Peptidylarginine Deiminase Enzymes Research Articles

SP0161 Functional Consequences of Anti-Pad-Antibodies in RA

Peptidylarginine deiminase (PAD) enzymes have emerged as key participants in the pathogenesis of rheumatoid arthritis (RA) due to their expression in inflamed RA synovium and generation of citrullinated autoantigens, hallmark...

Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates.

Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca+2-regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma.Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

Cellular and humoral immunity in arthritis are profoundly influenced by the interaction between cigarette smoke effects and host HLA-DR and DQ genes

Individuals carrying DRB1*0401 who smoke cigarettes are at an increased risk of developing severe seropositive RA. To determine how cigarette smoke (CS) interacts with host genetic factors in the induction of RA-associated autoimmunity, we used transgenic mice carrying the RA-susceptible HLA genes DR4 and DQ8, but lacking all endogenous murine class II molecules. Cigarette smoke exposure augmented peptidylarginine deiminase (PAD) enzyme expression, and enhanced immune responses to citrullinated collagen and vimentin. Here we show for the first time that DQ molecules can present citrullinated peptides much more efficiently than native peptides. Interestingly, CS exposure suppressed collagen-induced arthritis (CIA) in DRB1*0401 mice although innate immune response was enhanced. On the other hand, CS exposure exacerbated CIA in DQ8 mice, which was accompanied by an increased expression of Th17 gene transcripts in lungs. These observations suggest that cigarette smoke promotes antigen-specific autoimmunity that is profoundly influenced by host genetic factors.

A6.8 Elevated antibody levels to porphyromonas gingivalis detected in rheumatoid arthritis patients with a specific anti-citrullinated protein/peptide antibody profile

Background and ObjectivesPorphyromonas gingivalis, a major cause of chronic periodontitis (PD), has been implicated also in the aetiology of rheumatoid arthritis (RA), specifically in anti-citrullinated protein/peptide antibody (ACPA) positive RA,...

Does a biological link exist between periodontitis and rheumatoid arthritis?

Periodontitis or Periodontal disease (PD) and Rheumatoid arthritis (RA) are two the most common chronic inflammatory diseases. Periodontitis is a biofilm associ ated destructive inflammatory dise ase of the periodontium caused by specific microorganisms. Rheumatoid arthritis is an autoimmune condition and is identified by elevated serum autoantibody titre directed against citrullinated peptides or rheumatoid factor. Periodontitis may involve some elements of autoimmunity. Recent studies have established that PD and RA show a common pathway and could be closely associated through a common dysregulation and dysfunction in inflamma tory mechanism. The enzyme peptidyl arginine deiminase (PAD), expressed by Porphyromonas gingivalis (P. gingivalis ) is responsible for the enzymatic deimination of arginine residuals to citrulline resulting in protein citrullination and its increased accumulation in RA. Citrullination by PAD may act as a putative biologic link between PD and RA. Association of Human leukocytic antigen-DR4 antigen has been established both with RA and PD. Several interleukins and inflammatory me diators (ILs) and Nuclear factor kappa beta ligand are linked to these common chronic inflammatory diseases. Antibodies directed against heat shock protein (hsp 70 ab) of P. gingivalis , P. melanogenicus and P. intermedia are raised in PD as well as RA. Both the conditions share many pathological and immunological similarities. Bacterial infection, genetic susceptibility, altered immune reaction and inflammatory mediators considered responsible for RA are also associated with PD. So it is plausible that a biological link may exist between PD and RA. Therapies aimed at modifying the expression and effect of inflammatory mediators and effector mol ecules such as matrix metalloproteinases, proinflamma tory cytokines and autoantibodies of structural proteins may probably reduce the severity of both RA and PD.

Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAbs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy donors. In conclusion, several of our mAbs proved useful in western blotting and immunohistochemistry, and the ELISA described here reliably measures PAD2 levels in blood. This allows investigation of PAD2 as a possible biomarker and further investigation of PAD2's involvement in various inflammatory diseases.

