Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.22.60 (caspase-7)
920 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The inhibitor of apoptosis proteins (IAPs) represent the only endogenous caspase inhibitors and are characterized by the presence of baculoviral IAP repeats (BIRs). Here, we report the crystal structure of the complex between human caspase-7 and XIAP (BIR2 and the proceeding linker). The structure surprisingly reveals that the linker is the only contacting element for the caspase, while the BIR2 domain is invisible in the crystal. The linker interacts with and blocks the substrate groove of the caspase in a backward fashion, distinct from substrate recognition. Structural analyses suggest that the linker is the energetic and specificity determinant of the interaction. Further biochemical characterizations clearly establish that the linker harbors the major energetic determinant, while the BIR2 domain serves as a regulatory element for caspase binding and Smac neutralization.
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PMID:Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain. 1125 31

The inhibitor of apoptosis proteins (IAP) regulates cell death by inhibiting caspases. The region of X-linked (X) IAP containing the second baculovirus IAP repeat domain (BIR2) is sufficient for inhibiting caspase-3 and -7. In this study, we found that the modes of inhibition of these two caspases were different: caspase-3 is inhibited in a competitive manner whereas caspase-7 inhibition occurs through a mixed competitive and noncompetitive mechanism. Binding assays revealed that the inhibition of caspase-3 by XIAP was totally dependent on the interaction between the active site of caspase-3 and the linker region between the BIR1 and BIR2 domains of XIAP. In contrast, the active site and the NH(2)-terminal region of caspase-7 bound to the linker region and the BIR2, respectively. Moreover the BIR2 with a mutated linker region, which inhibited caspase-3 very weakly, still bound to and inhibited caspase-7. Furthermore, a chimeric caspase-7/3 comprising the NH(2)-terminal portion of caspase-7 and COOH-terminal portion of caspase-3 was inhibited by XIAP by a mixed competitive and noncompetitive mechanism. Our results suggest that the linker region between BIR1 and BIR2 domains is responsible for active site-directed, competitive inhibition of both caspase-3 and -7, whereas the BIR2 itself is involved in noncompetitive inhibition of caspase-7.
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PMID:X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes. 1135 76

The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.
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PMID:Requirement of both the second and third BIR domains for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by Smac. 1451 14

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.
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PMID:XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs. 1565 Jul 47

Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP proteins inhibit caspase activity, whereas Smac/Diablo antagonizes IAP. XIAP, a ubiquitous IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for binding of the BIR3 domain of XIAP with caspase-9, whereas how Smac liberates effector caspases from XIAP inhibition is not clear. It is generally believed that binding of Smac with IAP generates a steric hindrance that prevents XIAP from inhibiting effector caspases, and therefore small molecule mimics of Smac are not able to reverse inhibition of the effector caspases. Surprisingly, we show here that binding of a dimeric Smac N-terminal peptide with the BIR2 domain of XIAP effectively antagonizes inhibition of caspase-3 by XIAP. Further, we defined the dynamic and cooperative interaction of Smac with XIAP: binding of Smac with the BIR3 domain anchors the subsequent binding of Smac with the BIR2 domain, which in turn attenuates the caspase-3 inhibitory function of XIAP. We also show that XIAP homotrimerizes via its C-terminal Ring domain, making its inhibitory activity toward caspase-3 more susceptible to Smac.
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PMID:A dimeric Smac/diablo peptide directly relieves caspase-3 inhibition by XIAP. Dynamic and cooperative regulation of XIAP by Smac/Diablo. 1772 22

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.
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PMID:Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains. 1802 97

The ability of the irreversibly denatured XIAP-BIR2 domain versus the native protein in inhibition of executioner caspase-3 and -7 was investigated. The denatured protein that lacked the physical characteristics of the native protein inhibited caspase-7, while failing to inhibit caspase-3. Furthermore, the kinetics of association of the denatured protein with caspase-7 decreased substantially suggesting that the exposure of the linker is reduced. This was further confirmed by the decreased level of proteolysis at the linker by trypsin for the denatured protein. These results suggest that the essential moiety of the XIAP involved in inhibition is the linker joining BIR1 to BIR2 and that the BIR2 plays a marginal role in inhibition.
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PMID:BIR2 domain of XIAP plays a marginal role in inhibition of executioner caspases. 2006 Apr 11

Caspases have been suggested to contribute to not only apoptosis regulation but also non-apoptotic cellular phenomena. Recently, we have reported the involvement of caspase-7 to the cell cycle progression at mitotic phase by knockdown of caspase-7 using small interfering RNAs and short hairpin RNA. Here we showed that chemically synthesized broad-spectrum caspase inhibitors, which have been used to suppress apoptosis, prevented the cell proliferation in a dose-dependent manner, and that the subtype-specific peptide-based caspase inhibitor for caspase-3 and -7, but not for caspase-9, inhibited cell proliferation. It was also indicated that the BIR2 domain of X-linked inhibitor of apoptosis protein, functioning as an inhibitor for caspase-3 and -7, but not the BIR3 domain which plays as a caspase-9 inhibitor, induced cell cycle arrest. Furthermore, flow cytometry revealed that the cells treated with caspase inhibitors arrested at G(2)/M phase. By using HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, the prevention of the cell proliferation by caspase inhibitors induced cell cycle arrest at mitotic phase accompanying the accumulation of the substrates for APC/C, suggesting the impairment of the APC/C activity at the transition from M to G(1) phases. These results indicate that caspase(s) contribute to the cell cycle regulation at mitotic phase.
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PMID:Contribution of caspase(s) to the cell cycle regulation at mitotic phase. 2147 77

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.
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PMID:Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target. 2674 18

Nimbolide is one of the major compounds from the leaves and flowers of the neem tree and exhibits antitumor properties on various cancer cells. However, no report has shown that nimbolide induces apoptosis in vitro and in vivo in human hepatocellular carcinoma cells. Our results indicated that it inhibited cell growth in Huh-7 and PLC/PRF/5 cells. We also found that nimbolide induced cell death through the induction of G2/M phase arrest and mitochondrial dysfunction, accompanied by the increased expression of cleaved caspase-7, caspase-9, caspase-3, caspase-PARP, and Bax and decreased expression of Mcl-1 and Bcl-2. A human apoptosis antibody array analysis demonstrated that inhibition of the apoptosis family proteins (XIAP, c-IAP1, and c-IAP2) was one of the major targets of nimbolide. Additionally, nimbolide sustained activation of ERK expression. Moreover, pretreatment with U0126 (MEK inhibitor) markedly abolished nimbolide-inhibited cell viability, induced cell apoptosis, ERK phosphorylation, cleaved caspase-9, caspase-3, cleaved-PARP activation, and increased c-IAP1 expression in Huh-7 cells. An in vivo study showed that nimbolide significantly reduced Huh-7 tumor growth and weight in a xenograft mouse model. This study indicated the antitumor potential of nimbolide in human hepatocellular carcinoma cells.
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PMID:Nimbolide induced apoptosis by activating ERK-mediated inhibition of c-IAP1 expression in human hepatocellular carcinoma cells. 2996 3


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