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Porphyromonas gingivalis is one of the major oral pathogens implicated in the widespread inflammatory disorder periodontitis. Moreover, in recent years, P. gingivalis has been associated with the autoimmune disease rheumatoid arthritis. The peptidylarginine deiminase enzyme of P. gingivalis (PPAD) is a major virulence factor that catalyzes the citrullination of both bacterial and host proteins, potentially contributing to production of anticitrullinated protein antibodies. Considering that these antibodies are very specific for rheumatoid arthritis, PPAD appears to be a link between P. gingivalis, periodontitis, and the autoimmune disorder rheumatoid arthritis. PPAD was thus far considered unique among prokaryotes, with P. gingivalis being the only bacterium known to produce and secrete it. To challenge this hypothesis, we investigated the possible secretion of PPAD by 11 previously collected Porphyromonas isolates from a dog, 2 sheep, 3 cats, 4 monkeys, and a jaguar with periodontitis. Our analyses uncovered the presence of secreted PPAD homologues in 8 isolates that were identified as Porphyromonas gulae (from a dog, monkeys, and cats) and Porphyromonas loveana (from sheep). In all 3 PPAD-producing Porphyromonas species, the dominant form of the secreted PPAD was associated with outer membrane vesicles, while a minor fraction was soluble. Our results prove for the first time that the citrullinating PPAD exoenzyme is not unique to only 1 prokaryotic species. Instead, we show that PPAD is produced by at least 2 other oral pathogens.

Background: Mammary cancer is highly prevalent in dogs and cats and results in a poor prognosis due to critically lacking viable treatment options. Recent human and mouse studies have suggested that inhibiting peptidyl arginine deiminase enzymes (PAD) may be a novel breast cancer therapy. Based on the similarities between human breast cancer and mammary cancer in dogs and cats, we hypothesized that PAD inhibitors would also be an effective treatment for mammary cancer in these animals., Methods: Canine and feline mammary cancer cell lines were treated with BB-Cl-Amidine (BB-CLA) and evaluated for viability and tumorigenicity. Endoplasmic reticulum stress was tested by western blot, immunofluorescence, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Canine and feline mammary cancer xenograft models were created using NOD scid gamma (NSG) mice, and were treated with BB-CLA for two weeks., Results: We found that BB-CLA reduced viability and tumorigenicity of canine and feline mammary cancer cell lines in vitro. Additionally, we demonstrated that BB-CLA activates the endoplasmic reticulum stress pathway in these cells by downregulating 78 kDa Glucose-regulated Protein (GRP78), a potential target in breast cancer for molecular therapy, and upregulating the downstream target gene DNA Damage Inducible Transcript 3 (DDIT3). Finally, we established a mouse xenograft model of both canine and feline mammary cancer in which we preliminarily tested the effects of BB-CLA in vivo., Conclusion: We propose that our established mouse xenograft models will be useful for the study of mammary cancer in dogs and cats, and furthermore, that BB-CLA has potential as a novel therapeutic for mammary cancer in these species.

Aim: To recognize genetic associations of hydrochlorothiazide-induced change in serum uric acid (SUA) concentration., Patients & Methods: We conducted a genome-wide association study on hydrochlorothiazide-induced change in SUA in 214 Finnish men from the GENRES study. Replication analyses were performed in 465 Finns from the LIFE study., Results: In GENRES, we identified 31 loci associated with hydrochlorothiazide-induced change in SUA at p < 5 × 10 -5 . rs1002976 near VEGFC associated with the change in GENRES and in LIFE. rs950569 near BRINP3 associated with the change in SUA in GENRES and LIFE. The analysis of previously reported SNPs and candidate genes provided some proof for PADI4 and ABCC4., Conclusion: We report genetic markers that may predict the increase in SUA concentration during thiazide treatment.

