Myeloid-Specific Deletion of Peptidylarginine Deiminase 4 Mitigates Atherosclerosis.
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ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC6094966 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveThe molecular mechanism of citrullination involves the calcium-dependent peptidylarginine deiminase (PAD) family of enzymes. These enzymes induce a stereochemical modification of normal proteins and transform them into autoantigens, which in rheumatoid arthritis trigger a complex cascade of joint inflammatory events followed by chronic synovitis, pannus formation, and finally, cartilage destruction. By hypothesizing that PAD2 and PAD4 enzymes produce autoantigens, we investigated five possible synovial protein targets of PAD enzymes.Material and methodsWe measured PAD2, PAD4, and citrullinated proteins in 10 rheumatoid and 10 osteoarthritis synovial biopsies and then assessed the post-translational modifications of fibrinogen, cytokeratin, tubulin, IgG, and vimentin proteins using a double-fluorescence assay with specific antibodies and an affinity-purified anti-citrullinated peptide (CCP) antibody. The degree of co-localization was analyzed, and statistical significance was determined by ANOVA, Fisher's exact test, and regression analysis.ResultsThe principal results of this study demonstrated that citrullinated proteins, such as fibrinogen, IgG, and other probed proteins, were targets of PAD2 and PAD4 activity in rheumatoid synovial biopsies, whereas osteoarthritis biopsies were negative for this enzyme (p<0.0001). An analysis of citrullination sites using the UniProtKB/Swiss-Prot data bank predicts that the secondary structure of the analyzed proteins displays most of the sites for citrullination; a discussion regarding its possible meaning in terms of pathogenesis is made.ConclusionOur results support the conclusion that the synovial citrullination of proteins is PAD2 and PAD4 dependent. Furthermore, there is a collection of candidate proteins that can be citrullinated.
| S-EPMC5042229 | biostudies-literature
Project description:Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H2O2 and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H2O2 at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H2O2 at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.
| S-EPMC6021033 | biostudies-literature
Project description:Peptidylarginine deiminase (PAD) modifies peptidylarginine and converts it to peptidylcitrulline in the presence of elevated calcium. Protein modification can lead to severe changes in protein structure and function, and aberrant PAD activity is linked to human pathologies. While PAD homologs have been discovered in vertebrates-as well as in protozoa, fungi, and bacteria-none have been identified in Drosophila melanogaster, a simple and widely used animal model for human diseases. Here, we describe the development of a human PAD overexpression model in Drosophila. We established fly lines harboring human PAD2 or PAD4 transgenes for ectopic expression under control of the GAL4/UAS system. We show that ubiquitous or nervous system expression of PAD2 or PAD4 have minimal impact on fly lifespan, fecundity, and the response to acute heat stress. Although we did not detect citrullinated proteins in fly homogenates, fly-expressed PAD4-but not PAD2-was active in vitro upon Ca2+ supplementation. The transgenic fly lines may be valuable in future efforts to develop animal models of PAD-related disorders and for investigating the biochemistry and regulation of PAD function.
| S-EPMC6961906 | biostudies-literature
Project description:Recent evidence suggests that enhanced neutrophil extracellular trap (NET) formation activates plasmacytoid dendritic cells and serves as a source of autoantigens in SLE. We propose that aberrant NET formation is also linked to organ damage and to the premature vascular disease characteristic of human SLE. Here, we demonstrate enhanced NET formation in the New Zealand mixed 2328 (NZM) model of murine lupus. NZM mice also developed autoantibodies to NETs as well as the ortholog of human cathelicidin/LL37 (CRAMP), a molecule externalized in the NETs. NZM mice were treated with Cl-amidine, an inhibitor of peptidylarginine deiminases (PAD), to block NET formation and were evaluated for lupus-like disease activity, endothelial function, and prothrombotic phenotype. Cl-amidine treatment inhibited NZM NET formation in vivo and significantly altered circulating autoantibody profiles and complement levels while reducing glomerular IgG deposition. Further, Cl-amidine increased the differentiation capacity of bone marrow endothelial progenitor cells, improved endothelium-dependent vasorelaxation, and markedly delayed time to arterial thrombosis induced by photochemical injury. Overall, these findings suggest that PAD inhibition can modulate phenotypes crucial for lupus pathogenesis and disease activity and may represent an important strategy for mitigating cardiovascular risk in lupus patients.
| S-EPMC3696545 | biostudies-literature
Project description:Peptidylarginine deiminase 4 (PAD4) is a homodimeric enzyme that catalyzes Ca²⁺-dependent protein citrullination, which results in the conversion of arginine to citrulline. This paper demonstrates the functional role of dimerization in the regulation of PAD4 activity. To address this question, we created a series of dimer interface mutants of PAD4. The residues Arg8, Tyr237, Asp273, Glu281, Tyr435, Arg544 and Asp547, which are located at the dimer interface, were mutated to disturb the dimer organization of PAD4. Sedimentation velocity experiments were performed to investigate the changes in the quaternary structures and the dissociation constants (K(d)) between wild-type and mutant PAD4 monomers and dimers. The kinetic data indicated that disrupting the dimer interface of the enzyme decreases its enzymatic activity and calcium-binding cooperativity. The K(d) values of some PAD4 mutants were much higher than that of the wild-type (WT) protein (0.45 µM) and were concomitant with lower k(cat) values than that of WT (13.4 s⁻¹). The K(d) values of the monomeric PAD4 mutants ranged from 16.8 to 45.6 µM, and the k(cat) values of the monomeric mutants ranged from 3.3 to 7.3 s⁻¹. The k(cat) values of these interface mutants decreased as the K(d) values increased, which suggests that the dissociation of dimers to monomers considerably influences the activity of the enzyme. Although dissociation of the enzyme reduces the activity of the enzyme, monomeric PAD4 is still active but does not display cooperative calcium binding. The ionic interaction between Arg8 and Asp547 and the Tyr435-mediated hydrophobic interaction are determinants of PAD4 dimer formation.
