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Jean-bernard Denault - One of the best experts on this subject based on the ideXlab platform.

  • Caspase-7 uses RNA to enhance proteolysis of poly(ADP-ribose) polymerase 1 and other RNA-binding proteins
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Alexandre Desroches, Jean-bernard Denault
    Abstract:

    To achieve swift cell demise during apoptosis, caspases cleave essential proteins for cell survival and removal. In addition to the binding of preferred amino acid sequences to its substrate-binding pocket, Caspase-7 also uses exosites to select specific substrates. 4 lysine residues (K38KKK) located in the N-terminal domain of Caspase-7 form such an exosite and promote the rapid proteolysis of the poly(ADP-ribose) polymerase 1 (PARP-1), but the mechanism of recognition remains mostly unknown. In this study, we show that the overall positive charge of the exosite is the critical feature of this evolutionarily conserved binding site. Additionally, interaction with the Caspase-7 exosite involves both the Zn3 and BRCT domains of PARP-1 and is mediated by RNA. Indeed, PARP-1 proteolysis efficacy is sensitive to RNase A and promoted by added RNA. Moreover, using affinity chromatography and gel shift assays, we demonstrate that Caspase-7, but not caspase-3 or a Caspase-7 with a mutated exosite, binds nucleic acids. Finally, we show that Caspase-7 prefers RNA-binding proteins (RNA-BPs) as substrates compared to caspase-3 and that RNA enhances proteolysis by Caspase-7 of many of these RNA-BPs. Thus, we have uncovered an unusual way by which Caspase-7 selects and cleaves specific substrates.

  • Characterization of Hsp90 Co-Chaperone p23 Cleavage by Caspase-7 Uncovers a Peptidase–Substrate Interaction Involving Intrinsically Disordered Regions
    Biochemistry, 2017
    Co-Authors: Cyrielle Martini, Mikaël Bédard, Pierre Lavigne, Jean-bernard Denault
    Abstract:

    Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, Caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure-activity relationship analyses using mutagenesis of the Caspase-7 NTD and kinetics show that residues 36-45 of Caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the Caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD142↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of Caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in Caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase-substrate pair to understand the modalities of substrate recognition by caspases.

  • Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Dave Boucher, Véronique Blais, Jean-bernard Denault
    Abstract:

    During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, Caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K(38)KKK) within the N-terminal domain of Caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7's N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by ∼30-fold. Cellular expression of Caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of Caspase-7 in promoting key substrate proteolysis.

  • Molecular determinants involved in activation of caspase 7.
    Bioscience reports, 2011
    Co-Authors: Dave Boucher, Véronique Blais, Marcin Drag, Jean-bernard Denault
    Abstract:

    During apoptosis, initiator caspases (8, 9 and 10) activate downstream executioner caspases (3, 6 and 7) by cleaving the IDC (interdomain connector) at two sites. Here, we demonstrate that both activation sites, site 1 and site 2, of caspase 7 are suboptimal for activation by initiator caspases 8 and 9 in cellulo, and in vitro using recombinant proteins and activation kinetics. Indeed, when both sites are replaced with the preferred motifs recognized by either caspase 8 or 9, we found an up to 36-fold improvement in activation. Moreover, cleavage at site 1 is preferred to site 2 because of its location within the IDC, since swapping sites does not lead to a more efficient activation. We also demonstrate the important role of Ile195 of site 1 involved in maintaining a network of contacts that preserves the proper conformation of the active enzyme. Finally, we show that the length of the IDC plays a crucial role in maintaining the necessity of proteolysis for activation. In fact, although we were unable to generate a caspase 7 that does not require proteolysis for activity, shortening the IDC of the initiator caspase 8 by four residues was sufficient to confer a requirement for proteolysis, a key feature of executioner caspases. Altogether, the results demonstrate the critical role of the primary structure of caspase 7's IDC for its activation and proteolytic activity.

