The Experts below are selected from a list of 209883 Experts worldwide ranked by ideXlab platform

Gerald M. Cohen - One of the best experts on this subject based on the ideXlab platform.

  • Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway.
    Cell death and differentiation, 2009
    Co-Authors: Satoshi Inoue, Gareth J. Browne, Gennaro Melino, Gerald M. Cohen
    Abstract:

    Caspases are a family of aspartate-specific cysteine proteases responsible for the biochemical and morphological changes that occur during the execution phase of apoptosis. The hierarchical ordering of caspases has been clearly established using dATP-activated cell lysates to model the intrinsic pathway induced by initial mitochondrial perturbation. In this model, Caspase-9, the apical caspase, directly processes and activates the effector caspases, caspase-3 and -7, and then active caspase-3 but not caspase-7, processes caspase-2 and -6, and subsequently the activated caspase-6 processes caspase-8 and -10. To address the possibility that this model in vitro system might not reflect the precise ordering of caspases in intact cells, we have examined this possibility in cells induced to undergo apoptosis by activation of the intrinsic pathway. We have used caspase deficient cells, small interference RNA for caspase-6 and -7, and a specific caspase-3 inhibitor. In contrast to the earlier in vitro studies, we now show that in intact cells caspase-7 can also directly process and activate caspase-2 and -6. The processing of caspase-2 and -6 occurs within the cytoplasm and active caspase-6 is then responsible for both the processing of caspase-8 and the cleavage of caspase-6 substrates, including lamin A/C.

  • xiap inhibition of caspase 3 preserves its association with the apaf 1 apoptosome and prevents cd95 and bax induced apoptosis
    Cell Death & Differentiation, 2002
    Co-Authors: Shawn B. Bratton, Michael Butterworth, Jennifer M Lewis, Colin S Duckett, Gerald M. Cohen
    Abstract:

    Ligation of death receptors or formation of the Apaf-1 apoptosome results in the activation of caspases and execution of apoptosis. We recently demonstrated that X-linked inhibitor-of-apoptosis protein (XIAP) associates with the apoptosome in vitro. By utilizing XIAP mutants, we now report that XIAP binds to the ‘native’ apoptosome complex via a specific interaction with the small p12 subunit of processed Caspase-9. Indeed, we provide the first direct evidence that XIAP can simultaneously bind active caspases-9 and -3 within the same complex and that inhibition of caspase-3 by the Linker-BIR2 domain prevents disruption of BIR3-Caspase-9 interactions. Recent studies suggest that inhibition of caspase-3 is dispensable for its anti-apoptotic effects. However, we clearly demonstrate that inhibition of caspase-3 is required to inhibit CD95 (Fas/Apo-1)-mediated apoptosis, whereas inhibition of either Caspase-9 or caspase-3 prevents Bax-induced cell death. Finally, we illustrate for the first time that XIAP mutants, which are incapable of binding to caspases-9 and -3 are completely devoid of anti-apoptotic activity. Thus, XIAP's capacity to maintain inhibition of Caspase-9 within the Apaf-1 apoptosome is influenced by its ability to simultaneously inhibit active caspase-3, and depending upon the apoptotic stimulus, inhibition of Caspase-9 or 3 is essential for XIAP's anti-apoptotic activity.

  • proteasome inhibitor induced apoptosis of b chronic lymphocytic leukaemia cells involves cytochrome c release and caspase activation accompanied by formation of an 700 kda apaf 1 containing apoptosome complex
    Leukemia, 2001
    Co-Authors: J B Almond, Kelvin Cain, Roger T Snowden, A Hunter, David Dinsdale, Gerald M. Cohen
    Abstract:

    Proteasome inhibitors, including lactacystin and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B cells from patients with B cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and -9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), did not prevent the release of cytochrome c or partial processing of Caspase-9 but prevented activation of effector caspases and the induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that Caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an 700 kDa caspase-activating apoptosome complex containing Apaf-1. We describe for the first time the formation of a similar 700 kDa caspase-activating apoptosome complex in B-CLL cells induced to undergo apoptosis by proteasome inhibitors.

  • recruitment activation and retention of caspases 9 and 3 by apaf 1 apoptosome and associated xiap complexes
    The EMBO Journal, 2001
    Co-Authors: Shawn B. Bratton, Emad S Alnemri, Srinivasa M Srinivasula, Xiao-ming Sun, Gail Walker, Michael Butterworth, Gerald M. Cohen
    Abstract:

    During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed Caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in Caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed Caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex.

