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Shawn B. Bratton - One of the best experts on this subject based on the ideXlab platform.
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the apaf 1 apoptosome induces formation of caspase 9 homo and heterodimers with distinct activities
Nature Communications, 2016Co-Authors: Sunhee Lee, Srinivas Malladi, Miao Der Chen, Nicholas J Mastrandrea, Zhiwen Zhang, Shawn B. BrattonAbstract:According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that Procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, Procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated Procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.
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the apaf 1 Procaspase 9 apoptosome complex functions as a proteolytic based molecular timer
The EMBO Journal, 2009Co-Authors: Srinivas Malladi, Howard O. Fearnhead, Madhavi Challamalladi, Shawn B. BrattonAbstract:During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of Procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector Procaspase‐3, Procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, Procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of Procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, Procaspase‐9 per se contributed little to the activation of Procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of Procaspase‐9 sets the overall duration of the timer, Procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate Procaspase‐3) dictates how fast the timer ‘ticks’ over.
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The Apaf‐1•Procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer
The EMBO journal, 2009Co-Authors: Srinivas Malladi, Madhavi Challa-malladi, Howard O. Fearnhead, Shawn B. BrattonAbstract:During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of Procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector Procaspase‐3, Procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, Procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of Procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, Procaspase‐9 per se contributed little to the activation of Procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of Procaspase‐9 sets the overall duration of the timer, Procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate Procaspase‐3) dictates how fast the timer ‘ticks’ over.
Emad S. Alnemri - One of the best experts on this subject based on the ideXlab platform.
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Oligomerization is a general mechanism for the activation of apoptosis initiator and inflammatory Procaspases.
The Journal of biological chemistry, 2003Co-Authors: David W. Chang, Srinivasa M. Srinivasula, Emad S. Alnemri, Dara Ditsworth, Hongtu Liu, Xiaolu YangAbstract:Proteolytic activation of initiator Procaspases is a crucial step in the cellular commitment to apoptosis. Alternative models have been postulated for the activation mechanism, namely the oligomerization or induced proximity model and the allosteric regulation model. While the former holds that Procaspases become activated upon proper oligomerization by an adaptor protein, the latter states that the adaptor is an allosteric regulator for Procaspases. The allosteric regulation model has been applied for the activation of Procaspase-9 by apoptotic protease-activating factor (Apaf-1) in an oligomeric complex known as the apoptosome. Using approaches that allow for controlled oligomerization, we show here that aggregation of multiple Procaspase-9 molecules can induce their activation independent of the apoptosome. Oligomerization-induced Procaspase-9 activation, both within the apoptosome and in artificial systems, requires stable homophilic association of the protease domains, raising the possibility that the function of Apaf-1 is not only to oligomerize Procaspase-9 but also to maintain the interaction of the caspase-9 protease domain after processing. In addition, we provide biochemical evidence that other apoptosis initiator caspases (caspase-2 and -10) as well as a Procaspase involved in inflammation (murine caspase-11) are also activated by oligomerization. Thus, oligomerization of precursor molecules appears to be a general mechanism for the activation of both apoptosis initiator and inflammatory Procaspases.
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Identification of Ipaf, a Human Caspase-1-activating Protein Related to Apaf-1
The Journal of biological chemistry, 2001Co-Authors: Jean-luc Poyet, Srinivasa M. Srinivasula, Teresa Fernandes-alnemri, Mehdi Tnani, Marjaneh Razmara, Emad S. AlnemriAbstract:Abstract Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of Procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of Procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of Procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of Procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli.
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negative regulation of cytochrome c mediated oligomerization of apaf 1 and activation of Procaspase 9 by heat shock protein 90
The EMBO Journal, 2000Co-Authors: Pramod Pandey, Srinivasa M. Srinivasula, Emad S. Alnemri, Ayman Saleh, Atsuko Nakazawa, Shailendra Kumar, Vijay Kumar, Ralph R Weichselbaum, Carlo Nalin, Donald KufeAbstract:The release of cytochrome c from mitochondria results in the formation of an Apaf-1–caspase-9 apoptosome and induces the apoptotic protease cascade by activation of Procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of Procaspase-9. We also show that Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of Procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90–Apaf-1 complex and relieves the inhibition of Procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade.