  • Engineered Hybrid Dimers: Tracking the Activation Pathway of Caspase-7
    Molecular cell, 2006
    Co-Authors: Jean-bernard Denault, Matthew Bogyo, Miklós Békés, Fiona L. Scott, Kelly B. Sexton, Guy S Salvesen
    Abstract:

    Caspase-7 is an obligate dimer of catalytic domains, with generation of activity requiring limited proteolysis within a region that separates the large and small chains of each domain. Using hybrid dimers we distinguish the relative contribution of each domain to catalysis by the whole molecule. We demonstrate that the zymogen arises from direct dimerization and not domain swapping. In contrast to previous conclusions, we show that only one of the catalytic domains must be proteolyzed to enable activation. The processed domain of this singly cleaved zymogen has the same catalytic activity as a domain of fully active Caspase-7. A transient intermediate of singly cleaved dimeric Caspase-7 can be found in a cell-free model of apoptosis induction. However, we see no evidence for an analogous intermediate of the related executioner caspase-3. Our study demonstrates the efficiency by which the executioner caspases are activated in vivo.

Jurg Tschopp - One of the best experts on this subject based on the ideXlab platform.

  • Inflammasome-Activated Caspase 7 Cleaves PARP1 to Enhance the Expression of a Subset of NF-κB Target Genes
    Molecular cell, 2012
    Co-Authors: Suheda Erener, Jurg Tschopp, Virginie Pétrilli, Ingrid Kassner, Roberta Minotti, Rosa Castillo, Raffaella Santoro, Paul O. Hassa, Michael O. Hottiger
    Abstract:

    Caspase 1 is part of the inflammasome, which is assembled upon pathogen recognition, while caspases 3 and/or 7 are mediators of apoptotic and nonapoptotic functions. PARP1 cleavage is a hallmark of apoptosis yet not essential, suggesting it has another physiological role. Here we show that after LPS stimulation, caspase 7 is activated by caspase 1, translocates to the nucleus, and cleaves PARP1 at the promoters of a subset of NF-κB target genes negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes. These findings propose an apoptosis-independent regulatory role for caspase 7-mediated PARP1 cleavage in proinflammatory gene expression and provide insight into inflammasome signaling.

  • inflammatory caspases linking an intracellular innate immune system to autoinflammatory diseases
    Cell, 2004
    Co-Authors: Fabio Martinon, Jurg Tschopp
    Abstract:

    Abstract Caspases not only play an essential role during apoptotic cell death, but a subfamily of them—the inflammatory caspases—are associated with immune responses to microbial pathogens. Activation of inflammatory caspases, such as caspase-1 and caspase-5, occurs upon assembly of an intracellular complex, designated the inflammasome. This results in the cleavage and activation of the proinflammatory cytokines IL-1β and IL-18. Mutations in one of the scaffold proteins of the inflammasome, NALP3/Cryopyrin, are associated with autoinflammatory disorders underscoring the importance of regulating inflammatory caspase activation.

  • The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.
    Molecular Cell, 2002
    Co-Authors: Fabio Martinon, Kimberly Burns, Jurg Tschopp
    Abstract:

    Generation of Interleukin (IL)-1beta via cleavage of its proform requires the activity of caspase-1 (and caspase-11 in mice), but the mechanism involved in the activation of the proinflammatory caspases remains elusive. Here we report the identification of a caspase-activating complex that we call the inflammasome. The inflammasome comprises caspase-1, caspase-5, Pycard/Asc, and NALP1, a Pyrin domain-containing protein sharing structural homology with NODs. Using a cell-free system, we show that proinflammatory caspase activation and proIL-1beta processing is lost upon prior immunodepletion of Pycard. Moreover, expression of a dominant-negative form of Pycard in differentiated THP-1 cells blocks proIL-1beta maturation and activation of inflammatory caspases induced by LPS in vivo. Thus, the inflammasome constitutes an important arm of the innate immunity.

