Publications by authors named "Dave Boucher"

18 Publications

  • Page 1 of 1

Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria.

Nat Commun 2020 06 24;11(1):3276. Epub 2020 Jun 24.

Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066, Epalinges, Switzerland.

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling.
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http://dx.doi.org/10.1038/s41467-020-16889-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314798PMC
June 2020

Caspase-1 cleaves Bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD.

Life Sci Alliance 2020 06 28;3(6). Epub 2020 Apr 28.

Department of Biochemistry, University of Lausanne, Epalinges, Switzerland

Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). deficiency, however, only delays cell lysis, indicating that caspase-1 controls alternative cell death pathways. Here, we show that in the absence of GSDMD, caspase-1 activates apoptotic initiator and executioner caspases and triggers a rapid progression into secondary necrosis. GSDMD-independent cell death required direct caspase-1-driven truncation of Bid and generation of caspase-3 p19/p12 by either caspase-8 or caspase-9. tBid-induced mitochondrial outer membrane permeabilization was also required to drive SMAC release and relieve inhibitor of apoptosis protein inhibition of caspase-3, thereby allowing caspase-3 auto-processing to the fully active p17/p12 form. Our data reveal that cell lysis in inflammasome-activated -deficient cells is caused by a synergistic effect of rapid caspase-1-driven activation of initiator caspases-8/-9 and Bid cleavage, resulting in an unusually fast activation of caspase-3 and immediate transition into secondary necrosis. This pathway might be advantageous for the host in counteracting pathogen-induced inhibition of GSDMD but also has implications for the use of GSDMD inhibitors in immune therapies for caspase-1-dependent inflammatory disease.
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http://dx.doi.org/10.26508/lsa.202000735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190276PMC
June 2020

Isolation of Neutrophil Nuclei for Use in NETosis Assays.

Authors:
Dave Boucher

Bio Protoc 2019 Sep 5;9(17):e3357. Epub 2019 Sep 5.

Institut de Biochimie, Université de Lausanne, Epalinges, Switzerland.

Neutrophils are critical immune cells that protect our body against invading pathogens. They generate antibacterial DNA structures called neutrophil extracellular traps (NET). Recently we identified a new mechanism that enables NET formation. We observed that following recognition of lipopolysaccharides, inflammatory caspases cleave Gasdermin D and enable NET generation ( Chen , 2018 ). This protocol describes how we purify neutrophil nuclei to visualize NET formation by live microscopy. After neutrophil purification from murine bone marrow, neutrophils are lysed in a hypotonic buffer using a nitrogen cavitation device to prevent lysis of neutrophil granules and subsequent contamination by granules proteases. Lysed neutrophils are then centrifuged, and nuclei are counted. The protocol described here is straightforward and enables the study of early changes happening in the nuclei of neutrophils undergoing NETosis with limited contamination by granule proteases.
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http://dx.doi.org/10.21769/BioProtoc.3357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854059PMC
September 2019

MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.

Nat Chem Biol 2019 06 13;15(6):556-559. Epub 2019 May 13.

Institute for Molecular Bioscience and IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Queensland, Australia.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.
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http://dx.doi.org/10.1038/s41589-019-0277-7DOI Listing
June 2019

Variation in hemolysin A expression between uropathogenic isolates determines NLRP3-dependent -independent macrophage cell death and host colonization.

FASEB J 2019 06 14;33(6):7437-7450. Epub 2019 Mar 14.

Centre for Inflammation and Disease Research, Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland, Australia.

Uropathogenic (UPEC) is the major cause of urinary tract infections (UTIs). The multidrug-resistant sequence type 131 (ST131) clone is a serious threat to human health, yet its effects on immune responses are not well understood. Here we screened a panel of ST131 isolates, finding that only strains expressing the toxin hemolysin A (HlyA) killed primary human macrophages and triggered maturation of the inflammasome-dependent cytokine IL-1β. Using a representative strain, the requirement for the gene in these responses was confirmed. We also observed considerable heterogeneity in levels of cell death initiated by different HlyA ST131 isolates, and this correlated with secreted HlyA levels. Investigation into the biological significance of this variation revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partly dependent on the nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, with this response being associated with a host-protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonization was significantly increased. Thus, variation in HlyA expression in UPEC affects mechanisms by which macrophages die, as well as host susceptibility resistance to colonization.-Murthy, A. M. V., Sullivan, M. J., Nhu, N. T. K., Lo, A. W., Phan, M.-D., Peters, K. M., Boucher, D., Schroder, K., Beatson, S. A., Ulett, G. C., Schembri, M. A., Sweet, M. J. Variation in hemolysin A expression between uropathogenic isolates determines NLRP3-dependent -independent macrophage cell death and host colonization.
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http://dx.doi.org/10.1096/fj.201802100RDOI Listing
June 2019

Divide to conquer: NLRP3 is activated on dispersed trans-Golgi network.

Cell Res 2019 03;29(3):181-182

Department of Biochemistry, University of Lausanne, CH-1066, Epalinges, Switzerland.

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http://dx.doi.org/10.1038/s41422-018-0138-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460435PMC
March 2019

Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome.

