleaves DFF45, which causes the release of DFF40 from the DFF45/DFF40 complex and triggers DNA fragmentation and nuclear condensation. The three-dimensional structure of the N-terminal domain of the proapoptotic CIDE-B protein was determined (35Lugovskoy A.A. Zhou P. Chou J.J. McCarty J.S. Li P. Wagner G. Solution structure of the CIDE-N domain of CIDE-B and a model for CIDE-N/CIDE-N interactions in the DNA fragmentation pathway of apoptosis.Cell. 1999; 99: 747-755Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The structure consists of a twisted, five-stranded β sheet and two α helices arranged in an α/β roll fold that is very different from the structure of the DD, DED, and CARDs that also participate in homophilic interactions in cell death signaling. On the basis of the structure, a model was proposed for the homophilic interactions involving the N-terminal CIDE domains involving complementary neutralization of their charged surfaces. The family of cysteine proteases called caspases play a central role in the execution of programmed cell death by cleaving a wide variety of substrates (e.g., DFF45), leading to the characteristic morphological changes associated with apoptosis. Caspases can be divided into two types—those with large prodomains that function upstream as initiators of the death cascade and those with a small prodomain that act downstream as effectors. As described above, the prodomains of the initiator caspases contain either a DED or CARD. These domains interact with other signaling proteins that contain these modules through homophilic interactions. This binding event triggers the activation of the initiator caspases by bringing together the proenzymes, allowing autolytic processing in trans as a result of their induced proximity (47Salvesen G.S. Dixit V.M. Caspase activation the induced-proximity model.Proc. Natl. Acad. Sci. USA. 1999; 96: 10964-10967Crossref PubMed Scopus (742) Google Scholar). All caspases have a distinct substrate specificity that requires cleavage after an aspartic acid at the P1 position N-terminal to the cleavage site. The preference of amino acids further to the N terminus of peptide substrates at the P2–P4 positions differs among caspase family members and defines their substrate specificity (58Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. et al.A combinatorial approach defines specificities of members of the caspase family of granzyme b.J. Biol. Chem. 1997; 272: 17907-17911Crossref PubMed Scopus (1786) Google Scholar). The initiator caspases such as caspase-8 and caspase-9 prefer (V,L)EXD-containing substrates like those found in the cleavage sites used to process caspase zymogens into active enzymes. In contrast, the effector caspases cleave DEXD-containing substrates such as those found in structural proteins that are cleaved during apoptosis, resulting in the typical morphological changes in the cell. X-ray crystal structures of caspase-1, (65Walker N.P.C. Talanian R.V. Brady K.D. Dang L.C. Bump N.J. Ferenz C.R. Franklin S. Ghayur T. Hackett M.C. Hammill L.D. et al.Crystal structure of the cysteine protease interleukin-1β-converting enzyme a (p20/p10)2 homodimer.Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (512) Google Scholar, 67Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Structure and mechanism of interleukin-1β converting enzyme.Nature. 1994; 370: 270-275Crossref PubMed Scopus (731) Google Scholar) caspase-3 (46Rotonda J. Nicholson D.W. Fazil K.M. Gallant M. Gareau Y. Labelle M. Peterson E.P. Rasper D.M. Ruel R. Vaillancourt J.P. et al.The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis.Nature Struct. Biol. 1996; 3: 619-625Crossref PubMed Scopus (393) Google Scholar, 39Mittl P.R.E. Di Marco S. Krebs J.F. Bai X. Karanewsky D.S. Priestle J.P. Tomaselli K.J. Grütter M.G. Structure of recombinant human CPP32 in complex with the tetrapeptide Acetyl-Asp-Val-Ala-Asp fluoromethyl ketone.J. Biol. Chem. 1997; 272: 6539-6547Crossref PubMed Scopus (219) Google Scholar), and caspase-8 (4Blanchard H. Kodandapani L. Mittl P.R.E. Di Marco S. Krebs J.F. Wu J.C. Tomaselli K.J. Grütter M.G. The three-dimensional structure of caspase-8 an initiator enzyme in apoptosis.Structure. 1999; 7: 1125-1133Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 66Watt W. Koeplinger K.A. Mildner A.M. Heinrikson R.L. Tomasselli A.G. Watenpaugh K.D. The atomic-resolution structure of human caspase-8, a key activator of apoptosis.Structure. 1999; 7: 1135-1143Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) covalently attached to irreversible peptidic inhibitors have been determined. The overall architecture of these three caspases is similar and consists of two heterodimers composed of a large and small subunit that form a tetramer (Figure 5). The mature enzymes are derived by proteolytically removing the N-terminal prodomains and cleaving the proenzymes between the large and small subunits. In the X-ray structures of the processed enzymes, the C terminus of the large subunit (Figure 5, blue) is far away from the N terminus of the small subunit (magenta) in the heterodimer, but is very close to the N terminus of the small subunit (orange) in the adjacent protein. This observation suggests that the large subunit in the heterodimer may have been linked before processing to the small subunit in the adjacent heterodimer. Alternatively, a large conformational change may occur upon autoprocessing. In addition to providing information on the possible mechanisms of