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Jurg Tschopp - One of the best experts on this subject based on the ideXlab platform.
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Inflammatory Caspases and inflammasomes: master switches of inflammation
Cell Death & Differentiation, 2007Co-Authors: Fabio Martinon, Jurg TschoppAbstract:Fifteen years have passed since the cloning and characterization of the interleukin-1 β -converting enzyme (ICE/Caspase-1), the first identified member of a family of proteases currently known as Caspases. Caspase-1 is the prototypical member of a subclass of Caspases involved in cytokine maturation termed inflammatory Caspases that also include Caspase-4 Caspase -5, Caspase -11 and Caspase -12. Efforts to elucidate the molecular mechanisms involved in the activation of these proteases have uncovered an important role for the NLR family members, NALPs, NAIP and IPAF. These proteins promote the assembly of multiprotein complexes termed inflammasomes, which are required for activation of inflammatory Caspases. This article will review some evolutionary aspects, biochemical evidences and genetic studies, underlining the role of inflammasomes and inflammatory Caspases in innate immunity against pathogens, autoinflammatory syndromes and in the biology of reproduction.
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inflammatory Caspases linking an intracellular innate immune system to autoinflammatory diseases
Cell, 2004Co-Authors: Fabio Martinon, Jurg TschoppAbstract: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.
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inflammatory Caspases linking an intracellular innate immune system to autoinflammatory diseases
Cell, 2004Co-Authors: Fabio Martinon, Jurg TschoppAbstract: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-1beta 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.
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The inflammasome: a molecular platform triggering activation of inflammatory Caspases and processing of proIL-beta.
Molecular Cell, 2002Co-Authors: Fabio Martinon, Kimberly Burns, Jurg TschoppAbstract: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.
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the inflammasome a molecular platform triggering activation of inflammatory Caspases and processing of proil beta
Molecular Cell, 2002Co-Authors: Fabio Martinon, Kimberly Burns, Jurg TschoppAbstract: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.
Fabio Martinon - One of the best experts on this subject based on the ideXlab platform.
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Inflammatory Caspases and inflammasomes: master switches of inflammation
Cell Death & Differentiation, 2007Co-Authors: Fabio Martinon, Jurg TschoppAbstract:Fifteen years have passed since the cloning and characterization of the interleukin-1 β -converting enzyme (ICE/Caspase-1), the first identified member of a family of proteases currently known as Caspases. Caspase-1 is the prototypical member of a subclass of Caspases involved in cytokine maturation termed inflammatory Caspases that also include Caspase-4 Caspase -5, Caspase -11 and Caspase -12. Efforts to elucidate the molecular mechanisms involved in the activation of these proteases have uncovered an important role for the NLR family members, NALPs, NAIP and IPAF. These proteins promote the assembly of multiprotein complexes termed inflammasomes, which are required for activation of inflammatory Caspases. This article will review some evolutionary aspects, biochemical evidences and genetic studies, underlining the role of inflammasomes and inflammatory Caspases in innate immunity against pathogens, autoinflammatory syndromes and in the biology of reproduction.
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inflammatory Caspases linking an intracellular innate immune system to autoinflammatory diseases
Cell, 2004Co-Authors: Fabio Martinon, Jurg TschoppAbstract: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.
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inflammatory Caspases linking an intracellular innate immune system to autoinflammatory diseases
Cell, 2004Co-Authors: Fabio Martinon, Jurg TschoppAbstract: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-1beta 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.
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The inflammasome: a molecular platform triggering activation of inflammatory Caspases and processing of proIL-beta.
Molecular Cell, 2002Co-Authors: Fabio Martinon, Kimberly Burns, Jurg TschoppAbstract: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.
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the inflammasome a molecular platform triggering activation of inflammatory Caspases and processing of proil beta
Molecular Cell, 2002Co-Authors: Fabio Martinon, Kimberly Burns, Jurg TschoppAbstract: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.
Kimberly Burns - One of the best experts on this subject based on the ideXlab platform.
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The inflammasome: a molecular platform triggering activation of inflammatory Caspases and processing of proIL-beta.
Molecular Cell, 2002Co-Authors: Fabio Martinon, Kimberly Burns, Jurg TschoppAbstract: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.
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the inflammasome a molecular platform triggering activation of inflammatory Caspases and processing of proil beta
Molecular Cell, 2002Co-Authors: Fabio Martinon, Kimberly Burns, Jurg TschoppAbstract: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.
Guy S Salvesen - One of the best experts on this subject based on the ideXlab platform.
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Exploring the prime site in Caspases as a novel chemical strategy for understanding the mechanisms of cell death: a proof of concept study on necroptosis in cancer cells
Cell Death & Differentiation, 2020Co-Authors: Katarzyna Groborz, Scott J Snipas, Guy S Salvesen, Monica L. Gonzalez Ramirez, Marcin Drąg, Marcin PorębaAbstract:Caspases participate in regulated cell death mechanisms and are divided into apoptotic and proinflammatory Caspases. The main problem in identifying the unique role of a particular Caspase in the mechanisms of regulated cell death is their overlapping substrate specificity; Caspases recognize and hydrolyze similar peptide substrates. Most studies focus on examining the non-prime sites of the Caspases, yet there is a need for novel and more precise chemical tools to identify the molecular participants and mechanisms of programmed cell death pathways. Therefore, we developed an innovative chemical approach that examines the prime area of the Caspase active sites. This method permits the agile parallel solid-phase synthesis of Caspase inhibitors with a high yield and purity. Using synthesized compounds we have shown the similarities and differences in the prime area of the Caspase active site and, as a proof of concept, we demonstrated the exclusive role of Caspase-8 in necroptosis.