Impaired apoptosis of rheumatoid arthritis (RA)‑fibroblast‑like synoviocytes (FLS) is pivotal in the process of RA. Peptidyl arginine deiminase type IV (PADI4) is associated with autoantibody regulation via histone citrullination in RA. The present study aimed to investigate the role of PADI4 in the apoptosis of RA‑FLS. FLS were isolated from patients with RA and a rat model. The effects of PADI4 on RA‑FLS were investigated in vitro and in vivo. Hypoxia‑induced autophagy was induced by 1% O2 and was detected by immunohistochemical and immunofluorescence analysis; in addition, apoptosis was detected by flow cytometry. RA‑FLS obtained from RA rat model exhibited significant proliferation under severe hypoxia conditions. Hypoxia also significantly induced autophagy and elevated the expression of PADI4. Subsequently, short hairpin RNA‑mediated PADI4 knockdown was demonstrated to significantly inhibit hypoxia‑induced autophagy and promote apoptosis in RA‑FLS. The results of these in vitro and in vivo studies suggested that PADI4 may be closely associated with hypoxia‑induced autophagy, and the inhibition of hypoxia‑induced autophagy by PADI4 knockdown may contribute to an increase in the apoptosis of RA‑FLS.

Background: Many studies have analyzed the association between peptidylarginine deiminase 4 (PADI4) -104C/T polymorphism and rheumatoid arthritis (RA). However, the results are inconsistent. This meta-analysis, based on different populations, updated and reevaluated the possible associations between PADI4 -104C/T polymorphism and the susceptibility to RA., Methods: A literature search was performed on PubMed and related Chinese databases up to April 2017. The association between PADI4 -104C/T polymorphism and RA risk was evaluated by calculating pooled odds ratios (ORs) and 95% confidence intervals (CIs)., Results: A total of seventeen studies, including 5,756 RA cases and 4,987 controls, were screened out. In the overall population, PADI -104C/T polymorphism was significantly associated with an increased RA risk. In this meta-analysis stratified by ethnicity, a significant association between PADI -104C/T polymorphism and RA risk was established in China and Japan., Conclusions: Our study indicated a significantly increased association between PADI -104C/T polymorphism and RA in Chinese and Japanese populations. Because most included populations in this meta-analysis were Asian, further studies are needed to elucidate whether the PADI4 -104C/T gene confers RA in other ethnic groups.

Objectives: Antibodies directed to citrullinated proteins are highly specific for rheumatoid arthritis (RA). Citrullination is catalyzed by peptidylarginine deiminase (PAD) enzymes. The current study examined the mRNA expression of PADI2 and PADI4 in bone marrow (BM) CD34+ cells from RA patients., Methods: CD34+ cells were purified from BM samples obtained from 48 RA patients and from 30 osteoarthritis (OA) patients during joint operations via aspiration from the iliac crest. The expression of mRNAs for PADI2, PADI4 and Sp1 was examined by quantitative reverse transcription PCR., Results: The expression of mRNA for PADI2 was significantly higher in RA BM CD34+ cells than OA BM CD34+ cells. The expression of mRNAs for PADI4 and Sp1 in RA BM CD34+ cells appeared to be increased compared to OA BM CD34+ cells, although it did not reach the statistical significance. The levels of mRNAs for PADI2, PADI4 and Sp1 were not correlated with serum C-reactive protein or with the administration of methotrexate or oral steroids. Finally, the level of PADI2 mRNA as well as that of PADI4 mRNA was significantly correlated with the level of Sp1 mRNA in RA BM CD34+ cells., Conclusions: These results indicate that the mRNA expression of PADI2, PADI4 and Sp1 is upregulated in RA BM CD34+ cells independently of the systemic inflammation or treatment regimen. Moreover, the data suggest that the enhanced mRNA expression of PADI2 and PADI4 in BM CD34+ cells might be a result of the enhanced expression of Sp1 gene in RA BM CD34+ cells.