  • caspase activation involves the formation of the aposome a large 700 kda caspase activating complex
    Journal of Biological Chemistry, 1999
    Co-Authors: Kelvin Cain, David Brown, Claudia Langlais, Gerald M. Cohen
    Abstract:

    In mammals, apoptotic protease-activating factor 1 (Apaf-1), cytochrome c, and dATP activate Caspase-9, which initiates the postmitochondrial-mediated caspase cascade by proteolytic cleavage/activation of effector caspases to form active approximately 60-kDa heterotetramers. We now demonstrate that activation of caspases either in apoptotic cells or following dATP activation of cell lysates results in the formation of two large but different sized protein complexes, the "aposome" and the "microaposome". Surprisingly, most of the DEVDase activity in the lysate was present in the aposome and microaposome complexes with only small amounts of active caspase-3 present as its free approximately 60-kDa heterotetramer. The larger aposome complex (M(r) = approximately 700,000) contained Apaf-1 and processed Caspase-9, -3, and -7. The smaller microaposome complex (M(r) = approximately 200,000-300,000) contained active caspase-3 and -7 but little if any Apaf-1 or active Caspase-9. Lysates isolated from control THP.1 cells, prior to caspase activation, showed striking differences in the distribution of key apoptotic proteins. Apaf-1 and procaspase-7 may be functionally complexed as they eluted as an approximately 200-300-kDa complex, which did not have caspase cleavage (DEVDase) activity. Procaspase-3 and -9 were present as separate and smaller 60-90-kDa (dimer) complexes. During caspase activation, Apaf-1, Caspase-9, and the effector caspases redistributed and formed the aposome. This resulted in the processing of the effector caspases, which were then released, possibly bound to other proteins, to form the microaposome complex.

Emad S Alnemri - One of the best experts on this subject based on the ideXlab platform.

  • Mechanism of XIAP-Mediated Inhibition of Caspase-9
    Molecular cell, 2003
    Co-Authors: Eric N. Shiozaki, Emad S Alnemri, Srinivasa M Srinivasula, Daniel J. Rigotti, Robert Fairman, Jijie Chai, Stefan J. Riedl, Yigong Shi
    Abstract:

    The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator Caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of Caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 A resolution. The structure reveals that the BIR3 domain forms a heterodimer with a Caspase-9 monomer. Strikingly, the surface of Caspase-9 that interacts with BIR3 also mediates its homodimerization. We demonstrate that monomeric Caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters Caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases.

  • recruitment activation and retention of caspases 9 and 3 by apaf 1 apoptosome and associated xiap complexes
    The EMBO Journal, 2001
    Co-Authors: Shawn B. Bratton, Emad S Alnemri, Srinivasa M Srinivasula, Xiao-ming Sun, Gail Walker, Michael Butterworth, Gerald M. Cohen
    Abstract:

    During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed Caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in Caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed Caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex.

  • molecular determinants of the caspase promoting activity of smac diablo and its role in the death receptor pathway
    Journal of Biological Chemistry, 2000
    Co-Authors: Srinivasa M Srinivasula, Pinaki Datta, Xuejun Fan, Teresa Fernandesalnemri, Ziwei Huang, Emad S Alnemri
    Abstract:

    Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes cytochrome c-dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs). We provide evidence that Smac/DIABLO functions at the levels of both the Apaf-1-Caspase-9 apoptosome and effector caspases. The N terminus of Smac/DIABLO is absolutely required for its ability to interact with the baculovirus IAP repeat (BIR3) of XIAP and to promote cytochrome c-dependent caspase activation. However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the effector caspases in the death receptor pathway of Type II cells.

  • ordering the cytochrome c initiated caspase cascade hierarchical activation of caspases 2 3 6 7 8 and 10 in a caspase 9 dependent manner
    Journal of Cell Biology, 1999
    Co-Authors: Elizabeth A Slee, Mary T Harte, Ruth M Kluck, Beni B Wolf, Carlos A Casiano, Donald D Newmeyer, John C Reed, Donald W Nicholson, Honggang Wang, Emad S Alnemri
    Abstract:

    Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of Caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that Caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of Caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that Caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of Caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving Caspase-9.

  • iaps block apoptotic events induced by caspase 8 and cytochrome c by direct inhibition of distinct caspases
    The EMBO Journal, 1998
    Co-Authors: Quinn Deveraux, Henning R Stennicke, Guy S Salvesen, Emad S Alnemri, Srinivasa M Srinivasula, Natalie Roy, Todd Van Arsdale, Qiao Zhou, John C Reed
    Abstract:

    Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-Caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-Caspase-9 as a new target for IAP-mediated inhibition of apoptosis.