activation, the X-ray structures of caspase/inhibitor complexes revealed the structural basis for the observed substrate specificity. The requirement for an aspartic acid at the P1 position in all substrates and substrate-based inhibitors could be explained by the favorable interaction of this aspartic acid with two highly conserved arginines and a glutamine. In caspase-3, these residues correspond to Arg64, Arg207, and Gln161 (Figure 6). The differences in substrate specificity within the caspase family can also be explained from the X-ray structures. In caspase-1, the P4 binding site (S4 subsite) is a large hydrophobic pocket that can accommodate several residues. In contrast, caspase-3 has a relatively narrow pocket that forms hydrogen bonds with the aspartic acid preferred at the P4 position (Figure 6). Caspase-8 differs from the other caspases in both the S3 and S4 specificity pockets, resulting in the preference for glutamic acid at P3 and for small hydrophobic amino acids at P4. Based on the location of the amino acids in the active site observed in the X-ray structures, an enzymatic mechanism was proposed for the caspases (Figure 7). Like other cysteine proteases, the sulfur of the active site cysteine acts as a nucleophile to form a tetrahedral intermediate (Figure 7B). A nearby histidine increases the nucleophilicity of the sulfur by acting as a base and abstracting a proton (Figure 7A). Some investigators have postulated that a Cys/His dyad is involved in the enzymatic mechanism whereas others have proposed a catalytic triad. The high resolution (1.2 Å) structure of a caspase-8/inhibitor complex supports a catalytic triad in which a clear interaction is observed between the backbone carbonyl of Arg258 to His317 that enhances the basicity of the histidine. Arg258 in caspase-8 is equivalent to Pro177 in caspase-1 and Thr62 in caspase-3. Thus, it appears that the identity of the third residue of the catalytic triad is irrelevant, which is consistent with the observed interaction involving a backbone carbonyl. In addition to the importance of the histidine as a general base in the reaction mechanism, the same histidine is critical for protonating the α-amino group of the scissile bond that favors the release of the leaving group and increases the nucleophilicity of a water molecule to aid in the cleavage of the thiol ester (Figure 7C and Figure 7D). Several proteins have been discovered that regulate apoptosis. In cancer, the levels of these proteins are often altered to allow cancer cells to stay alive even though the cell death pathways have been triggered. Moreover, viruses have engineered mimics of these proteins that allow the host cell to stay alive long enough for the virus to replicate. Thus, these proteins serve as possible targets for the regulation of apoptosis. The inhibitor of apoptosis (IAP) family of proteins represent an important class of regulators of programmed cell death. They were initially discovered in baculoviruses where they were found to inhibit apoptosis in host cells during viral infection (12Crook N.E. Clem R.J. Miller L.K. An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar). Subsequently, IAPs have been found in other viruses, yeast, flies, worms, and mammals (59Uren A.G. Coulson E.J. Vaux D.L. Conservation of baculovirus inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts.TIBS. 1998; 23: 159-162PubMed Google Scholar). All IAPs contain one to three baculovirus IAP repeat (BIR) domains which are composed of about 70 amino acids and have a characteristic signature sequence (CX2CX16HX6C). Some of the IAPs also contain a C-terminal ring finger. Several functions have been attributed to the IAPs. In Drosophila, IAPs have been shown to interact with the proapoptotic proteins REAPER, HID, and GRIM (63Vucic D. Kaiser W.J. Miller L.K. Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophil a proteins HID and GRIM.Mol. Cell. Biol. 1998; 18: 3300-3309Crossref PubMed Scopus (186) Google Scholar, 21Goyal L. McCall K. Agapite J. Hartwieg E. Steller H. Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function.EMBO J. 2000; 19: 589-597Crossref PubMed Scopus (365) Google Scholar). The mammalian IAPs, cIAP-1 (MIHB) and cIAP-2 (MIHC), bind to TNF-receptor associated factors (TRAFS) 1 and 2, while survivin (1Ambrosini G. Adida C. Altieri D.C. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nature Med. 1997; 3: 917-921Crossref PubMed Scopus (2913) Google Scholar), has been implicated in the cell cycle (29Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Control of apoptosis and mitotic spindle checkpoint by survivin.Nature. 1998; 396 (a): 580-584Crossref PubMed Scopus (1685) Google Scholar). Some IAPs have also been shown to potently inhibit caspases. For example, human XIAP inhibits caspases-3 and -7 with Ki's of 0.2–0.7 nM (14Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. X-linked IAP is a direct inhibitor of cell-death proteases.Nature. 1997; 388: 300-304Crossref PubMed Scopus (1665) Google Scholar) and also inhibits caspase-9 (16Deveraux Q.L. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases.EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (664) Google Scholar). The portion of XIAP responsible for inhibiting caspase-3 was found to contain the BIR2 domain (55Takahashi R. Deveraux Q. Tamm I. Welsh K. Assa-Munt N. Salvesen G.S. Reed J.C. A single BIR domain of XIAP sufficient for inhibiting caspases.J. Biol. Chem. 1998; 273: 7787-7790Crossref PubMed Scopus