Molecular characterization of balanced chromosomal abnormalities constitutes a powerful tool in understanding the pathogenic mechanisms of complex genetic disorders. Here we report a male with severe global developmental delay in the presence of a complex karyotype and normal microarray and exome studies. The subject, referred to as DGAP294, has two de novo apparently balanced translocations involving chromosomes 1 and 14, and chromosomes 4 and 10, disrupting several different transcripts of adhesion G protein-coupled receptor L2 (ADGRL2) and protocadherin 15 (PCDH15). In addition, a maternally inherited inversion disrupts peptidyl arginine deiminase types 3 and 4 (PADI3 and PADI4) on chromosome 1. None of these gene disruptions explain the patient's phenotype. Using genome regulatory annotations and chromosome conformation data, we predict a position effect ~370 kb upstream of a translocation breakpoint located at 14q12. The position effect involves forkhead box G1 (FOXG1), mutations in which are associated with the congenital form of Rett syndrome and FOXG1 syndrome. We believe the FOXG1 position effect largely accounts for the clinical phenotype in DGAP294, which can be classified as FOXG1 syndrome like. Our findings emphasize the significance of not only analyzing disrupted genes by chromosomal rearrangements, but also evaluating potential long-range position effects in clinical diagnoses.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that is characterized by chronic, destructive, and debilitating arthritis. Peptidyl arginine deiminase type 4 (PADI4) polymorphisms (PADI4&#95;94) have been reported to play a vital role in the disease. Nevertheless, the results were inconclusive. An electronic search of Embase, PubMed, CNKI, and WANFANG Date was performed to the identification of relevant studies published from 2003 to 2017. A total of 20 studies were enrolled as being eligible for analysis. In overall analysis, we had found a significant correlation between the PADI4&#95;94 polymorphisms and RA under T vs. C (OR = 1.05, 95% CI 1.01-1.08, P = 0.01). Nevertheless, there were no significant associations under other four genetic models. In the subgroup analysis, we had observed the similar results in Asian population (T vs. C: OR = 1.11, 95% CI 1.05-1.17, P = 0.001), whereas no significant difference were observed in Caucasian population under any genetic model (P > 0.05). In conclusion, our meta-analysis demonstrated that the PADI4&#95;94 polymorphisms might contribute to RA susceptibility especially in Asian populations but not in Caucasian populations. More well-designed studies with larger sample size are still required to further elucidate the relationship.

We previously demonstrated that renal tubular peptidylarginine deiminase-4 (PAD4) is induced after ischemia-reperfusion (IR) injury and this induction of PAD4 exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation and neutrophil infiltration. However, the mechanisms of renal tubular PAD4 induction after IR remain unknown. Here, we tested the hypothesis that ATP, a proinflammatory danger-associated molecular pattern (DAMP) ligand released from necrotic cells after IR injury, induces renal tubular PAD4 and exacerbates ischemic AKI via P2 purinergic receptor activation. ATP as well as ATPγS (a nonmetabolizable ATP analog) induced PAD4 mRNA, protein, and activity in human and mouse renal proximal tubule cells. Supporting the hypothesis that ATP induces renal tubular PAD4 via P2X7 receptor activation, A804598 (a selective P2X7 receptor antagonist) blocked the ATP-mediated induction of renal tubular PAD4 whereas BzATP (a selective P2X7 receptor agonist) mimicked the effects of ATP by inducing renal tubular PAD4 expression and activity. Moreover, ATP-mediated calcium influx in renal proximal tubule cells was blocked by A804598 and was mimicked by BzATP. P2X7 activation by BzATP also induced PAD4 expression and activity in mouse kidney in vivo. Finally, supporting a critical role for PAD4 in P2X7-mediated exacerbation of renal injury, BzATP exacerbated ischemic AKI in PAD4 wild-type mice but not in PAD4-deficient mice. Taken together, our studies show that ATP induces renal tubular PAD4 via P2X7 receptor activation to exacerbate renal tubular inflammation and injury after IR.

Wnt (wingless)/β-catenin signaling is critical for tumor progression and is frequently activated in colorectal cancer as a result of the mutation of adenomatous polyposis coli (APC); however, therapeutic agents targeting this pathway for clinical use are lacking. Here we report that nitazoxanide (NTZ), a clinically approved antiparasitic drug, efficiently inhibits Wnt signaling independent of APC. Using chemoproteomic approaches, we have identified peptidyl arginine deiminase 2 (PAD2) as the functional target of NTZ in Wnt inhibition. By targeting PAD2, NTZ increased the deamination (citrullination) and turnover of β-catenin in colon cancer cells. Replacement of arginine residues disrupted the transcriptional activity, and NTZ induced degradation of β-catenin. In Wnt-activated colon cancer cells, knockout of either PAD2 or β-catenin substantially increased resistance to NTZ treatment. Our data highlight the potential of NTZ as a modulator of β-catenin citrullination for the treatment of cancer patients with Wnt pathway mutations.

