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      Functional and Evolutionary Analyses Identify Proteolysis as a General Mechanism for NLRP1 Inflammasome Activation

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          Abstract

          Inflammasomes are cytosolic multi-protein complexes that initiate immune responses to infection by recruiting and activating the Caspase-1 protease. Human NLRP1 was the first protein shown to form an inflammasome, but its physiological mechanism of activation remains unknown. Recently, specific variants of mouse and rat NLRP1 were found to be activated upon N-terminal cleavage by the anthrax lethal factor protease. However, agonists for other NLRP1 variants, including human NLRP1, are not known, and it remains unclear if they are also activated by proteolysis. Here we demonstrate that two mouse NLRP1 paralogs (NLRP1A B6 and NLRP1B B6) are also activated by N-terminal proteolytic cleavage. We also demonstrate that proteolysis within a specific N-terminal linker region is sufficient to activate human NLRP1. Evolutionary analysis of primate NLRP1 shows the linker/cleavage region has evolved under positive selection, indicative of pathogen-induced selective pressure. Collectively, these results identify proteolysis as a general mechanism of NLRP1 inflammasome activation that appears to be contributing to the rapid evolution of NLRP1 in rodents and primates.

          Author Summary

          Hosts and their pathogens often engage in evolutionary ‘arms races’, iterative cycles of adaptation, in which each opponent evolves strategies to overcome the other. For example, the anthrax bacterium overcomes the host immune response by producing lethal factor, a proteolytic enzyme that specifically cleaves and inactivates host immunity proteins called MAP kinases. Rodents counteract this strategy by producing a sensor protein called NLRP1 that is cleaved by anthrax lethal factor. Upon cleavage, NLRP1 activates a potent anti-bacterial immune response that compensates for the loss of the MAP kinase response. Humans also produce NLRP1, but human NLRP1 is neither cleaved nor activated by lethal factor. Thus, the mechanism of human NLRP1 activation and its function in immunity remains unknown. In our study, we show that human NLRP1, like rodent NLRP1, can be activated by proteolytic cleavage. Interestingly, evolutionary analysis supports the hypothesis that primate NLRP1 is rapidly evolving to be cleaved by (and thereby detect) pathogen-encoded proteases. Our results elucidate a general mechanism for NLRP1 activation and suggest that host immunity proteins may evolve toward recognition by bacterial proteases to engage in evolutionary arms races with pathogens.

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          Most cited references38

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          Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection.

          Nod2 activates the NF-kappaB pathway following intracellular stimulation by bacterial products. Recently, mutations in Nod2 have been shown to be associated with Crohn's disease, suggesting a role for bacteria-host interactions in the etiology of this disorder. We show here that Nod2 is a general sensor of peptidoglycan through the recognition of muramyl dipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria. Moreover, the 3020insC frameshift mutation, the most frequent Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP. Together, these results impact on the understanding of Crohn's disease development. Additionally, the characterization of Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well known biological activities of this immunomodulatory compound.
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            The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus.

            Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity. © 2011 Macmillan Publishers Limited. All rights reserved
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              Innate immune recognition of bacterial ligands by NAIPs dictates inflammasome specificity

              Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the CASPASE1 (CASP1) protease 1,2 . Although genetic data support a critical role for inflammasomes in immune defense and inflammatory diseases 3 , the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing C4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin 4,5 and PrgJ 6 , a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homolog of NLRC4, NAIP5 (NLR family, Apoptosis Inhibitory Protein 5), has been implicated in activation of NLRC4 7–11 , but is widely assumed to play only an auxiliary role 1,2 , since NAIP5 is often dispensable for NLRC4 activation 7,8 . However, Naip5 is a member of a small multigene family 12 , raising the possibility of redundancy and functional specialization among Naip genes. Indeed, we show here that different NAIP paralogs dictate the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that NAIP2/NLRC4 physically associates with PrgJ but not flagellin, whereas NAIP5/NLRC4 associates with flagellin but not PrgJ. Taken together, our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP/NLRC4 inflammasomes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                7 December 2016
                December 2016
                : 12
                : 12
                : e1006052
                Affiliations
                [1 ]Division of Immunology & Pathogenesis, Department of Molecular & Cell Biology, and Cancer Research Laboratory, University of California, Berkeley, California, United States of America
                [2 ]Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, California, United States of America
                [3 ]Howard Hughes Medical Institute, University of California, Berkeley, California, United States of America
                Portland VA Medical Center, Oregon Health and Science University, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                • Conceived and designed the experiments: JCS PSM AMH MDD REV.

                • Performed the experiments: JCS PSM AMH MDD.

                • Analyzed the data: JCS PSM AMH MDD REV.

                • Wrote the paper: JCS PSM MDD REV.

                Author information
                http://orcid.org/0000-0003-0769-2268
                http://orcid.org/0000-0002-4879-9603
                http://orcid.org/0000-0002-6686-3912
                Article
                PPATHOGENS-D-16-01267
                10.1371/journal.ppat.1006052
                5142783
                27926929
                c9c142e6-69d9-411f-95ae-00fc2eeae623
                © 2016 Chavarría-Smith et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 3 June 2016
                : 9 November 2016
                Page count
                Figures: 5, Tables: 0, Pages: 22
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: AI075039
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: AI063302
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000861, Burroughs Wellcome Fund;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100001033, Jane Coffin Childs Memorial Fund for Medical Research;
                Award Recipient :
                This work was supported in part by grants (AI063302 and AI075039) from the National Institutes of Health ( https://www.nih.gov) to REV. REV is an Investigator of the Howard Hughes Medical Institute ( http://www.hhmi.org) and a Burroughs Wellcome Fund ( http://www.bwfund.org) Investigator in the Pathogenesis of Infectious Disease. JCS was a recipient of an National Science Foundation ( http://www.nsf.gov) Graduate Research Fellowship. PSM is a recipient of a Jane Coffin Childs Memorial Research Fund Postdoctoral Fellowship ( http://www.jccfund.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Biology and Life Sciences
                Immunology
                Immune System Proteins
                Inflammasomes
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                Infectious disease & Microbiology
                Infectious disease & Microbiology

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