John C Reed - One of the best experts on this subject based on the ideXlab platform.

  • cellular biochemical and genetic analysis of mechanism of small molecule iap inhibitors
    Journal of Biological Chemistry, 2004
    Co-Authors: Zhiliang Wang, Kate Welsh, Michael Cuddy, Temesgen Samuel, Aaron D Schimmer, Farid Hanaii, Richard A Houghten, Clemencia Pinilla, John C Reed
    Abstract:

    Abstract XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25–35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease Caspase-9 from XIAP in vitro and restored caspase-3 but not Caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-XL and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis doseresponse of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.

  • apaf 1 cytochrome c independent and smac dependent induction of apoptosis in multiple myeloma mm cells
    Journal of Biological Chemistry, 2001
    Co-Authors: Dharminder Chauhan, John C Reed, Teru Hideshima, Steven D Rosen, Surender Kharbanda, Kenneth C Anderson
    Abstract:

    Abstract Smac, a second mitochondria-derived activator of caspases, promotes caspase activation in the cytochrome c(cyto-c)/Apaf-1/Caspase-9 pathway. Here, we show that treatment of multiple myeloma (MM) cells with dexamethasone (Dex) triggers the release of Smac from mitochondria to cytosol and activates Caspase-9 without concurrent release of cyto-c and Apaf-1 oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein) and thereby, at least in part, eliminates its inhibitory effect on Caspase-9. Interleukin-6, a growth factor for MM, blocks Dex-induced apoptosis and prevents release of Smac. Taken together, these findings demonstrate that Smac plays a functional role in mediating Dex-induced Caspase-9 activation and apoptosis in MM cells.

  • ordering the cytochrome c initiated caspase cascade hierarchical activation of caspases 2 3 6 7 8 and 10 in a caspase 9 dependent manner
    Journal of Cell Biology, 1999
    Co-Authors: Elizabeth A Slee, Mary T Harte, Ruth M Kluck, Beni B Wolf, Carlos A Casiano, Donald D Newmeyer, John C Reed, Donald W Nicholson, Honggang Wang, Emad S Alnemri
    Abstract:

    Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of Caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that Caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of Caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that Caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of Caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving Caspase-9.

  • iaps block apoptotic events induced by caspase 8 and cytochrome c by direct inhibition of distinct caspases
    The EMBO Journal, 1998
    Co-Authors: Quinn Deveraux, Henning R Stennicke, Guy S Salvesen, Emad S Alnemri, Srinivasa M Srinivasula, Natalie Roy, Todd Van Arsdale, Qiao Zhou, John C Reed
    Abstract:

    Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-Caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-Caspase-9 as a new target for IAP-mediated inhibition of apoptosis.

  • a single bir domain of xiap sufficient for inhibiting caspases
    Journal of Biological Chemistry, 1998
    Co-Authors: Ryosuke Takahashi, Guy S Salvesen, Quinn Deveraux, Ingo Tamm, Kate Welsh, Nuria Assamunt, John C Reed
    Abstract:

    Abstract 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.

Howard O. Fearnhead - One of the best experts on this subject based on the ideXlab platform.

  • apoptosome dependent myotube formation involves activation of caspase 3 in differentiating myoblasts
    Cell Death and Disease, 2020
    Co-Authors: Mahshid H Dehkordi, Amin Tashakor, Enda Oconnell, Howard O. Fearnhead
    Abstract:

    : Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the apoptosome primarily control cell fusion.

  • A non-apoptotic role for Caspase-9 in muscle differentiation.
    Journal of Cell Science, 2008
    Co-Authors: Thomas V.a. Murray, Jill Mcmahon, Breege A Howley, Alanna Stanley, Thomas Ritter, Andrea Mohr, Ralf M. Zwacka, Howard O. Fearnhead
    Abstract:

    Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that Caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of Caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits Caspase-9 activation, had the same effect on muscle differentiation as knockdown of Caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of Caspase-9 activation during muscle differentiation.

  • Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation
    Apoptosis, 2007
    Co-Authors: Afshin Samali, Jill Mcmahon, Martin O’mahoney, Janice Reeve, Susan Logue, Eva Szegezdi, Howard O. Fearnhead
    Abstract:

    By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of Caspase-9 activation in extracts from heart cells. Unlike extracts from other cell types, heart extracts were found to activate caspases poorly. This could be explained by the low levels of Apaf-1 in heart cells. However, subsequent testing showed that heart extracts contained an inhibitor of caspase activation that could block caspase activation in extracts from different cell types. Subsequent purification of the inhibitor of caspase activation from these extracts identified ATP. Caspase-9 is activated by recruitment into a multi-protein complex, the apoptosome, which then activates downstream caspases that kill the cell. Importantly, size exclusion chromatography showed that ATP inhibits apoptosome formation at physiologically relevant concentrations. Together these data support the hypothesis that intracellular ATP concentration is a critical factor in determining whether an apoptotic stimulus can induce apoptosome formation. Thus, the well described fall in intracellular ATP apoptosis is not an epiphenomenon but may be a pro-apoptotic event contributing to cell death.