  • the inflammasome a molecular platform triggering activation of inflammatory caspases and processing of proil beta
    Molecular Cell, 2002
    Co-Authors: Fabio Martinon, Kimberly Burns, Jurg Tschopp
    Abstract:

    Abstract Generation of Interleukin (IL)-1β via cleavage of its proform requires the activity of caspase-1 (and caspase-11 in mice), but the mechanism involved in the activation of the proinflammatory caspases remains elusive. Here we report the identification of a caspase-activating complex that we call the inflammasome. The inflammasome comprises caspase-1, caspase-5, Pycard/Asc, and NALP1, a Pyrin domain-containing protein sharing structural homology with NODs. Using a cell-free system, we show that proinflammatory caspase activation and proIL-1β processing is lost upon prior immunodepletion of Pycard. Moreover, expression of a dominant-negative form of Pycard in differentiated THP-1 cells blocks proIL-1β maturation and activation of inflammatory caspases induced by LPS in vivo. Thus, the inflammasome constitutes an important arm of the innate immunity.

Katsuhiko Mikoshiba - One of the best experts on this subject based on the ideXlab platform.

  • Inositol 1,4,5-Trisphosphate Receptor Type 1 Is a Substrate for Caspase-3 and Is Cleaved during Apoptosis in a Caspase-3-dependent Manner
    The Journal of biological chemistry, 1999
    Co-Authors: Junji Hirota, Teiichi Furuichi, Katsuhiko Mikoshiba
    Abstract:

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.

  • inositol 1 4 5 trisphosphate receptor type 1 is a substrate for caspase 3 and is cleaved during apoptosis in a caspase 3 dependent manner
    Journal of Biological Chemistry, 1999
    Co-Authors: Junji Hirota, Teiichi Furuichi, Katsuhiko Mikoshiba
    Abstract:

    Next Section Abstract The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, modulates intracellular Ca2+signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP3R type 1 (IP3R1) was found to be cleaved. IP3R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH2DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP3R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP3R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888DEVD*1892R (mouse IP3R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP3R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-α- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP3R1 degradation upon apoptotic stimuli. Therefore IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP3R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP3R1 was digested, indicating that caspase-3 inactivates IP3R1 channel functions.

Guy G Poirier - One of the best experts on this subject based on the ideXlab platform.

  • Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of Caspase-7.
    The Journal of biological chemistry, 1999
    Co-Authors: Marc Germain, Guy S Salvesen, El Bachir Affar, Damien D’amours, Vishva M. Dixit, Guy G Poirier
    Abstract:

    The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by Caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, Caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, Caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, Caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is Caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.

  • involvement of caspase dependent activation of cytosolic phospholipase a2 in tumor necrosis factor induced apoptosis
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: D Wissing, Helle Mouritzen, Mikala Egeblad, Guy G Poirier, Marja Jäättelä
    Abstract:

    Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1β-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A2 (cPLA2) and that activated cPLA2 contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32/caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA2 and apoptosis. TNF-induced activation of cPLA2 was accompanied by a cleavage of the 100-kDa cPLA2 to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32/caspase-3. Interestingly, specific inhibition of cPLA2 enzyme activity by arachidonyl trifluoromethylketone (AACOCF3) partially inhibited TNF-induced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA2 during apoptosis and identify cPLA2 as a mediator of TNF-induced cell death acting downstream of caspases.

Junji Hirota - One of the best experts on this subject based on the ideXlab platform.

  • Inositol 1,4,5-Trisphosphate Receptor Type 1 Is a Substrate for Caspase-3 and Is Cleaved during Apoptosis in a Caspase-3-dependent Manner
    The Journal of biological chemistry, 1999
    Co-Authors: Junji Hirota, Teiichi Furuichi, Katsuhiko Mikoshiba
    Abstract:

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.

  • inositol 1 4 5 trisphosphate receptor type 1 is a substrate for caspase 3 and is cleaved during apoptosis in a caspase 3 dependent manner
    Journal of Biological Chemistry, 1999
    Co-Authors: Junji Hirota, Teiichi Furuichi, Katsuhiko Mikoshiba
    Abstract:

    Next Section Abstract The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, modulates intracellular Ca2+signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP3R type 1 (IP3R1) was found to be cleaved. IP3R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH2DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP3R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP3R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888DEVD*1892R (mouse IP3R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP3R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-α- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP3R1 degradation upon apoptotic stimuli. Therefore IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP3R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP3R1 was digested, indicating that caspase-3 inactivates IP3R1 channel functions.