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      Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

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          Abstract

          Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

          Author Summary

          A number of successful pathogens have evolved mechanisms to evade host defenses, thus establishing persistent and chronic infections. Although membrane-bound innate immune receptors including Toll-like receptors play a role in inflammation in response to the common oral pathogen Porphyromonas gingivalis, it is not known how intracellular host defense mechanisms to this pathogen contribute to persistent infection and resulting chronic inflammation. In this study, we have defined a novel strategy by which P. gingivalis modulates the levels of key intracellular proteins involved in cell death and host defense responses. We demonstrate that the lysine-specific bacterial cysteine protease of P. gingivalis (Kgp) induces the proteolysis of receptor interacting protein kinase 1 (RIPK1), RIPK2 and poly (ADP-ribose) polymerase (PARP). Although endogenous host mechanisms may contribute to this process, activation of innate immune signaling cascades, caspases, or apoptosis alone were not sufficient to drive proteolysis. These findings support a role for pathogen-mediated modification of cellular kinases as a strategy for bacterial persistence within target host cells. Together with other recently described mechanisms for P. gingivalis host immune evasion, our work supports the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.

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          Most cited references93

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          Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE.

          Recent studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis. Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis. prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.
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            Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis.

            Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. We report here that the death domain kinase RIP, a key component of the TNF signaling complex, was cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site was mapped to the aspartic acid at position 324 of RIP. We demonstrated that the cleavage of RIP resulted in the blockage of TNF-induced NF-kappaB activation. RIPc, one of the cleavage products, enhanced interaction between TRADD and FADD/MORT1 and increased cells' sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protected cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Further more, RIP cleavage was also detected in other death receptor-mediated apoptosis. Therefore, our study provides a potential mechanism to convert cells from life to death in death receptor-mediated apoptosis.
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              Virus inhibition of RIP3-dependent necrosis.

              Viral infection activates cytokine expression and triggers cell death, the modulation of which is important for successful pathogenesis. Necroptosis is a form of programmed necrosis dependent on two related RIP homotypic interaction motif (RHIM)-containing signaling adaptors, receptor-interacting protein kinases (RIP) 1 and 3. We find that murine cytomegalovirus infection induces RIP3-dependent necrosis. Whereas RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis, virus-induced death proceeds independently of RIP1 and is therefore distinct from TNFalpha-dependent necroptosis. Viral M45-encoded inhibitor of RIP activation (vIRA) targets RIP3 during infection and disrupts RIP3-RIP1 interactions characteristic of TNFalpha-induced necroptosis, thereby suppressing both death pathways. Importantly, attenuation of vIRA mutant virus in wild-type mice is normalized in RIP3-deficient mice. Thus, vIRA function validates necrosis as central to host defense against viral infections and highlights the benefit of multiple virus-encoded cell-death suppressors that inhibit not only apoptotic, but also necrotic mechanisms of virus clearance. Copyright 2010 Elsevier Inc. All rights reserved.
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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, USA )
                1553-7366
                1553-7374
                June 2012
                June 2012
                7 June 2012
                : 8
                : 6
                : e1002723
                Affiliations
                [1]Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America
                Tufts University School of Medicine, United States of America
                Author notes

                Conceived and designed the experiments: AGM CAG. Performed the experiments: AGM KB GP. Analyzed the data: AGM KB GP. Contributed reagents/materials/analysis tools: AGM KB GP CAG. Wrote the paper: AGM KB GP CAG.

                Article
                PPATHOGENS-D-11-02610
                10.1371/journal.ppat.1002723
                3369954
                22685397
                772597e4-9038-4af9-aaf5-b5961cbb6ddd
                Madrigal 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
                : 22 November 2011
                : 13 April 2012
                Page count
                Pages: 19
                Categories
                Research Article
                Biology
                Immunology
                Immunity
                Immune Defense
                Immunity to Infections
                Innate Immunity
                Microbiology
                Bacterial Pathogens
                Gram Negative
                Host-Pathogen Interaction
                Medical Microbiology
                Medicine
                Cardiovascular
                Atherosclerosis
                Oral Medicine
                Oral Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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