Interaction with XIAP prevents full caspase‐3/‐7 activation in proliferating human T lymphocytes (original) (raw)
Related papers
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.
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.
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).
Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants
Biotechnology and Bioengineering, 2002
Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iaprepeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 704–716, 2002; DOI 10.1002/bit.10154
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.
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.
Targeting the XIAP/caspase-7 complex selectively kills caspase-3–deficient malignancies
Journal of Clinical Investigation, 2013
Caspase-3 downregulation (CASP3/DR) in tumors frequently confers resistance to cancer therapy and is significantly correlated with a poor prognosis in cancer patients. Because CASP3/DR cancer cells rely heavily on the activity of caspase-7 (CASP7) to initiate apoptosis, inhibition of activated CASP7 (p19/p12-CASP7) by X-linked inhibitor of apoptosis protein (XIAP) is a potential mechanism by which apoptosis is prevented in those cancer cells. Here, we identify the pocket surrounding the Cys 246 residue of p19/p12-CASP7 as a target for the development of a protein-protein interaction (PPI) inhibitor of the XIAP:p19/p12-CASP7 complex. Interrupting this PPI directly triggered CASP7-dependent apoptotic signaling that bypassed the activation of the apical caspases and selectively killed CASP3/DR malignancies in vitro and in vivo without adverse side effects in nontumor cells. Importantly, CASP3/DR combined with p19/p12-CASP7 accumulation correlated with the aggressive evolution of clinical malignancies and a poor prognosis in cancer patients. Moreover, targeting of this PPI effectively killed cancer cells with multidrug resistance due to microRNA let-7a-1-mediated CASP3/DR and resensitized cancer cells to chemotherapy-induced apoptosis. These findings not only provide an opportunity to treat CASP3/DR malignancies by targeting the XIAP:p19/p12-CASP7 complex, but also elucidate the molecular mechanism underlying CASP3/DR in cancers. Introduction Caspase-3 (CASP3) is a major executioner protein of proteolytic degradation during apoptosis. Most cancer therapies, including radiotherapy, chemotherapy, and targeted therapy, induce extrinsic death receptor/CASP8/10-dependent and intrinsic mitochondria/CASP9dependent apoptotic signals, which ultimately converge to activate CASP3 and promote cancer cell apoptosis. CASP3 downregulation (CASP3/DR), a progressive phenomenon that enables cancer cells to survive cancer therapy-induced apoptosis, has been observed in many malignancies and correlates significantly with poor survival in patients with solid tumors (1-11) and leukemia (12). In fact, insufficient induction of the apoptotic machinery is observed in CASP3/ DR cancer cells treated with anticancer agents. Because these malignant cancer cells often develop drug resistance (9, 12, 13), an effective strategy to combat CASP3/DR in malignancies is urgently needed. X-linked inhibitor of apoptosis protein (XIAP) belongs to the IAP family and tightly regulates the apoptotic and nonapoptotic caspase functions via interaction with the activated forms of the executioner caspases-namely, CASP3 and CASP7-in mammalian cells (14). Under normal circumstances, IAPs ensure that low-level caspase activity does not erroneously initiate an apoptotic response either through incidental activation or as a consequence of nonapoptotic functions, such as proliferation (15), differentiation (16), and cytoskeletal remodeling (17). Once cells are committed to apoptosis, the mitochondria release the second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI (SMAC/DIABLO) to relieve the XIAP-mediated inhibition of activated CASP3. Subsequently, this reaction triggers activation of CASP7 to promote apoptotic proteolysis (18). In normal cells, XIAP predominantly inhibits CASP3 activation because it both mediates caspase-associated cellular functions (19) and regulates CASP7 activation (20). However, cancer cells that downregulate CASP3 expression to escape from apoptosis may upregulate the structurally and functionally similar CASP7 (18, 21, 22) to achieve cellular homeostasis (23, 24), although the mechanism remains unclear. Staurosporine (STS), an inducer of the intrinsic mitochondrial apoptotic pathway, appears to inefficiently elevate intracellular CASP7 activity and induce apoptosis in embryonic fibroblasts derived from Casp3-knockout mice (14) and CASP3-null MCF-7 breast cancer cells (25), even in the presence of SMAC/DIABLO. These results are consistent with those from in vitro binding assays demonstrating that SMAC/DIABLO is unable to block the proteinprotein interaction (PPI) between XIAP and the Ala-Lys-Pro (AKP) motif of activated CASP7 (composed of the p19 and p12 subunits; referred to herein as p19/p12-CASP7) (26). Because the XIAP:p19/ p12-CASP7 complex accumulates in STS-treated MCF-7 cells, the PPI between XIAP and p19/p12-CASP7 is thought to be the mechanism of chemoresistance in CASP3/DR cancer cells (25). Although recent studies have used MCF-7 cells as a model to assess new therapeutic strategies for overcoming CASP3/DR-associated chemoresistance (27-29), our present study is the first to propose a strategy to specifically inhibit the PPI between XIAP and p19/p12-CASP7 to achieve anticancer activity against CASP3/DR malignancies.