Background/aims: Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting up to 1% of the population worldwide. The aim of the present study was to investigate whether miRNA-146a rs2910164, miRNA-499 rs3746444, IRAK1 rs3027898 and PADI4 rs1748033 polymorphisms are associated with susceptibility to RA in Egyptians and whether they influence disease severity and activity., Methods: The study was performed on 104 unrelated RA patients and 112 healthy subjects. RA patients were further subdivided into active and inactive RA groups. Polymorphisms were genotyped by using real-time polymerase chain reaction with TaqMan allelic discrimination assay., Results: Significant differences in the frequency of miRNA-146a rs2910164, miRNA-499 rs3746444, IRAK1 rs3027898 and PADI4 rs1748033 alleles and genotypes were observed between RA patients and controls. Only CA and AA genotypes of IRAK1 rs3027898 shows a significant difference between active and inactive subgroups. MiRNA-146a rs2910164 and IRAK1 rs3027898 polymorphisms were a risk factor for predisposition to RA in codominant and dominant tested inheritance models, while, the miRNA-499 rs3746444 and PADI4 rs1748033 polymorphisms were a risk factor in codominant and recessive one. CG and GG genotypes of miRNA-146a rs2910164 were associated with positive erosions. CA genotype of IRAK1 rs3027898 was associated with low disease activity and negative erosions, while, the AA genotype was associated with high disease activity. CC genotype of PADI4 rs1748033 was associated with negative rheumatoid factor., Conclusion: The 4 studied SNPs were likely to play an important role in the susceptibility to RA and can influence disease severity and activity in Egyptian population., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Mechanisms driving acute food allergic reactions have not been fully characterized. We profile the dynamic transcriptome of acute peanut allergic reactions using serial peripheral blood samples obtained from 19 children before, during, and after randomized, double-blind, placebo-controlled oral challenges to peanut. We identify genes with changes in expression triggered by peanut, but not placebo, during acute peanut allergic reactions. Network analysis reveals that these genes comprise coexpression networks for acute-phase response and pro-inflammatory processes. Key driver analysis identifies six genes (LTB4R, PADI4, IL1R2, PPP1R3D, KLHL2, and ECHDC3) predicted to causally modulate the state of coregulated networks in response to peanut. Leukocyte deconvolution analysis identifies changes in neutrophil, naive CD4 + T cell, and macrophage populations during peanut challenge. Analyses in 21 additional peanut allergic subjects replicate major findings. These results highlight key genes, biological processes, and cell types that can be targeted for mechanistic study and therapeutic targeting of peanut allergy.

Importance: Alteration of ex vivo magnetic resonance imaging transverse relaxation is associated with late-life cognitive decline even after controlling for common neuropathologic conditions. However, the underlying neurobiology of this association is unknown., Objective: To investigate the association between brain gene expression, DNA methylation, and alteration of magnetic resonance imaging transverse relaxation in late-life cognitive decline., Design, Setting, and Participants: Data came from 2 community-based longitudinal cohort studies of aging and dementia, the Religious Orders Study, which began in 1993, and the Rush Memory and Aging Project, which began in 1997. All participants agreed to undergo annual clinical evaluations and to donate their brains after death. By October 24, 2016, a total of 1358 individuals had died and had brain autopsies that were approved by board-certified neuropathologists. Of those, 552 had undergone ex vivo imaging. The gene expression analysis was limited to 174 individuals with both imaging and brain RNA sequencing data. The DNA methylation analysis was limited to 225 individuals with both imaging and brain methylation data., Main Outcomes and Measures: Maps of ex vivo magnetic resonance imaging transverse relaxation were generated using fast spin echo imaging. The target was a composite measure of the transverse relaxation rate (R2) that was associated with cognitive decline after controlling for common neuropathologic conditions. Next-generation RNA sequencing and DNA methylation data were generated using frozen tissue from the dorsolateral prefrontal cortex. Genome-wide association analysis was used to investigate gene expression and, separately, DNA methylation for signals associated with the R2 measure., Results: Of the 552 individuals with ex vivo imaging data, 394 were women and 158 were men, and the mean (SD) age at death was 90.4 (6.0) years. Four co-expressed genes (PADI2 [Ensembl ENSG00000117115], ZNF385A [Ensembl ENSG00000161642], PSD2 [Ensembl ENSG00000146005], and A2ML1 [Ensembl ENSG00000166535]) were identified, of which higher expressions were associated with slower R2. The association of R2 with cognitive decline was attenuated when the gene expression signals were added to the model, such that the mean (SE) coefficient of association was reduced from 0.028 (0.008) (P < .001) to 0.019 (0.009) (P = .03). The DNA methylation scan did not detect a genome-wide significant signal, but it revealed an anticorrelation between R2 and DNA methylation in many of the cytosine-guanine dinucleotides., Conclusions and Relevance: Brain gene expression and DNA methylation dysregulations are implicated in the alteration of brain tissue properties associated with late-life cognitive decline above and beyond the influence of common neuropathologic conditions.