Life Sci Alliance 2018 Dec 6;1(6):e201800237. Epub 2018 Dec 6.

Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Australia.

Caspase-11 is a cytosolic sensor and protease that drives innate immune responses to the bacterial cell wall component, LPS. Caspase-11 provides defence against cytosolic Gram-negative bacteria; however, excessive caspase-11 responses contribute to murine endotoxic shock. Upon sensing LPS, caspase-11 assembles a higher order structure called the non-canonical inflammasome that enables the activation of caspase-11 protease function, leading to gasdermin D cleavage and cell death. The mechanism by which caspase-11 acquires protease function is, however, poorly defined. Here, we show that caspase-11 dimerization is necessary and sufficient for eliciting basal caspase-11 protease function, such as the ability to auto-cleave. We further show that during non-canonical inflammasome signalling, caspase-11 self-cleaves at site (D285) within the linker connecting the large and small enzymatic subunits. Self-cleavage at the D285 site is required to generate the fully active caspase-11 protease (proposed here to be p32/p10) that mediates gasdermin D cleavage, macrophage death, and NLRP3-dependent IL-1β production. This study provides a detailed molecular mechanism by which LPS induces caspase-11-driven inflammation and cell death to provide host defence against cytosolic bacterial infection.
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http://dx.doi.org/10.26508/lsa.201800237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284101PMC
December 2018

The Salmonella pathogenicity island-2 subverts human NLRP3 and NLRC4 inflammasome responses.

J Leukoc Biol 2019 02 4;105(2):401-410. Epub 2018 Oct 4.

Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Queensland, Australia.

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella-derived molecules is extensively studied in murine myeloid cells. Salmonella-induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island-2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium-induced inflammasome responses from primary human macrophages, resulting in strong IL-1β production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD-domain containing protein 4 (NLRC4) and NLR family PYRIN-domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL-1β and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1-induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species-specific immune evasion mechanism.
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http://dx.doi.org/10.1002/JLB.MA0318-112RRDOI Listing
February 2019

Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps.

Sci Immunol 2018 08;3(26)

Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, University of Queensland, St Lucia Brisbane 4072, Queensland, Australia.

Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis) provides host defense against extracellular pathogens, whereas macrophage death by pyroptosis enables defense against intracellular pathogens. We report the unexpected discovery that gasdermin D (GSDMD) connects these cell death modalities. We show that neutrophil exposure to cytosolic lipopolysaccharide or cytosolic Gram-negative bacteria ( Δ and ) activates noncanonical (caspase-4/11) inflammasome signaling and triggers GSDMD-dependent neutrophil death. GSDMD-dependent death induces neutrophils to extrude antimicrobial NETs. Caspase-11 and GSDMD are required for neutrophil plasma membrane rupture during the final stage of NET extrusion. Unexpectedly, caspase-11 and GSDMD are also required for early features of NETosis, including nuclear delobulation and DNA expansion; this is mediated by the coordinate actions of caspase-11 and GSDMD in mediating nuclear membrane permeabilization and histone degradation. In vivo application of deoxyribonuclease I to dissolve NETs during murine Δ challenge increases bacterial burden in wild-type but not in and mice. Our studies reveal that neutrophils use an inflammasome- and GSDMD-dependent mechanism to activate NETosis as a defense response against cytosolic bacteria.
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http://dx.doi.org/10.1126/sciimmunol.aar6676DOI Listing
August 2018

Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion.

Cell Rep 2018 08;24(6):1425-1433

Institute for Molecular Bioscience (IMB), and IMB Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia. Electronic address:

IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion.
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http://dx.doi.org/10.1016/j.celrep.2018.07.027DOI Listing
August 2018

Cutting Edge: Blockade of Inhibitor of Apoptosis Proteins Sensitizes Neutrophils to TNF- but Not Lipopolysaccharide-Mediated Cell Death and IL-1β Secretion.

J Immunol 2018 05 16;200(10):3341-3346. Epub 2018 Apr 16.

Centre for Inflammation and Disease Research, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia;

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death-inducing complex, termed the ripoptosome, which can trigger caspase-8-dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1β maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1β maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling.
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http://dx.doi.org/10.4049/jimmunol.1701620DOI Listing
May 2018

Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity.

J Exp Med 2018 03 6;215(3):827-840. Epub 2018 Feb 6.

Centre for Inflammation and Disease Research, Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia

Host-protective caspase-1 activity must be tightly regulated to prevent pathology, but mechanisms controlling the duration of cellular caspase-1 activity are unknown. Caspase-1 is activated on inflammasomes, signaling platforms that facilitate caspase-1 dimerization and autoprocessing. Previous studies with recombinant protein identified a caspase-1 tetramer composed of two p20 and two p10 subunits (p20/p10) as an active species. In this study, we report that in the cell, the dominant species of active caspase-1 dimers elicited by inflammasomes are in fact full-length p46 and a transient species, p33/p10. Further p33/p10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20/p10 from the inflammasome, whereupon the tetramer becomes unstable in cells and protease activity is terminated. The inflammasome-caspase-1 complex thus functions as a holoenzyme that directs the location of caspase-1 activity but also incorporates an intrinsic self-limiting mechanism that ensures timely caspase-1 deactivation. This intrinsic mechanism of inflammasome signal shutdown offers a molecular basis for the transient nature, and coordinated timing, of inflammasome-dependent inflammatory responses.
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http://dx.doi.org/10.1084/jem.20172222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839769PMC
March 2018

Quantifying Caspase-1 Activity in Murine Macrophages.