| S-EPMC3120853 | biostudies-literature
Project description:Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.
| S-EPMC2493360 | biostudies-literature
Project description:Ulcerative Colitis (UC) has been reported to be related to Porphyromonas gingivalis (P. gingivalis). Porphyromonas gingivalis peptidylarginine deiminase (PPAD), a virulence factor released by P. gingivalis, is known to induce inflammatory responses. To explore the pathological relationships between PPAD and UC, we used homologous recombination technology to construct a P. gingivalis strain in which the PPAD gene was deleted (Δppad) and a Δppad strain in which the PPAD gene was restored (comΔppad). C57BL/6 mice were orally gavaged with saline, P. gingivalis, Δppad, or comΔppad twice a week for the entire 40 days (days 0-40), and then, UC was induced by dextran sodium sulfate (DSS) solution for 10 days (days 31-40). P. gingivalis and comΔppad exacerbated DDS-induced colitis, which was determined by assessing the parameters of colon length, disease activity index, and histological activity index, but Δppad failed to exacerbate DDS-induced colitis. Flow cytometry and ELISA revealed that compared with Δppad, P. gingivalis, and comΔppad increased T helper 17 (Th17) cell numbers and interleukin (IL)-17 production but decreased regulatory T cells (Tregs) numbers and IL-10 production in the spleens of mice with UC. We also cocultured P. gingivalis, Δppad, or comΔppad with T lymphocytes in vitro and found that P. gingivalis and comΔppad significantly increased Th17 cell numbers and decreased Treg cell numbers. Immunofluorescence staining of colon tissue paraffin sections also confirmed these results. The results suggested that P. gingivalis exacerbated the severity of UC in part via PPAD.
| S-EPMC8484350 | biostudies-literature
Project description:RATIONALE:Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question. OBJECTIVE:We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis. METHODS AND RESULTS:We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE-/- background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE-/-/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. CONCLUSIONS:Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.
| S-EPMC6375743 | biostudies-literature
Project description:The keystone oral pathogen Porphyromonas gingivalis is associated with severe periodontitis. Intriguingly, this bacterium is known to secrete large amounts of an enzyme that converts peptidylarginine into citrulline residues. The present study was aimed at identifying possible functions of this citrullinating enzyme, named Porphyromonas peptidylarginine deiminase (PPAD), in the periodontal environment. The results show that PPAD is detectable in the gingiva of patients with periodontitis, and that it literally neutralizes human innate immune defenses at three distinct levels, namely bacterial phagocytosis, capture in neutrophil extracellular traps (NETs), and killing by the lysozyme-derived cationic antimicrobial peptide LP9. As shown by mass spectrometry, exposure of neutrophils to PPAD-proficient bacteria reduces the levels of neutrophil proteins involved in phagocytosis and the bactericidal histone H2. Further, PPAD is shown to citrullinate the histone H3, thereby facilitating the bacterial escape from NETs. Last, PPAD is shown to citrullinate LP9, thereby restricting its antimicrobial activity. The importance of PPAD for immune evasion is corroborated in the infection model Galleria mellonella, which only possesses an innate immune system. Together, the present observations show that PPAD-catalyzed protein citrullination defuses innate immune responses in the oral cavity, and that the citrullinating enzyme of P. gingivalis represents a new type of bacterial immune evasion factor.IMPORTANCE Bacterial pathogens do not only succeed in breaking the barriers that protect humans from infection, but they also manage to evade insults from the human immune system. The importance of the present study resides in the fact that protein citrullination is shown to represent a new bacterial mechanism for immune evasion. In particular, the oral pathogen P. gingivalis employs this mechanism to defuse innate immune responses by secreting a protein-citrullinating enzyme. Of note, this finding impacts not only the global health problem of periodontitis, but it also extends to the prevalent autoimmune disease rheumatoid arthritis, which has been strongly associated with periodontitis, PPAD activity, and loss of tolerance against citrullinated proteins, such as the histone H3.
| S-EPMC6212822 | biostudies-literature
Project description:Periodontitis is an infective process that ultimately leads to destruction of the soft and hard tissues that support the teeth (the periodontium). Periodontitis has been proposed as a candidate risk factor for development of the autoimmune disease rheumatoid arthritis (RA). Porphyromonas gingivalis, a major periodontal pathogen, is the only known prokaryote expressing a peptidyl arginine deiminase (PAD) enzyme necessary for protein citrullination. Antibodies to citrullinated proteins (anti-citrullinated protein antibodies, ACPA) are highly specific for RA and precede disease onset. Objective of this study was to assess P. gingivalis PAD (PPAD) gene expression and citrullination patterns in representative samples of P. gingivalis clinical isolates derived from periodontitis patients with and without RA and in related microbes of the Porphyromonas genus. Our findings indicate that PPAD is omnipresent in P. gingivalis, but absent in related species. No significant differences were found in the composition and expression of the PPAD gene of P. gingivalis regardless of the presence of RA or periodontal disease phenotypes. From this study it can be concluded that if P. gingivalis plays a role in RA, it is unlikely to originate from a variation in PPAD gene expression.
| S-EPMC4585897 | biostudies-literature