Peptidylarginine Deiminase Type Research Articles

Hypoxia‑induced autophagy is inhibited by PADI4 knockdown, which promotes apoptosis of fibroblast‑like synoviocytes in rheumatoid arthritis

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.

The relationship of PADI4_94 polymorphisms with the morbidity of rheumatoid arthritis in Caucasian and Asian populations: a meta-analysis and system review.

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_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_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_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.

Affinity maturation shapes the function of agonistic antibodies to peptidylarginine deiminase type 4 in rheumatoid arthritis

ObjectivesThe citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is the target of a polyclonal group of autoantibodies in patients with rheumatoid arthritis (RA). A subgroup of such antibodies, initially identified...

An in silico analysis of primary and secondary structure specificity determinants for human peptidylarginine deiminase types 2 and 4

Human peptidylarginine deiminases (hPADs) are a family of five calcium-dependent enzymes that facilitate citrullination, which is the post-translational modification of peptidyl arginine to peptidyl citrulline. The isozymes hPAD2 and hPAD4 have been implicated in the development and progression of several autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. To better characterize the primary and secondary structure determinants of citrullination specificity, we mined the literature for protein sequences susceptible to citrullination by hPAD2 or hPAD4. First, protein secondary structure classification (α-helix, β-sheet, or coil) was predicted using the PSIPRED software. Next, we used motif-x and pLogo to extract and visualize statistically significant motifs within each data set. Within the data sets of peptides predicted to lie in coil regions, both hPAD2 and hPAD4 appear to favor citrullination of glycine-containing motifs, while distinct hydrophobic motifs were identified for hPAD2 citrullination sites predicted to reside within α-helical and β-sheet regions. Additionally, we identified potential substrate overlap between coil region citrullination and arginine methylation. Together, these results confirm the importance and offer some insight into the role of secondary structure elements for citrullination specificity, and provide biological context for the existing hPAD specificity and arginine post-translational modification literature.

Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice.

Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases.

PADI4 polymorphisms and the functional haplotype are associated with increased rheumatoid arthritis susceptibility: A replication study in a Southern Mexican population

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_89 (rs11203366), PADI4_90 (rs11203367) and PADI4_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_89∗G, PADI4_90∗T and PADI4_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.

High‐Titer Rheumatoid Arthritis Antibodies Preferentially Bind Fibrinogen Citrullinated by Peptidylarginine Deiminase 4

Most patients with rheumatoid arthritis (RA) harbor antibodies to citrullinated autoantigens such as citrullinated fibrinogen. Two isoforms of peptidylarginine deiminase (PAD), PAD type 2 (PAD2) and PAD4, which catalyze citrullination with different substrate specificities, can be detected in the synovium of RA patients. This study was undertaken to determine whether RA antibodies preferentially bind PAD2- or PAD4-citrullinated fibrinogen. RA patient and normal donor plasma specimens were tested for binding to PAD2- or PAD4-citrullinated fibrinogen, native fibrinogen, or citrullinated fibrinogen peptides in various dilutions by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Bands corresponding to masses demonstrating RA antibody reactivity by Western blotting were excised and analyzed by mass spectrometry. At low antibody titers (1:40 and 1:100), there was no significant difference between RA antibody reactivity to PAD2- and PAD4-citrullinated fibrinogen. When plasma was further diluted to 1:250 and 1:1,000, RA patient plasma bound PAD4-citrullinated fibrinogen significantly more than PAD2-citrullinated fibrinogen, as measured by ELISA and Western blotting. An increased antibody titer was associated with increased avidity for both PAD2- and PAD4-citrullinated fibrinogen. Both enzymes hypercitrullinated fibrinogen, but PAD4 citrullinated arginines more intermittently, generating a mix of citrullinated and noncitrullinated arginines. Peptide ELISA and preadsorption assays confirmed that the region of intermittent citrullination accounts for the majority of RA antibody binding to the β-chain of citrullinated fibrinogen. At high titers, RA antibodies preferentially bind fibrinogen modified by PAD4, because intermittent citrullination offers a more diverse assortment of citrullinated epitopes.

PADI4 and the HLA-DRB1 shared epitope in juvenile idiopathic arthritis.