Effect of Porphyromonas gingivalis on Citrullination of Proteins by Macrophages In Vitro

Citrullination of proteins within inflamed periodontal tissues may provide an important link between periodontitis and rheumatoid arthritis. The aim of this study is to determine whether the presence of Porphyromonas gingivalis peptidylarginine deiminase (PPAD) can influence citrullination of proteins by either increasing the amount of local citrullinated protein or influencing the peptidylarginine deiminase (PAD) enzymes found in the monocyte/macrophage population. Human peripheral blood monocytes and macrophages were incubated in the presence of live or heat-killed P. gingivalis. Expression of PAD2 and PAD4, PPAD, and citrullinated proteins were assessed by either a combination of real-time polymerase chain reaction, Western blotting, or a colorimetric assay. PPAD was detected only in mononuclear cells incubated in the presence of live P. gingivalis and resulted in increased extracellular citrullination. Endogenous PAD (mRNA and protein) expression was detected in monocytes and macrophages but was not affected by P. gingivalis. Although P. gingivalis produces a PAD that can citrullinate extracellular proteins and may contribute to the citrullinated protein load in gingival tissues, it does not appear to affect PAD expression or citrullination by host monocytes or macrophages.

Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?

The discovery of neutrophil extracellular traps in 2004 opened a fascinating new chapter in immune-mediated microbial killing. Brinkman etal. demonstrated that neutrophils, when catastrophically stimulated, undergo a novel form of programmed cell death (neutrophil extracellular trap formation) whereby they decondense their entire nuclear chromatin/DNA and release the resulting structure into the cytoplasm to mix with granule-derived antimicrobial peptides before extruding these web-like structures into the extracellular environment. The process requires the activation of the granule enzyme peptidyl arginine deiminase-4, the formation of reactive oxygen species (in particular hypochlorous acid), the neutrophil microtubular system and the actin cytoskeleton. Recent work by Yousefi etal. demonstrated that exposure to different agents for shorter stimulation periods resulted in neutrophil extracellular trap release from viable granulocytes, and that such neutrophil extracellular traps comprised mitochondrial DNA rather than nuclear DNA and were also capable of microbial entrapment and destruction. Deficiency in NADPH-oxidase production (as found in patients with chronic granulomatous disease) results in an inability to produce neutrophil extracellular traps and, along with their failure to produce antimicrobial reactive oxygen species, these patients suffer from severe, and sometimes life-threatening, infections. However, conversely the release of nuclear chromatin into tissues is also potentially autoimmunogenic and is now associated with the generation of anti-citrullinated protein antibodies in seropositive rheumatoid arthritis. Other neutrophil-derived nuclear and cytoplasmic contents are also pathogenic, either through direct effects on tissues or via autoimmune processes (e.g. autoimmune vasculitis). In this review, we discuss the plant origins of a highly conserved innate immune method of microbial killing, the history and biology of neutrophil extracellular traps and their role in defence and in human diseases. We attempt to resolve areas of controversy and propose roles for excess neutrophil extracellular trap release from hyperactive/reactive neutrophils and for the unique peptidyl arginine deiminase enzyme of Porphyromonas gingivalis in the pathogenesis of periodontitis, and subsequently a role for periodontitis/the peptidyl arginine deiminase enzyme of P.gingivalis in the causal pathway of autoimmune diseases such as rheumatoid arthritis. We propose that neutrophil extracellular trap and peptidyl arginine deiminase release may propagate tissue-destructive mechanisms rather than provide protection in susceptible individuals and that release of host-derived DNase may play an important role in the digestion and removal of neutrophil extracellular traps within tissues.

AB0181 Padi4 haplotype a potential modulator of antigen expression in an immunological environment

BackgroundPeptidil arginine deiminase IV (PAD 4) is the responsible enzyme for a posttranslational modification called citrullination, originating the antigenic determinant recognized by anti-cyclic citrullinated peptide antibodies (aCCP)1. Four SNPs (single...

AB0033 Association between radiographic joint damage and a PADI4 SNP in japanese RA patients

BackgroundPeptidyl arginine deiminase, type IV (PADI4) gene has been reported to be associated with susceptibility to rheumatoid arthritis (RA) mainly in Asian populations (1, 2).ObjectivesTo investigate the impact of peptidyl...