Rheumatoid arthritis (RA) is a chronic inflammatory articular disease that is characterized by synovial hyperplasia. A number of signaling pathways are associated with the development and induced symptoms of RA. Notably, patients with RA have increased protein citrullination and generation of auto‑antibodies against citrullinated proteins. Genome wide association studies have revealed that peptidyl‑arginine deiminase 4 (PADI4) is an enzyme implicated in citrullination in the RA synovium. Autoantibodies targeting citrullinated proteins are used as diagnostic markers in patients with RA. The functions associated with citrullinated proteins are thought to induce autoimmunity, however, the regulatory mechanisms of citrullination via PADI4 are unclear. The group has previously cloned an E3 ubiquitin ligase, synoviolin (SYVN1), from the RA synovium, demonstrating that SYVN1 serves critical roles in synovial hyperplasia. The data indicated that the endoplasmic reticulum (ER) associated degradation system, which involves SYVN1, may have important roles in the proliferation of synoviocytes. In addition, ubiquitination by SYVN1 is associated with fibrosis, inflammation and cytokine production via the regulation of ER stress signals and quality control of proteins. The present study investigated the crosstalk between the representative post‑translational signaling processes, citrullination and ubiquitination. The results revealed that PADI4 interacted with SYVN1 directly and that overexpression of PADI4 suppressed the ubiquitination of proteins. Thus, a reduction in ER stress induced by PADI4 may abrogate the initiation of chronic RA by suppressing the proliferative signals of RA synoviocytes.

We previously found that transgenic mice overexpressing MMTV-FLAG-hPAD2 (PAD2OE) developed spontaneous skin lesions, with a subset of these lesions progressing to invasive squamous cell carcinoma (SCC). The goal of this report was to better understand the potential mechanisms by which PAD2 overexpression promotes skin cancer. Here, PAD2OE mice were treated with the carcinogen, 9,10-dimethyl-1,2-benzanthracene and with O-tetradecanoylphorbol-13-acetate and then scored for papilloma formation. Additionally, tumor sections were evaluated for evidence of tumor cell invasion and inflammation. We found that the total number of papillomas was significantly increased in PAD2OE mice compared to controls. Histopathologic analysis of the lesions found that in PAD2OE skin tumors progressed to invasive SCC more frequently than controls. Additionally, we found that PAD2OE lesions were highly inflamed, with a dense inflammatory cell infiltrate and an associated increase in nuclear phospho-STAT3 (signal transducer and activator of transcription 3) in the transgenic tumors. These data suggest that overexpression of the hPAD2 transgene in the epidermis increases the malignant conversion rate of benign tumors by promoting an inflammatory microenvironment.