ObjectiveBoth genetic and environmental factors are associated with susceptibility to juvenile idiopathic arthritis (JIA). Many studies have reported that both a ‘shared epitope’ (SE) encoded by several HLA-DRB1 alleles and the peptidyl arginine deiminase type 4 (PADI4) gene polymorphisms are associated with susceptibility to rheumatoid arthritis (RA). However, it is uncertain whether JIA and RA share the latter genetic risk factor. Therefore, here we investigated relationships between HLA-SE and PADI4 polymorphisms with clinical subtypes of JIA.MethodsJIA patients (39 oligoarthritis, 48 RF-positive polyarthritis, 19 RF-negative polyarthritis and 82 systemic) and 188 healthy controls were genotyped for HLA-DRB1 by PCR-sequence-specific oligonucleotide probe methodology. Three PADI4 gene single nucleotide polymorphisms (SNPs), rs2240340, rs2240337 and rs1748033, were genotyped using TaqMan SNP Genotyping Assays.ResultsFrequencies of the HLA-SE were higher in RF-positive polyarticular JIA than in healthy controls. RF-positive polyarticular JIA was associated with HLA-SE (OR = 5.3, 95% CI = 2.5–11.9, pc < 0.001). No associations were found between clinical subtypes of JIA and PADI4 allele frequency. Nonetheless, rs2240337 in the PADI4 gene was significantly associated with anti-cyclic citrullinated peptide antibody (ACPA)-positivity in JIA. The A allele at rs2240337 was a significant risk factor for ACPA positivity in JIA (OR = 5.6, 95% CI = 1.71–23.7 pc = 0.03).ConclusionPADI4 gene polymorphism is associated with ACPA-positivity in JIA. The association of HLA-SE with RF-positive polyarticular JIA as well as RA is confirmed in Japanese. Thus, HLA-SE and PADI4 status both influence JIA clinical manifestations.

PADI4 Epigenetically Suppresses p21 Transcription and Inhibits Cell Apoptosis in Fibroblast-like Synoviocytes from Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is characterized by synovial lining hyperplasia, which involves abnormal growth of fibroblast-like synoviocytes (FLSs). This study aimed to investigate the function and molecular mechanism of peptidylarginine deiminase type 4 (PADI4) in FLSs isolated from RA patients (RA-FLSs). FLSs were isolated from RA patients and transfected with small interfering RNAs (siRNAs) or PADI4 overexpression plasmid. FLSs were treated by Adriamycin (ADR) to induce apoptosis, and apoptotic cells were detected by flow cytometry. The expression of PADI4, p53 and p21 was detected by qRT-PCR and Western blot analysis. The recruitment of PADI4 and histone H3 arginine modifications to p21 promoter was measured by chromatin immunoprecipitation. The results showed that knockdown of PADI4 promoted the apoptosis of RA-FLSs and the expression of p53 and p21. Ectopic expression of PADI4 inhibited ADR-induced apoptosis of RA-FLSs, and down-regulated the expression of p53 and p21. In RA-FLSs, global H3 citrullination (CitH3) and H3 arginine 17 methylation levels were dynamically changed by PADI4 and ADR treatment. PADI4 and H3 could bind p21 promoter region to regulate p21 expression. In conclusion, PADI4 contributes to the pathogenesis of RA by protecting FLSs from apoptosis. PADI4 suppresses p21 transcription through altering histone H3 arginine modifications on p21 promoter region. Our study provides new insight into the anti-apoptotic role of PADI4 in RA development.

A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination.

NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, which have been erroneously classified as “NETosis.” While these NETosis mimics share morphological similarities with NETosis (i.e., extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE. Distinguishing NETosis from LTH and defective mitophagy is paramount to understanding the role of neutrophil damage in immunity and the pathogenesis of human diseases. This provides a framework to design specific inhibitors of these distinct biological processes in human disease.

Association of anti-peptidyl arginine deiminase antibodies with radiographic severity of rheumatoid arthritis in African Americans.