Methods Mol Biol 2018 ;1725:163-176

Institute for Molecular Bioscience and IMB Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, Australia.

The caspase-1 protease is a core component of multiprotein inflammasome complexes, which play a critical role in regulating the secretion of mature, bioactive pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The activity of caspase-1 is often measured indirectly, by monitoring cleavage of cellular caspase-1 substrates, processing of caspase-1 itself, or by quantifying cell death. Here we describe methods for eliciting caspase-1 activity in murine macrophages, via activation of the NLRP3, NAIP/NLRC4 or AIM2 inflammasomes. We then describe a simple fluorogenic assay for directly quantifying cellular caspase-1 activity.
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http://dx.doi.org/10.1007/978-1-4939-7568-6_14DOI Listing
January 2019

Salmonella-induced inflammasome activation in humans.

Mol Immunol 2017 06 11;86:38-43. Epub 2016 Dec 11.

Institute for Molecular Bioscience, and Centre for Inflammation and Disease Research, The University of Queensland, St. Lucia, 4072, Australia. Electronic address:

Inflammasomes are macromolecular complexes that assemble upon recognition of pathogen- or danger-associated molecular patterns. Inflammasome assembly is nucleated by the oligomerisation of specific, activated pattern recognition receptors within the cytosol. Inflammasomes function as platforms for the activation of the caspase-1 protease, which in turn triggers the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18, and initiates pyroptosis, a highly inflammatory form of lytic cell death. Recently, additional inflammatory caspases (murine caspase-11, and human caspase-4/5) were also reported to be activated upon a pyroptosis-inducing 'non-canonical inflammasome' by direct recognition of lipopolysaccharide (LPS), a pathogen-associated molecular pattern. Here we review and discuss recent advances in our understanding of inflammasome-mediated host defence against Salmonella particularly in human cells, and their implications for cellular survival and cytokine secretion.
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http://dx.doi.org/10.1016/j.molimm.2016.11.009DOI Listing
June 2017

NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5.

Eur J Immunol 2015 Oct 24;45(10):2918-26. Epub 2015 Aug 24.

Inflammation Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

Humans encode two inflammatory caspases that detect cytoplasmic LPS, caspase-4 and caspase-5. When activated, these trigger pyroptotic cell death and caspase-1-dependent IL-1β production; however the mechanism underlying this process is not yet confirmed. We now show that a specific NLRP3 inhibitor, MCC950, prevents caspase-4/5-dependent IL-1β production elicited by transfected LPS. Given that both caspase-4 and caspase-5 can detect cytoplasmic LPS, it is possible that these proteins exhibit some degree of redundancy. Therefore, we generated human monocytic cell lines in which caspase-4 and caspase-5 were genetically deleted either individually or together. We found that the deletion of caspase-4 suppressed cell death and IL-1β production following transfection of LPS into the cytoplasm, or in response to infection with Salmonella typhimurium. Although deletion of caspase-5 did not confer protection against transfected LPS, cell death and IL-1β production were reduced after infection with Salmonella. Furthermore, double deletion of caspase-4 and caspase-5 had a synergistic effect in the context of Salmonella infection. Our results identify the NLRP3 inflammasome as the specific platform for IL-1β maturation, downstream of cytoplasmic LPS detection by caspase-4/5. We also show that both caspase-4 and caspase-5 are functionally important for appropriate responses to intracellular Gram-negative bacteria.
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http://dx.doi.org/10.1002/eji.201545655DOI Listing
October 2015

General in vitro caspase assay procedures.

Methods Mol Biol 2014 ;1133:3-39

Institute of Molecular Bioscience, University of Queensland, St. Lucia, QLD, Australia.

One of the most valuable tools that have been developed for the study of apoptosis is the availability of recombinant active caspases. The determination of caspase substrate preference, the design of sensitive substrates and potent inhibitors, the resolution of caspase structures, the elucidation of their activation mechanisms, and the identification of their substrates were made possible by the availability of sufficient amounts of enzymatically pure caspases. The current chapter describes at length the expression, purification, and basic enzymatic characterization of apoptotic caspases.
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http://dx.doi.org/10.1007/978-1-4939-0357-3_1DOI Listing
October 2014

Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

Proc Natl Acad Sci U S A 2012 Apr 26;109(15):5669-74. Epub 2012 Mar 26.

Department of Pharmacology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4.

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.
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http://dx.doi.org/10.1073/pnas.1200934109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326497PMC
April 2012

Molecular determinants involved in activation of caspase 7.

Biosci Rep 2011 Aug;31(4):283-94

Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4.

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.
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http://dx.doi.org/10.1042/BSR20100111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485920PMC
August 2011