Onset of castration-resistance prostate cancer (CRPC) after long-term androgen deprivation therapy remains a major obstacle in the treatment of prostate cancer. The peptidylarginine deiminase PADI2 has been implicated in chronic inflammatory diseases and cancer. Here we show that PADI2 is an androgen-repressed gene and is upregulated in CRPC. PADI2 expression was required for survival and cell-cycle progression of prostate cancer cells, and PADI2 promoted proliferation of prostate cancer cells under androgen-deprived or castration conditions in vitro and in vivo Cytoplasmic PADI2 protected the androgen receptor (AR) against proteasome-mediated degradation and facilitated AR binding to its target genes after nuclear translocation and citrullination of histone H3 amino acid residue R26. In contrast, mutant PADI2 D180A failed to affect AR stability, nuclear translocation, or transcriptional activity. PADI2 mediated AR control in a manner dependent on its enzymatic activity and nuclear localization, as correlated with increased histone H3 citrullination. Notably, coadministration of the PADI inhibitor Cl-Amidine and the AR signaling inhibitor enzalutamide synergized in inhibiting CRPC cell proliferation in vitro and tumor growth in vivo Overall, our results establish PADI2 as a key mediator for AR in prostate cancer progression, especially CRPC, and they suggest PADI as novel therapeutic targets in this disease setting. Cancer Res; 77(21); 5755-68. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Rheumatoid arthritis (RA) is a common autoimmune disease with a complex genetic background. The peptidyl arginine deiminase type IV (PADI4) gene has been associated with RA susceptibility in several populations. We addressed the relationship between three exonic PADI4 gene single nucleotide polymorphisms (SNPs) PADI4&#95;89 (rs11203366), PADI4&#95;90 (rs11203367) and PADI4&#95;92 (rs874881) and related haplotypes with RA in a population from Southern México. This study included 200 RA patients and 200 control subjects. The SNPs were evaluated using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) technique, and antibodies to cyclic citrullinated peptides (anti-CCP) were measured by enzyme-linked immunosorbent assay (ELISA). In this population, the minor alleles of PADI4&#95;89∗G, PADI4&#95;90∗T and PADI4&#95;92∗G gene polymorphisms were associated with RA susceptibility (OR=1.34, p=0.04; OR=1.35, p=0.03; OR=1.34, p=0.04; respectively). The GTG haplotype was also significantly associated with RA (OR=2.27 95%CI=1.18-4.41; p=0.008), but did not show association with levels of anti-CCP antibodies and clinical parameters. In conclusion, our replication study in a Southern Mexican population suggests that PADI4 individual polymorphisms and the related susceptibility haplotype (GTG) are also genetic risk markers for RA., (Copyright © 2017 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Many studies have evaluated the correlation between peptidylarginine deiminase 4 (PADI4) -92C/G polymorphism and rheumatoid arthritis (RA), but the results remain inconclusive. Therefore, we performed a meta-analysis in the Chinese population to provide comprehensive data on the association between PADI4 -92C/G polymorphism and RA. Eligible studies published before May 2016 were identified in PubMed and Chinese databases. The strengths of these associations were assessed by pooled odds ratios (OR) and 95% confidence interval (CI). Eight studies documenting a total of 1351 RA cases and 1585 controls were included in this meta-analysis. In the overall analysis, a significant association between the PADI4 -92C/G polymorphism and RA was found in the Chinese population (G vs C: OR=1.32, 95%CI=1.02-1.71; GG+CG vs CC: OR=1.75, 95%CI=1.20-2.53). The subgroup analyses stratified by geographic area(s) and source of controls revealed significant results in South China, in hospital-based studies and population-based studies. In summary, this meta-analysis suggested that PADI4 -92C/G polymorphism may be associated with the RA incidence in the Chinese population, especially for South China. Further studies conducted on other ethnic groups are required for definite conclusions.