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An Optimized Activity-Based Probe for the Study of Caspase-6 Activation
Chemistry & Biology, 2012Co-Authors: Laura E Edgington, Bram J. Van Raam, Christoph Wierschem, Martijn Verdoes, Guy S Salvesen, Matthew BogyoAbstract:Summary Although significant efforts have been made to understand the mechanisms of Caspase activation during apoptosis, many questions remain regarding how and when executioner Caspases get activated. We describe the design and synthesis of an activity-based probe that labels Caspase-3/-6/-7, allowing direct monitoring of all executioner Caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that Caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that Caspase-6 activation does not require active Caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that Caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.
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Caspase assays: identifying Caspase activity and substrates in vitro and in vivo.
Methods in enzymology, 2008Co-Authors: Cristina Pop, Guy S Salvesen, Fiona L. ScottAbstract:The measurement of general Caspase activity and the quantification of purified recombinant Caspases in vitro can be accomplished with relative ease. But the determination of which Caspases are active in a cellular context is much more challenging. This is because commercially available small molecule substrates and inhibitors do not display sufficient specificity to dissect the complex interplay of Caspase pathways. Here we describe procedures that can be used to validate which Caspases are active in cell culture models and determine which Caspases are responsible for specific cleavage events. We also recommend methods for working with recombinant initiator Caspases in vitro and suggest ways to accurately assess the cleavage efficiency of natural Caspase substrates.
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Caspase activation.
Biochemical Society symposium, 2003Co-Authors: Kelly M Boatright, Guy S SalvesenAbstract:Caspase activation is the 'point of no return' commitment to cell death. Synthesized as inactive zymogens, it is essential that the Caspases remain inactive until the death signal is received. It is known for the downstream executioner Caspases-3 and -7 that the activation event is proteolytic cleavage, and this had been assumed to apply to the initiator Caspases as well. However, recent studies conducted on Caspases-2, -8 and -9 have challenged this tenet of Caspase activation. In this review we focus on the molecular details of Caspase activation, with emphasis on recent work that provides a pleasing explanation for the differential requirements for the activation of executioner and initiator Caspases.
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calpain functions in a Caspase independent manner to promote apoptosis like events during platelet activation
Blood, 1999Co-Authors: Beni B Wolf, Joshua C Goldstein, Henning R Stennicke, Helen M Beere, Gustavo P Amarantemendes, Guy S Salvesen, Douglas R GreenAbstract:Apoptosis and platelet activation share common morphological and biochemical features. Because Caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained Caspase-9, Caspase-3, and the Caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of proCaspase-9 and proCaspase-3 and subsequent proteolysis of Caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of proCaspase-9, proCaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without Caspase activation or release of mitochondrial cytochrome c, suggesting a role for a nonCaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented Caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant proCaspase-9 and proCaspase-3 without activating either Caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of Caspases and suggest that calpain, not Caspases, promotes apoptosis-like events during platelet activation.
Lisa Bouchier-hayes - One of the best experts on this subject based on the ideXlab platform.
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Detection of Initiator Caspase Induced Proximity in Single Cells by Caspase Bimolecular Fluorescence Complementation.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Melissa J. Parsons, Sara R. Fassio, Lisa Bouchier-hayesAbstract:The Caspase family of proteases includes key regulators of apoptosis and inflammation. The Caspases can be divided into two groups, the initiator Caspases and the executioner Caspases. Initiator Caspases include Caspase-2, Caspase-8, and Caspase-9 and are activated by proximity-induced dimerization upon recruitment to large molecular weight protein complexes called activation platforms. This protocol describes an imaging-based technique called Caspase Bimolecular Fluorescence Complementation (BiFC) that measures induced proximity of initiator Caspases. This method uses nonfluorescent fragments of the fluorescent protein Venus fused to initiator Caspase monomers. When the Caspase is recruited to its activation platform, the resulting induced proximity of the Caspase monomers facilitates refolding of the Venus fragments into the full molecule, reconstituting its fluorescence. Thus, the assembly of initiator Caspase activation platforms can be followed in single cells in real time. Induced proximity is the most apical step in the activation of initiator Caspases, and therefore, Caspase BiFC is a robust and specific method to measure initiator Caspase activation.
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Imaging-Based Methods for Assessing Caspase Activity in Single Cells
CSH Protocols, 2015Co-Authors: Melissa J. Parsons, Markus Rehm, Lisa Bouchier-hayesAbstract:: Caspases, a family of proteases that are essential mediators of apoptosis, are divided into two groups: initiator Caspases and executioner Caspases. Each initiator Caspase is activated at the apex of its respective pathway, which generally leads to the cleavage and activation of executioner Caspases. Executioner Caspases in turn cleave numerous substrates in the cell, leading to its demise. Initiator Caspases are activated when inactive monomers undergo induced proximity to form an active Caspase. In contrast, executioner Caspases are activated by cleavage. Based on this key difference, different imaging techniques have been developed to measure Caspase activation and activity on a single-cell basis. Bimolecular fluorescence complementation (BiFC) is used to measure induced proximity of initiator Caspases, whereas Forster resonance energy transfer (FRET) permits the investigation of Caspase-mediated substrate cleavage in real time. Because many of the events in apoptosis, including Caspase activation, are asynchronous in nature, these single-cell imaging techniques have proven to be immensely powerful in ordering and dissecting Caspase pathways. When coupled with parallel detection of additional hallmark events of apoptosis, they provide detailed and quantitative kinetic and positional insights into the signal transduction pathways that regulate cell death. Here we provide a brief introduction into BiFC- and FRET-based imaging of Caspase activation and activity in single cells.