Abstract 54: Peptidylarginine Deiminase 4-dependent Generation of Neutrophil Extracellular Traps is Crucial for Deep Vein Thrombosis in Mice

Introduction Histone hypercitrullination by the enzyme peptidylarginine deiminase 4 (PAD4) leads to nuclear chromatin decondensation that is needed for neutrophil extracellular trap (NET) formation. NETs consist of chromatin and granule proteins that are released into the extracellular environment. NETs were shown to be involved in thrombosis by promoting coagulation and platelet adhesion and were identified in the thrombus scaffold in animal models of deep vein thrombosis (DVT). Objective Whether NETs are involved in the pathogenesis of DVT or whether they are merely a consequence of neutrophil recruitment to the thrombus is unknown. We hypothesized that NET formation would be impaired in PAD4-deficient mice during deep vein thrombosis and that this may affect thrombus formation and/or stability. Methods PAD4-deficient mice are incapable of citrullinating histones and therefore fail to decondense chromatin during NETosis. We performed the inferior vena cava stenosis model of DVT in wild-type or PAD4-/- mice. Intravital microscopy was done to assess leukocyte vessel wall interaction in PAD4 deficiency. Results We induced NET formation in isolated peripheral blood mouse neutrophils with ionomycin and found that PAD4-/- neutrophils had a complete inability to produce NETs (WT, 20.65±2.61% NETs; PAD4-/-, not detected. n=4). Leukocyte-endothelial interactions in PAD4-/- mice were not impaired upon induction of systemic Weibel-Palade body release (WT, 55.2±11.8; PAD4-/-, 62.0±17.5 cells/min, n=5-6). In the DVT model, while a majority (9/10) of wild-type mice formed a thrombus 48 hours after stenosis, only 1 of 11 PAD4-/- mice formed a thrombus. Thrombus formation could be rescued by infusions of isolated WT bone marrow neutrophils into PAD4-/- mice, and extracellular H3Cit+ areas were seen within these thrombi. This data suggests that neutrophil PAD4 was essential for thrombus formation in deep veins. Conclusion NETs comprise a crucial part of the pathologic thrombus scaffold, and here we report that the lack of NETs inhibits pathological thrombosis. Chromatin decondensation initiated by PAD4 in neutrophils is a key player in the formation of deep vein thrombi and targeting neutrophil histone modification could be a new way to prevent DVT.

Retinal deimination and PAD2 levels in retinas from donors with age-related macular degeneration (AMD)

Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the association of deimination with the pathogenesis of age-related macular degeneration (AMD), we studied protein deimination and PAD2 levels in retinas of AMD donor eyes compared to age-matched non-AMD retinas. Eyes from non-AMD and AMD donors were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. Retina and retinal pigment epithelium (RPE) from donor eyes were processed for immunohistochemical detection and western blotting using antibodies to PAD2 and citrulline residues. The ganglion cell, inner plexiform, inner nuclear and outer nuclear layers were labeled by both PAD2 and citrulline antibodies. Changes in the localization of deiminated residues and PAD2 were evident as the retinal layers were remodeled coincident with photoreceptor degeneration in AMD retinas. Immunodetection of either PAD2 or citrulline residues could not be evaluated in the RPE layer due to the high autofluorescence levels in this layer. Interestingly, higher deimination immunoreactivity was detected in AMD retinal lysates. However, no significant changes in PAD2 were detected in the AMD and non-AMD retinas and RPE lysates. Our observations show increased levels of protein deimination but not PAD2 in AMD retinas and RPE, suggesting a reduced rate of turnover of deiminated proteins in these AMD retinas.