Studies have shown that the genetic risk factors for rheumatoid arthritis (RA) differ substantially between Asian and Caucasian populations. Even among Asian populations, the genetic contributions of NLRP1, PTPN22 and PADI4 have been controversial. Consequently, we sought to address these separate findings and determine whether any of these proposed risk variants are associated with RA susceptibility, onset, DAS activity and erosion in a Singaporean Chinese cohort. We genotyped five SNPs within NLRP1 (rs878329 and rs6502867), PTPN22 (rs2488457 and rs6665194), and PADI4 (rs2240340) in 500 anti-cyclic citrullinated peptide antibody-positive (ACPA) patients with RA and 500 healthy controls using TaqMan assays. The CC genotype of NLRP1 rs878329 and TT genotype of PADI4 rs2240340 were associated with RA susceptibility. The risk association of the T allele of PADI4 rs2240340 with RA was confirmed through a meta-analysis based on previous reports in Asian populations. The GG genotype of PTPN22 rs6665194 (-3508A>G) was associated with significantly reduced risk of RA. No significant association was found for NLRP1 rs6502867 T/C and PTPN22 rs2488457 G/C polymorphisms. None of the five SNPs was associated with RA's clinical features. This work supports the association of the T allele of PADI4 rs2240340 with RA in Asians. The roles of NLRP1 rs878329 G/C and PTPN22 rs6665194 A/G polymorphisms were demonstrated for the first time. We also propose rs6665194 to be a promising candidate for RA risk evaluation between ethnicities.

Periodontitis, an inflammatory disease initiated by Gram-negative bacteria such as Porphyromonas gingivalis ( Pg), is considered as a risk factor for rheumatoid arthritis (RA). Our study aimed to determine the effect of Pg and its LPS on the expression of peptidyl arginine deiminase isotypes (PADs) in human primary chondrocytes (HC). HCs were infected with Pg and activated by its LPS (LPS- Pg). The mRNA expression levels of human PADs (1, 2, 3, 4 and 6) and bacterial enzyme (PADPg) were quantified by RT-qPCR. Cellular extracts served to measure the enzymatic activities of PADs and PADPg and to visualize the profiles of citrullinated proteins/peptides by Western blotting. Our data showed significant inhibitions of mRNA expressions of human PAD-2, PAD-3 and PAD-4 during infection of HC with live Pg. Activation of HC by LPS- Pg increased mRNA expressions of human PAD-2 and PAD-3. The PADPg enzymatic activity was significantly increased in only infected HC. Analysis of citrullinated proteins/peptides profiles revealed the occurrence of low molecular bands only in cellular extracts from HC infected with Pg. Our data showed that Pg and its LPS differentially regulate the expression of PADs in human chondrocytes and that Pg favors the apparition of new citrullinated proteins/peptides.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis., (Published by Elsevier Ltd.)

Our recent studies of peptidylarginine deiminase 4 (PAD4) demonstrate that its non-catalytic Ca 2+ -binding sites play a crucial role in the assembly of the correct geometry of the enzyme. Here, we examined the folding mechanism of PAD4 and the role of Ca 2+ ions in the folding pathway. Multiple mutations were introduced into the calcium-binding sites, and these mutants were termed the Ca1&#95;site, Ca2&#95;site, Ca3&#95;site, Ca4&#95;site and Ca5&#95;site mutants. Our data indicate that during the unfolding process, the PAD4 dimer first dissociates into monomers, and the monomers then undergo a three-state denaturation process via an intermediate state formation. In addition, Ca 2+ ions assist in stabilizing the folding intermediate, particularly through binding to the Ca3&#95;site and Ca4&#95;site to ensure the correct and active conformation of PAD4. The binding of calcium ions to the Ca1&#95;site and Ca2&#95;site is directly involved in the catalytic action of the enzyme. Finally, this study proposes a model for the folding of PAD4. The nascent polypeptide chains of PAD4 are first folded into monomeric intermediate states, then continue to fold into monomers, and ultimately assemble into a functional and dimeric PAD4 enzyme, and cellular Ca 2+ ions may be the critical factor governing the interchange.

Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should be a dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis.

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFN γ , or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group ( p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro . PADs citrullinated Ro60 and La ribonucleoproteins in vivo . Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.

