Inhibitor of Apoptosis Proteins in Eukaryotic Evolution and Development: A Model of Thematic Conservation (original) (raw)
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XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs
The EMBO Journal, 2005
The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.
Mechanism of XIAP-Mediated Inhibition of Caspase-9
Molecular Cell, 2003
Department of Molecular Biology the caspase activation cascades. For example, active caspase-9 cleaves and activates caspase-3 and cas-Lewis Thomas Laboratory Princeton University pase-7. Thus, the activation and inhibition of the initiator caspases constitute a central regulatory step in cellular Princeton, New Jersey 08544 2 Department of Biology physiology. The inhibitor of apoptosis (IAP) family of proteins sup-370 Lancaster Avenue Haverford College presses apoptosis by inhibiting the enzymatic activity of both the initiator and the effector caspases (Deveraux Haverford, Pennsylvania 19041 3 Kimmel Cancer Center and Reed, 1999; Salvesen and Duckett, 2002; Shi, 2002b). At least eight members of the mammalian IAPs 233 S. 10 th Street Thomas Jefferson University have been identified, including X-linked IAP (XIAP), c-IAP1, c-IAP2, and Livin/ML-IAP. Each IAP protein con-Philadelphia, Pennsylvania 19107 tains one to three copies of the 80 residue zinc binding baculoviral IAP repeat (BIR). The different BIR domains Summary and segments in the same IAP protein appear to exhibit distinct functions. For example, the third BIR domain The inhibitor of apoptosis (IAP) proteins potently in-(BIR3) of XIAP potently inhibits the activity of the prohibit the catalytic activity of caspases. While profound cessed caspase-9 whereas the linker region between insight into the inhibition of the effector caspases has BIR1 and BIR2 selectively targets the active caspase-3 been gained in recent years, the mechanism of how or -7 (Fesik and Shi, 2001). The IAP-mediated inhibition the initiator caspase-9 is regulated by IAPs remains of all caspases can be effectively removed by the mitoenigmatic. This paper reports the crystal structure of chondrial protein Smac/DIABLO, which is released into caspase-9 in an inhibitory complex with the third bacuthe cytoplasm during apoptosis (Chai et al., 2000; Du loviral IAP repeat (BIR3) of XIAP at 2.4 Å resolution. et al., 2000; Verhagen et al., 2000). The proapoptotic The structure reveals that the BIR3 domain forms a activity of Smac/DIABLO depends on a 4 amino acid heterodimer with a caspase-9 monomer. Strikingly, IAP binding motif located at the N terminus of the mature the surface of caspase-9 that interacts with BIR3 also protein (Liu et al., 2000; Shi, 2002a; Wu et al., 2000).
Journal of Cell Biology, 2002
he X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/ Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. Ѩ RING point mutants T of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/ Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that IAP antagonism is required for apoptosis to proceed following UV irradiation. *Abbreviations used in this paper: IAP, inhibitor of apoptosis protein; BIR, baculoviral IAP repeat; TNF, tumor necrosis factor; TRAF, TNF receptor associated factors; XIAP, X-linked mammalian inhibitor of apoptosis protein.
Biochemical …, 2005
Several IAP (inhibitor of apoptosis) proteins regulate cell fate decisions, and the X-linked IAP (XIAP) does so in part by inhibiting caspases, proteases that execute the apoptotic pathway. A tissue-specific homologue of XIAP, known as ILP2 (IAP-like protein 2), has previously been implicated in the control of apoptosis in the testis by direct inhibition of caspase 9. In examining this protein we found that the putative caspase 9 interaction domain is a surprisingly weak inhibitor and is also conformationally unstable. Comparison with the equivalent domain in XIAP demonstrated that the instability is due to the lack of a linker segment N-terminal to the inhibitory BIR (baculovirus IAP repeat) domain. Fusion of a 9-residue linker from XIAP to the N-terminus of ILP2 restored tight caspase 9 inhibition, dramatically increased conformational stability and allowed crystallization of the ILP2 BIR domain in a form strikingly similar to the XIAP third BIR domain. We conclude that ILP2 is an unstable protein, and cannot inhibit caspase 9 in a physiological way on its own. We speculate that ILP2 requires assistance from unidentified cellular factors to be an effective inhibitor of apoptosis in vivo.
IAPS : More than just inhibitors of apoptosis proteins
Cell Cycle, 2008
Inhibitors of apoptosis proteins (IAPs) are a conserved family of proteins identified in species ranging from virus, yeasts, nematodes, fishes, flies and mammals. The common structural feature is the presence of at least one Baculovirus IAP Repeat (BIR) domain. Hence, IAPs are also known as BIR-containing proteins (BIRCs). Most of them display anti-apoptotic properties when overexpressed. In drosophila, IAPs are sufficient and necessary to promote cell survival through a direct regulation of apoptotic proteases called caspases. In mammals, BIRC4/XIAP, the most studied IAP member can directly inhibit the activity of caspase-3, 7 and 9. However, this activity is not conserved in other IAPs and physiological relevancies of such anti-caspase activities are still discussed. A detailed analysis of IAP-deficient mice or derived cells, deletion experiments performed in drosophila and zebrafish, or research of protein partners have revealed the importance of IAPs in adaptive response to cellular stress, in cell proliferation, differentiation, signaling, motility and in immune response. This review discusses recent data that help understanding of cellular functions of IAPs.
A single BIR domain of XIAP sufficient for inhibiting caspases
Journal of Biological …, 1998
The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or-7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (K i) for BIR2-mediated inhibition of caspases-3 and-7 were 2-5 nM, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.
Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family
Embo Reports, 2006
Several of the inhibitor of apoptosis protein (IAP) family members regulate apoptosis in response to various cellular assaults. Some members are also involved in cell signalling, mitosis and targeting proteins to the ubiquitin-proteasome degradation machinery. The most intensively studied family member, X-linked IAP (XIAP), is a potent inhibitor of caspase activity; hence, it is generally assumed that direct caspase inhibition is an important conserved function of most members of the family. Biochemical and structural studies have precisely mapped the elements of XIAP required for caspase inhibition. Intriguingly, these elements are not conserved among IAPs. Here, we review current knowledge of the caspaseinhibitory potential of the human IAPs and show that XIAP is probably the only bona fide caspase inhibitor, suggesting that the other family members never gained the ability to directly inhibit caspase activity.
2007
During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp 330 , and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp 330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp 330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp 315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp 315 , suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
Regulation of Apoptosis by Inhibitors of Apoptosis (IAPs)
2013
Inhibitors of Apoptosis (IAPs) are a family of proteins with various biological functions including regulation of innate immunity and inflammation, cell proliferation, cell migration and apoptosis. They are characterized by the presence of at least one N-terminal baculoviral IAP repeat (BIR) domain involved in protein-protein interaction. Most of them also contain a C-terminal RING domain conferring an E3-ubiquitin ligase activity. In drosophila, IAPs are essential to ensure cell survival, preventing the uncontrolled activation of the apoptotic protease caspases. In mammals, IAPs can also regulate apoptosis through controlling caspase activity and caspase-activating platform formation. Mammalian IAPs, mainly X-linked IAP (XIAP) and cellular IAPs (cIAPs) appeared to be important determinants of the response of cells to endogenous or exogenous cellular injuries, able to convert the survival signal into a cell death-inducing signal. This review highlights the role of IAP in regulating apoptosis in Drosophila and Mammals.