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Negative regulation of cytochrome c‐mediated oligomerization of Apaf‐1 and activation of Procaspase‐9 by heat shock protein 90
The EMBO journal, 2000Co-Authors: Pramod Pandey, Srinivasa M. Srinivasula, Emad S. Alnemri, Ayman Saleh, Atsuko Nakazawa, Shailendra Kumar, Vijay Kumar, Ralph R Weichselbaum, Carlo Nalin, Donald KufeAbstract:The release of cytochrome c from mitochondria results in the formation of an Apaf-1–caspase-9 apoptosome and induces the apoptotic protease cascade by activation of Procaspase-3. The present studies demonstrate that heat shock protein 90 (Hsp90) forms a cytosolic complex with Apaf-1 and thereby inhibits the formation of the active complex. Immunodepletion of Hsp90 depletes Apaf-1 and thereby inhibits cytochrome c-mediated activation of caspase-9. Addition of purified Apaf-1 to Hsp90-depleted cytosolic extracts restores cytochrome c-mediated activation of Procaspase-9. We also show that Hsp90 inhibits cytochrome c-mediated oligomerization of Apaf-1 and thereby activation of Procaspase-9. Furthermore, treatment of cells with diverse DNA-damaging agents dissociates the Hsp90–Apaf-1 complex and relieves the inhibition of Procaspase-9 activation. These findings provide the first evidence for a negative cytosolic regulator of cytochrome c-dependent apoptosis and for involvement of a chaperone in the caspase cascade.
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cytochrome c and datp mediated oligomerization of apaf 1 is a prerequisite for Procaspase 9 activation
Journal of Biological Chemistry, 1999Co-Authors: Ayman Saleh, Srinivasa M. Srinivasula, Samir Acharya, Richard Fishel, Emad S. AlnemriAbstract:Abstract To elucidate the mechanism of activation of Procaspase-9 by Apaf-1, we produced recombinant full-length Apaf-1 and purified it to complete homogeneity. Here we show using gel filtration that full-length Apaf-1 exists as a monomer that can be transformed to an oligomeric complex made of at least eight subunits after binding to cytochrome c and dATP. Apaf-1 binds to cytochromec in the absence of dATP but does not form the oligomeric complex. However, when dATP is added to the cytochromec-bound Apaf-1 complex, complete oligomerization occurs, suggesting that oligomerization is driven by hydrolysis of dATP. This was supported by the observation that ATP, but not the nonhydrolyzable adenosine 5′-O-(thiotriphosphate), can induce oligomerization of the Apaf-1-cytochrome c complex. Like the spontaneously oligomerizing Apaf-530, which lacks its WD-40 domain, the oligomeric full-length Apaf-1-cytochrome c complex can bind and process Procaspase-9 in the absence of additional dATP or cytochrome c. However, unlike the truncated Apaf-530 complex, the full-length Apaf-1 complex can release the mature caspase-9 after processing. Once released, mature caspase-9 can process Procaspase-3, setting into motion the caspase cascade. These observations indicate that cytochrome c and dATP are required for oligomerization of Apaf-1 and suggest that the WD-40 domain plays an important role in oligomerization of full-length Apaf-1 and the release of mature caspase-9 from the Apaf-1 oligomeric complex.
Srinivas Malladi - One of the best experts on this subject based on the ideXlab platform.
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the apaf 1 apoptosome induces formation of caspase 9 homo and heterodimers with distinct activities
Nature Communications, 2016Co-Authors: Sunhee Lee, Srinivas Malladi, Miao Der Chen, Nicholas J Mastrandrea, Zhiwen Zhang, Shawn B. BrattonAbstract:According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that Procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, Procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated Procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.
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the apaf 1 Procaspase 9 apoptosome complex functions as a proteolytic based molecular timer
The EMBO Journal, 2009Co-Authors: Srinivas Malladi, Howard O. Fearnhead, Madhavi Challamalladi, Shawn B. BrattonAbstract:During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of Procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector Procaspase‐3, Procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, Procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of Procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, Procaspase‐9 per se contributed little to the activation of Procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of Procaspase‐9 sets the overall duration of the timer, Procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate Procaspase‐3) dictates how fast the timer ‘ticks’ over.
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The Apaf‐1•Procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer
The EMBO journal, 2009Co-Authors: Srinivas Malladi, Madhavi Challa-malladi, Howard O. Fearnhead, Shawn B. BrattonAbstract:During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of Procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector Procaspase‐3, Procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, Procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of Procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, Procaspase‐9 per se contributed little to the activation of Procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of Procaspase‐9 sets the overall duration of the timer, Procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate Procaspase‐3) dictates how fast the timer ‘ticks’ over.
Howard O. Fearnhead - One of the best experts on this subject based on the ideXlab platform.
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the apaf 1 Procaspase 9 apoptosome complex functions as a proteolytic based molecular timer
The EMBO Journal, 2009Co-Authors: Srinivas Malladi, Howard O. Fearnhead, Madhavi Challamalladi, Shawn B. BrattonAbstract:During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of Procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector Procaspase‐3, Procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, Procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of Procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, Procaspase‐9 per se contributed little to the activation of Procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of Procaspase‐9 sets the overall duration of the timer, Procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate Procaspase‐3) dictates how fast the timer ‘ticks’ over.