Srinivasa M Srinivasula - One of the best experts on this subject based on the ideXlab platform.

  • Mechanism of XIAP-Mediated Inhibition of Caspase-9
    Molecular cell, 2003
    Co-Authors: Eric N. Shiozaki, Emad S Alnemri, Srinivasa M Srinivasula, Daniel J. Rigotti, Robert Fairman, Jijie Chai, Stefan J. Riedl, Yigong Shi
    Abstract:

    The inhibitor of apoptosis (IAP) proteins potently inhibit the catalytic activity of caspases. While profound insight into the inhibition of the effector caspases has been gained in recent years, the mechanism of how the initiator Caspase-9 is regulated by IAPs remains enigmatic. This paper reports the crystal structure of Caspase-9 in an inhibitory complex with the third baculoviral IAP repeat (BIR3) of XIAP at 2.4 A resolution. The structure reveals that the BIR3 domain forms a heterodimer with a Caspase-9 monomer. Strikingly, the surface of Caspase-9 that interacts with BIR3 also mediates its homodimerization. We demonstrate that monomeric Caspase-9 is catalytically inactive due to the absence of a supporting sequence element that could be provided by homodimerization. Thus, XIAP sequesters Caspase-9 in a monomeric state, which serves to prevent catalytic activity. These studies, in conjunction with other observations, define a unified mechanism for the activation of all caspases.

  • recruitment activation and retention of caspases 9 and 3 by apaf 1 apoptosome and associated xiap complexes
    The EMBO Journal, 2001
    Co-Authors: Shawn B. Bratton, Emad S Alnemri, Srinivasa M Srinivasula, Xiao-ming Sun, Gail Walker, Michael Butterworth, Gerald M. Cohen
    Abstract:

    During apoptosis, release of cytochrome c initiates dATP-dependent oligomerization of Apaf-1 and formation of the apoptosome. In a cell-free system, we have addressed the order in which apical and effector caspases, caspases-9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed Caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 via an interaction between the active site cysteine (C287) in Caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhibitor-of-apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c/dATP-activated lysates. XIAP associates with oligomerized Apaf-1 and/or processed Caspase-9 and influences the activation of caspase-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase-3 within the apoptosome and by preventing release of active caspase-3 from the complex.

  • molecular determinants of the caspase promoting activity of smac diablo and its role in the death receptor pathway
    Journal of Biological Chemistry, 2000
    Co-Authors: Srinivasa M Srinivasula, Pinaki Datta, Xuejun Fan, Teresa Fernandesalnemri, Ziwei Huang, Emad S Alnemri
    Abstract:

    Smac/DIABLO is a mitochondrial protein that is released along with cytochrome c during apoptosis and promotes cytochrome c-dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs). We provide evidence that Smac/DIABLO functions at the levels of both the Apaf-1-Caspase-9 apoptosome and effector caspases. The N terminus of Smac/DIABLO is absolutely required for its ability to interact with the baculovirus IAP repeat (BIR3) of XIAP and to promote cytochrome c-dependent caspase activation. However, it is less critical for its ability to interact with BIR1/BIR2 of XIAP and to promote the activity of the effector caspases. Consistent with the ability of Smac/DIABLO to function at the level of the effector caspases, expression of a cytosolic Smac/DIABLO in Type II cells allowed TRAIL to bypass Bcl-xL inhibition of death receptor-induced apoptosis. Combined, these data suggest that Smac/DIABLO plays a critical role in neutralizing IAP inhibition of the effector caspases in the death receptor pathway of Type II cells.

  • iaps block apoptotic events induced by caspase 8 and cytochrome c by direct inhibition of distinct caspases
    The EMBO Journal, 1998
    Co-Authors: Quinn Deveraux, Henning R Stennicke, Guy S Salvesen, Emad S Alnemri, Srinivasa M Srinivasula, Natalie Roy, Todd Van Arsdale, Qiao Zhou, John C Reed
    Abstract:

    Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-Caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-Caspase-9 as a new target for IAP-mediated inhibition of apoptosis.