Lessons to be learned from periodontitis

This article reviews the link between periodontitis and rheumatoid arthritis (RA) with regard to similarities in genetic risk factors and immunopathogenesis. Emphasis is paid to the potential role of the periodontal pathogen Porphyromonas gingivalis in the etiopathogenesis of both periodontitis and RA, in particular by post-translational modification of arginine into citrulline. P. gingivalis, a major periodontal pathogen, is presently known as the only bacterium in the oral flora which contains a peptidyl arginine deiminase enzyme (PAD). This enzyme is necessary for citrullination. As a result, citrullinated proteins and P. gingivalis PAD, PAD2 and PAD4 (expressed by infiltrating neutrophils) are found in periodontal tissues. Autoantibodies directed to citrullinated proteins, so-called anticitrullinated protein antibodies (ACPAs), are found to be present in gingival crevicular fluid originating from inflamed gingival tissue. Furthermore, treatment studies have revealed that nonsurgical periodontal treatment, that is removal of sub-gingival calculus and biofilm deposits, is accompanied by a reduction in the severity of RA. In this study the similarities in immune response and tissue degradation between RA and periodontitis are reviewed. It is shown that the two diseases share the same environmental and genetic risk factors, apart from the fact that there is a link between both diseases via citrullination of proteins by human PAD and P. gingivalis PAD.

Novel Inhibitors of Protein Arginine Deiminase with Potential Activity in Multiple Sclerosis Animal Model

In multiple sclerosis (MS), myelin basic protein (MBP), critical for the maintenance of myelin compaction and protecting against degradation, is known to contain concentrations of the noncoded amino acid, "citrulline", in abnormal proportions. Peptidyl arginine deiminase (PAD) catalyzes the post-translational citrullination of proteins via the deimination of Arg residues. In the central nervous system, specifically PAD2 and PAD4, are the enzymes responsible for the citrullination. We used in silico screening of commercial libraries to find small molecules that would reversibly inhibit PAD4. An initial set of 10 diverse compounds was selected from the screen, and from these compounds, 3, 4, 6, and 8 showed promising inhibitory activities against PAD4 with Ki in the range of 115-153 μM. Compound 4 was selected to partake in an in vivo MOG EAE mouse model study to evaluate its effect in MS-like conditions. Results from the 24 day pilot mouse study showed an improved clinical outcome for mice being administered compound 4 compared to the control group. In brain, 4 treated mice showed a clear reduction in the CD3 +ve T cells. These results suggest that compound 4 may have potential utility and confirmed that noncovalent inhibitors of PAD enzymes can be developed as potential agents targeting MS pathology.

Citrullination: A Target for Disease Intervention in Multiple Sclerosis and other Inflammatory Diseases?

Citrullinated histone epitopes are involved in the very early stages of inflammatory responses. An important early event is the activation of neutrophils. It has been shown that Peptidyl Arginine Deiminase (PAD) expression levels increase upon pro-inflammatory signalling followed by activation of neutrophils. Subsequently, PAD enzymes cause histone citrullination in the activated neutrophils. Histone citrullination is involved in various processes. One of the most important is NETosis, which results in the release of citrullinated histones to the extracellular space. There, they are involved in Neutrophil Extracellular Trap (NET) formation, which intensifies the inflammatory response. The central role of citrullinated histones in early inflammation makes NETs an attractive target for inflammatory disease intervention. Moreover, the safety profile is expected to be superior to immune-suppressing biologicals, like anti- Tumour Necrosis Factor (TNF) drugs. It is anticipated that shielding citrullinated histone epitopes from the immune system, as well as interfering with their putative roles in the inflammatory response, will have a broad applicability in preventing and treating various inflammatory diseases, including multiple sclerosis.

Peptidylarginine Deiminase and Citrullination: Potential Therapeutic Targets for Inflammatory Diseases

The multiple post-translational modifications of proteins display specific gain- or loss-of-function under normal and abnormal conditions. These modifications are precisely regulated by post-translational modification enzymes. The altered molecular status perturbs the pattern of gene expression and decides on a direction to signal transduction cascades as well as intrinsic properties of the proteins. Ultimately, it strictly maintains intracellular environment or results in disease manifestations. Recently, it has become that enzyme-dependent modification of arginine residue to citrulline exerts an important role in the induction of autoimmunity including rheumatoid arthritis, multiple sclerosis, and cancer. The modification of arginine residue to citrulline on proteins is called citrullination or deimination and is regulated by the calcium-dependent enzyme peptidylarginine deiminase (PAD). Now many effective PAD inhibitors (for example, Cl-amidine) have developed that ameliorates disease phenotypes. In this review, we discuss crucial roles of PAD enzyme and citrullination, the effectiveness of PAD inhibitors, and the implication in pathology.

Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

SUMMARYMultiple sclerosis (MS) is the most common CNS-demyelinating disease of humans, showing clinical and pathological heterogeneity and a general resistance to therapy. We first discovered that abnormal myelin hypercitrullination, even in normal-appearing white matter, by peptidylarginine deiminases (PADs) correlates strongly with disease severity and might have an important role in MS progression. Hypercitrullination is known to promote focal demyelination through reduced myelin compaction. Here we report that 2-chloroacetamidine (2CA), a small-molecule, PAD active-site inhibitor, dramatically attenuates disease at any stage in independent neurodegenerative as well as autoimmune MS mouse models. 2CA reduced PAD activity and protein citrullination to pre-disease status. In the autoimmune models, disease induction uniformly induced spontaneous hypercitrullination with citrulline+ epitopes targeted frequently. 2CA rapidly suppressed T cell autoreactivity, clearing brain and spinal cord infiltrates, through selective removal of newly activated T cells. 2CA essentially prevented disease when administered before disease onset or before autoimmune induction, making hypercitrullination, and specifically PAD enzymes, a therapeutic target in MS models and thus possibly in MS.

Neutrophil activation and B-cell stimulation in the pathogenesis of Felty’s syndrome

Felty's syndrome (FS) is a severe arthritic disorder that features chronic neutrophil activation and progresses to neutropenia and susceptibility to unabated infections. The life‑threatening manifestations of FS have focused the attention of clinical experimenters who have made persistent efforts to find new and effective therapies. This review highlights important milestones in the research on FS and draws attention to recent studies on the antigen specificity of antibodies present in patients' sera. Recent data have indicated that immunoglobulins G (IgGs) in patients with FS bind selectively and specifically to deiminated histones and neutrophil extracellular chromatin traps (NETs). Deimination is the conversion of certain arginine residues in proteins to citrullines by the enzyme peptidylarginine deiminase 4. Earlier observations had indicated that IgGs in FS patients avidly bind to citrullinated peptides. These observations suggest that NETosis, the type of cell death that is defined by the release of NETs, provides autoantigens that stimulate B cell responses in this patient group. This insight parallels recent observations in other autoimmune conditions and lends support to the paradigm that NETosis plays a leading role in the pathogenesis of antiself immune responses.

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.

Effect of Rheumatoid Arthritis (RA) Susceptibility Genes on the Immune Response to Viral Citrullinated Peptides in RA

To the Editor: Anticitrullinated peptide/protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific antibodies that recognize various protein sequences containing citrulline, the deiminated form of arginine produced posttranslationally by peptidylarginine deiminase (PAD) enzymes. Recently, viral citrullinated peptides (VCP) derived from Epstein-Barr virus nuclear proteins EBNA-1 and EBNA-2 have been described as a target of ACPA in RA sera1,2. Genetic factors predisposing to RA, namely HLA-DRB1 shared epitope (SE) alleles and PTPN22 gene variants, as well as the TRAF1/C5 locus and PADI4 susceptible haplotype, have been found to be predominantly associated with ACPA-positive RA, even if the association with ACPA-negative RA cannot be excluded. We evaluated the influence of genes conferring susceptibility to RA on the production of anti-VCP antibodies and tested the interaction of VCP with HLA in vitro . We analyzed 172 patients with RA of French white origin3. All provided informed consent and the Ethics Committee of Hopital Bicetre (AP-HP, Paris, France) approved the study. HLA-DRB1 typing and subtyping were performed by a polymerase chain reaction (PCR)-based method, using a panel of sequence-specific oligonucleotide probes. PADI4 haplotypes were obtained by typing 3 single-nucleotide polymorphisms (SNP: rs1886303, PADI4\_92, PADI4\_96 – CGC haplotype) by PCR-restriction fragment-length polymorphism (RFLP)4. PTPN22 (R620W; rs24766601 – T variant) and C5/TRAF1 region polymorphisms (rs10818488 – A variant) were genotyped by PCR-RFLP5,6. … Address correspondence to Dr. P. Migliorini; E-mail: paola.migliorini{at}med.unipi.it