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The Apaf‐1•Procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer
The EMBO journal, 2009Co-Authors: Srinivas Malladi, Madhavi Challa-malladi, Howard O. Fearnhead, Shawn B. BrattonAbstract:During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of Procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector Procaspase‐3, Procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, Procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of Procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, Procaspase‐9 per se contributed little to the activation of Procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of Procaspase‐9 sets the overall duration of the timer, Procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate Procaspase‐3) dictates how fast the timer ‘ticks’ over.
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small molecule inhibitors of apaf 1 related caspase 3 9 activation that control mitochondrial dependent apoptosis
Cell Death & Differentiation, 2006Co-Authors: Gema Malet, Angel G Martin, Mar Orzaez, Maria J Vicent, Isabel Masip, Gloria Sanclimens, Antonio Ferrermontiel, Ismael Mingarro, Angel Messeguer, Howard O. FearnheadAbstract:Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and Procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of Procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of Procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis.
Gabriel Núñez - One of the best experts on this subject based on the ideXlab platform.
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Expression and Functional Analysis of Apaf-1 Isoforms: EXTRA WD-40 REPEAT IS REQUIRED FOR CYTOCHROME cBINDING AND REGULATED ACTIVATION OF Procaspase-9
The Journal of biological chemistry, 2000Co-Authors: Mary A. Benedict, Naohiro Inohara, Gabriel NúñezAbstract:Abstract Apaf-1 is an important apoptotic signaling molecule that can activate Procaspase-9 in a cytochromec/dATP-dependent fashion. Alternative splicing can create an NH2-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate Procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH2-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH2-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated Procaspase 9 in vitro in response to cytochrome c and dATP, while the NH2-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to Procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate Procaspase-9 in response to cytochrome c and dATP.
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Role of cytochrome c and dATP/ATP hydrolysis in Apaf‐1‐mediated caspase‐9 activation and apoptosis
The EMBO journal, 1999Co-Authors: Mary A. Benedict, Liyun Ding, Gabriel NúñezAbstract:Apaf-1 plays a critical role in apoptosis by binding to and activating Procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated Procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to Procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to Procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind Procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that Procaspase-9 can recruit Procaspase-3 to the Apaf-1-Procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associated with and activated Procaspase-9, but failed to recruit Procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in Procaspase-9-mediated Procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate Procaspase-9 and induce apoptosis.
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role of cytochrome c and datp atp hydrolysis in apaf 1 mediated caspase 9 activation and apoptosis
The EMBO Journal, 1999Co-Authors: Mary A. Benedict, Liyun Ding, Gabriel NúñezAbstract:Apaf-1 plays a critical role in apoptosis by binding to and activating Procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated Procaspase9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to Procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to Procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind Procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that Procaspase-9 can recruit Procaspase3 to the Apaf-1‐Procaspase-9 complex. Apaf-1(1‐570), a mutant lacking the WD-40 repeats, associated with and activated Procaspase-9, but failed to recruit Procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in Procaspase-9-mediated Procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate Procaspase-9 and induce apoptosis.
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WD-40 Repeat Region Regulates Apaf-1 Self-association and Procaspase-9 Activation
The Journal of biological chemistry, 1998Co-Authors: Liyun Ding, David M. Spencer, Gabriel NúñezAbstract:The casp9 protein plays a critical role in apoptosis induced by a variety of death stimuli. A regulator of apoptosis, Apaf-1, binds to and activates pro-casp9 in the presence of cytochrome c and dATP, a requirement that is bypassed by deletion of the WD-40 repeats located in the C-terminal half of Apaf-1. In this report, we used constitutively active Apaf-1 mutant lacking the WD-40 repeat region to study the mechanism and regulation of pro-casp9 activation. Mutational analysis revealed that only a small portion of the CED-4 homologous region (residues 456–559) could be deleted without destroying the ability of Apaf-1-(1–559) to activate pro-casp9. Apaf-1 can self-associate to form oligomers. Disruption of Apaf-1 self-association by deletion (Δ109–559) or mutation of the P-loop region (K149R) abrogated Apaf-1-mediated pro-casp9 activation. Forced oligomerization of the caspase recruitment domain of Apaf-1 was sufficient for pro-casp9 activation. Dimerization of chimeric Fpk-pro-casp9 protein with the dimerizer drug FK1012 induced pro-casp9 processing and apoptosis in cells. Significantly, the C-terminal region containing WD-40 repeats interacted with its N-terminal CED-4 homologous region, as determined by immunoprecipitation experiments. Importantly, expression of the WD-40 repeat region inhibited Apaf-1 self-association and proteolytic activation of pro-casp9. These studies provide a mechanism by which Apaf-1 promotes autoactivation of pro-casp9 through Apaf-1 self-association, a process that is negatively regulated by the WD-40 repeats.