ORCID Profile
0000-0003-0075-8893
Current Organisations
University of York
,
University of Queensland
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Publisher: Portland Press Ltd.
Date: 02-03-2011
DOI: 10.1042/BSR20100111
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.
Publisher: Annual Reviews
Date: 26-04-2022
DOI: 10.1146/ANNUREV-IMMUNOL-101220-030653
Abstract: Inflammasomes are inflammatory signaling complexes that provide molecular platforms to activate the protease function of inflammatory caspases. Caspases-1, -4, -5, and -11 are inflammatory caspases activated by inflammasomes to drive lytic cell death and inflammatory mediator production, thereby activating host-protective and pathological immune responses. Here, we comprehensively review the mechanisms that govern the activity of inflammatory caspases. We discuss inflammatory caspase activation and deactivation mechanisms, alongside the physiological importance of caspase activity kinetics. We also examine mechanisms of caspase substrate selection and how inflammasome and cell identities influence caspase activity and resultant inflammatory and pyroptotic cellular programs. Understanding how inflammatory caspases are regulated may offer new strategies for treating infection and inflammasome-driven disease.
Publisher: Springer Science and Business Media LLC
Date: 13-05-2019
DOI: 10.1038/S41589-019-0277-7
Abstract: 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.
Publisher: Springer New York
Date: 2018
DOI: 10.1007/978-1-4939-7568-6_14
Abstract: 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.
Publisher: Elsevier BV
Date: 08-2018
DOI: 10.1016/J.CELREP.2018.07.027
Abstract: 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.
Publisher: Wiley
Date: 14-03-2019
Publisher: Oxford University Press (OUP)
Date: 04-10-2018
Abstract: 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. The Gram-negative bacterium Salmonella Typhimurium suppresses human macrophage inflammasome responses via the Salmonella Pathogenicity Island 2.
Publisher: Elsevier
Date: 2023
Publisher: Springer New York
Date: 2012
Publisher: Life Science Alliance, LLC
Date: 12-2018
Abstract: 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 10) 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.
Publisher: Life Science Alliance, LLC
Date: 28-04-2020
Abstract: Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). 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 12 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 12 form. Our data reveal that cell lysis in inflammasome-activated Gsdmd -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.
Publisher: Springer Science and Business Media LLC
Date: 24-06-2020
DOI: 10.1038/S41467-020-16889-Z
Abstract: 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.
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-0357-3_1
Abstract: 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.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 03-08-2018
DOI: 10.1126/SCIIMMUNOL.AAR6676
Abstract: Neutrophils form gasdermin D pores and expel antimicrobial neutrophil extracellular traps to defend against cytosolic bacteria.
Publisher: Cold Spring Harbor Laboratory
Date: 22-09-2021
DOI: 10.1101/2021.09.19.461010
Abstract: Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we use two in vitro cellular models of LPS-induced tolerance (bone marrow-derived macrophages or BMDMs and RAW 264.7 cells), single-cell protein measurements, and mathematical modelling to explore how AIH underpins primary and secondary responses to LPS. We measure expression of TNF, IL-6, pro-IL-1β, and NOS2 and demonstrate that macrophage community AIH is dependent on LPS dose. We show that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical 3-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a 4-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS. Overall, our findings provide support for a critical role for phenotypically negative macrophage populations as an active component of AIH and primary and secondary responses to LPS. This reveals unappreciated aspects of cellular ecology and community dynamics associated with LPS-driven training of macrophages.
Publisher: Springer Science and Business Media LLC
Date: 21-01-2019
Publisher: Portland Press Ltd.
Date: 06-2021
DOI: 10.1042/BST20200986
Abstract: Innate immune responses are tightly regulated by various pathways to control infections and maintain homeostasis. One of these pathways, the inflammasome pathway, activates a family of cysteine proteases called inflammatory caspases. They orchestrate an immune response by cleaving specific cellular substrates. Canonical inflammasomes activate caspase-1, whereas non-canonical inflammasomes activate caspase-4 and -5 in humans and caspase-11 in mice. Caspases are highly specific enzymes that select their substrates through erse mechanisms. During inflammation, caspase activity is responsible for the secretion of inflammatory cytokines and the execution of a form of lytic and inflammatory cell death called pyroptosis. This review aims to bring together our current knowledge of the biochemical processes behind inflammatory caspase activation, substrate specificity, and substrate signalling.
Publisher: Rockefeller University Press
Date: 06-02-2018
DOI: 10.1084/JEM.20172222
Abstract: 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 10) 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 10. Further p33 10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20 10 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.
Publisher: Springer New York
Date: 2016
Publisher: Wiley
Date: 24-08-2015
Abstract: 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 in idually 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.
Publisher: Elsevier BV
Date: 06-2017
DOI: 10.1016/J.MOLIMM.2016.11.009
Abstract: 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.
Publisher: The American Association of Immunologists
Date: 15-05-2018
Abstract: 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.
Publisher: F1000 Research Ltd
Date: 25-07-2022
DOI: 10.12688/WELLCOMEOPENRES.17557.2
Abstract: Background: Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we aimed to develop mathematical models to explore theoretical cellular states underpinning the empirically observed responses of macrophages following lipopolysaccharide (LPS) challenge. Methods: We obtained empirical data following primary and secondary responses to LPS in two in vitro cellular models (bone marrow-derived macrophages or BMDMs, and RAW 264.7 cells) and single-cell protein measurements for four key inflammatory mediators: TNF, IL-6, pro-IL-1β, and NOS2, and used mathematical modelling to understand heterogeneity. Results: For these four factors, we showed that macrophage community AIH is dependent on LPS dose and that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical three-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a four-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS. Conclusions: Overall, we provide a mathematical model for studying macrophage ecology and community dynamics that can be used to elucidate the role of phenotypically negative macrophage populations in AIH and, primary and secondary responses to LPS.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-03-2012
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.
Location: United Kingdom of Great Britain and Northern Ireland
Location: Canada
No related grants have been discovered for Dave Boucher.