Aim: Rheumatoid arthritis (RA) is a chronic autoimmune disease which affects many tissues and organs, but majorly attacks synovial joints. Beyond the major histocompatibility complex (MHC) genes, Peptidyl arginine deiminase type IV (PADI4) has been suggested to be associated with RA susceptibility. Evidence regarding the association of PADI4 single nucleotide polymorphisms (SNP) and RA is controversial, thus we conducted this large-scale case-control study to assess the association of rs874881 and rs11203367 PADI4 SNPs with susceptibility to RA., Materials and Methods: Study population (including 665 RA patients and 392 sex-, age-, and ethnicity-matched healthy controls) were enrolled from Rheumatology Research Center of Tehran University of Medical Sciences, Shariati hospital., Results: Allele or genotype frequencies of the investigated PADI4 SNPs were not different between RA patients and healthy subjects; genotypes (expressed as odds ratios) of rs11203367 [TT 0.98 (0.68-1.4), CT 0.93 (0.71-1.24), P value > 0.05] and rs874881 [CC 1.02 (0.71-1.46), CG (0.70-1.39), p value > 0.05] did not affect RA risk. Disease severity score DAS28, RF and anti-CCP antibodies of RA patients were not different between various genotypes of PADI4 SNPs., Conclusions: These findings were similar for haplotypes and diplotypes of rs11203367 and rs874881 PADI4 SNPs. In conclusion, in this case-control study with sufficient sample size to detect associations, we observed that PADI4 SNPS rs11203367 and rs874881 do not significantly determine RA susceptibility; which is in line with studies of some European populations. It seems RA pathogenesis might be different among various ethnicities, which encourage us to consider these differences in developing therapeutic interventions for management of patients.

Objective: Lung diseases (LD) are common extra-articular manifestations in rheumatoid arthritis (RA). However, little is known about factors associated with susceptibility to rheumatoid arthritis-related lung diseases (RA-LD). The aim of the present study was to investigate whether the single nucleotide polymorphisms (SNPs) of PADI4 and HLA-DRB1 alleles were associated with RA-LD., Methods: Blood samples and clinical data were collected from 116 consecutive RA patients who satisfied the 1987 American College of Rheumatology classification criteria. RA-LD was diagnosed using high-resolution computed tomography of the chest. All patients were genotyped for SNPs of PADI4 and HLA-DRB1 alleles and analyzed for full amino acid sequence of the HLA protein corresponding to a 4-digit HLA typing. Data were analyzed by independent t test (or Mann-Whitney test) for continuous variables, Chi-square test (or Fisher's exact test) and trend test for categorical variables, and logistic regression analysis., Results: Ninety-four (81.0 %) RA patients had LD, of which eight (6.9 %) had interstitial lung disease (ILD) and 92 (79.3 %) had airway abnormalities in which 64 (55.2 %) showed bronchiectasis and 47 (40.5 %) revealed bronchial wall thickening. The recessive genotype of padi4&#95;92 was susceptible to airway abnormalities (OR = 2.22, 95 % CI = 1.05-4.49, p = 0.034). Tryptophan at position 9 of HLA-DRB1 sequence was associated with the susceptibility to RA-ILD (OR = 22.89, 95 % CI = 1.20-432.56, p = 0.037)., Conclusion: PADI4 polymorphisms and HLA-DRB1 alleles could attribute differently to the development of airway abnormalities and ILD, respectively, in RA.

PADI4 (peptidyl deiminase isoform 4) is overexpressed in many tumor tissues and converts arginine residues to citrulline residues. This study used an Illumina SNP microarray and a TaqMan assay to determine the possible association of the PADI4 gene with various tumor risks. Both genotyping methods demonstrated significant associations between the tag SNPs rs1635566 and rs882537 in the PADI4 locus with gastric carcinoma in two independent cohorts. Based on this genotyping result, we used the Cancer Pathway Finder, p53 Signaling, Signal Transduction and Tumor Metastasis PCR arrays to investigate the tumorigenic pathway of PADI4 in MNK-45 cells derived from gastric carcinoma. We detected significantly decreased expression levels of CXCR2, KRT14 and TNF-α in MNK-45 cells that were treated with anti-PADI4 siRNA. We also detected increased expression of these three genes in MNK-45 cells transfected with a pcDNA3.1 plasmid overexpressing PADI4. A highly similar result was also obtained for SGC 7901 cells, which also originate from gastric carcinoma. Our result indicates that the PADI4 gene has genetic susceptibility in gastric carcinoma. PADI4 contributes to gastric tumorigenesis by upregulating CXCR2, KRT14 and TNF-α expression, which are well known to activate angiogenesis, cell proliferation, cell migration and the immune microenvironment in tumors., Competing Interests: The authors declare no financial conflict of interest.