BackgroundEvidence suggests that the presence of peptidyl arginine deiminase type 4 (PAD4) antibodies is associated with radiographic-severity rheumatoid arthritis (RA) among Caucasian patients. The presence of anti-PAD4 antibodies that were cross-reactivity against PAD3 was associated with more aggressive erosive disease (compared with the presence of anti-PAD4 antibodies without anti-PAD3 crossreactivity) in Caucasian RA patients. The objectives of this study were to determine the prevalence of serum anti-PAD4 and anti-PAD4/PAD3 cross-reactive autoantibodies in African Americans with RA and whether these antibodies associate with radiographic severity and radiographic progression.MethodsSerum anti-PAD4 and anti-PAD4/PAD3 antibodies were measured by immunoprecipitation, and the temporal trends in titers were analyzed. We compared total radiographic scores among anti-PAD4-positive, anti-PAD4/PAD3-positive, and anti-PAD4-negative patients and used a zero-inflated negative binomial model to determine associations between radiographic severity and antibody status. Logistic regression was used to analyze radiographic progression.ResultsOf 192 African-American patients with RA, 73 % were anti-citrullinated peptide/protein antibody (ACPA)-positive, 46 out of 192 (24 %) of whom had serum anti-PAD4 antibodies. Median (interquartile range) total Sharp van der Heijde radiographic scores were 2 (1–97.5) in ACPA-positive patients and 0 (0–3) in ACPA-negative patients (P < 0.001). Of the 46 anti-PAD4-positive patients, 20 had anti-PAD4 antibodies that cross-reacted with PAD3. In patients with early RA, anti-PAD4 and anti-PAD4/PAD3 antibody titers increased over time (P = 0.006, P = 0.001, respectively). Median (interquartile range) total radiographic scores were higher for anti-PAD4-positive than for anti-PAD4-negative patients (3 (1–115) versus 2 (0–11), respectively; P = 0.005). Median (interquartile range) total radiographic score for anti-PAD4/PAD3-positive patients was 76 (3–117) (P < 0.001) versus anti-PAD4-negative patients. Only anti-PAD4/PAD3 antibodies associated with radiographic severity (incidence rate ratio = 2.81; 95 % confidence interval 1.23, 6.43).ConclusionThis analysis suggests that autoantibodies against PAD4 and PAD3 proteins may serve as biomarkers for identifying African-American patients with RA and higher radiographic severity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1126-7) contains supplementary material, which is available to authorized users.

Role of peptidylarginine deiminase type 4 in gastric cancer.

Peptidylarginine deiminase type 4 (PADI4) post-translationally converts peptidylarginine to citrulline, appearing to be overexpressed in numerous carcinomas. The current study aimed to investigate the expression of PADI4 in gastric cancer tissues and its effect on the biological activities of SGC-7901 and AGS tumor cell lines. The expression of PADI4 was determined in gastric cancer and normal gastric mucosa tissues using western blot analysis and reverse transcription-quantitative polymerase chain reaction. Gastric cancer cell lines were divided into the following groups: Mock group (subjected to transfection reagent); negative group [subjected to small interfering RNA (siRNA) transfection]; PADI4 siRNA group (subjected to PADI4 siRNA transfection); 5-fluorouracil (5-Fu) group (subjected to 5-Fu); and 5-Fu + siRNA transfection group (subjected to 5-Fu and PADI4 siRNA transfection). The effects of silencing PADI4 with the above measures on the proliferation and invasion of SGC-7901 and AGS cells were determined by MTT and Transwell chamber assays. In addition, propidium iodide staining was performed to detect the effects of PADI4 on the cell cycle. A significant increase in the expression of PADI4 mRNA in gastric cancer tissue compared with normal mucosa tissue was identified (P<0.05). The proliferation and invasion of SGC-7901 and AGS cells were significantly decreased in the PADI4 siRNA group. Furthermore, flow cytometry DNA analysis revealed that silencing PADI4 resulted in significant S phase arrest and marked decrease of cells in the G2/M phase. PADI4 siRNA coupled with 5-Fu significantly enhanced its inhibitory effect on the proliferation of gastric cancer cells. In conclusion, PADI4 demonstrated high expression in gastric cancer and served an important role in the biological activities of gastric cancer cells involving cell proliferation, invasion and cell cycle. As a result, PADI4 may be a valid cancer susceptibility gene and potential target for cancer therapy.

Monomeric Form of Peptidylarginine Deiminase Type I Revealed by X-ray Crystallography and Small-Angle X-ray Scattering

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca2+-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca2+ at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.

Study of Peptidyl Arginine Deiminases 4 (PAD 4) Antibodies in Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is one of the most commonly occurring form of inflammatory polyarthritis. If untreated,20%–30% of RA patients become debilitated within the first three years following initial diagnosis. The clinical examinationoften fail to identify patients with early RA partly due to heterogeneity of disease presentation and course. To meet the needfor improved diagnostic and prognostic tests, various serum biomarkers are being assessed, including a wide range ofautoantibodies. Autoantibodies against peptidyl arginine deiminase type 4 (PAD-4) have recently been described as a specificbiomarker in subjects with clinically apparent RA. In this study, we tested the presence of anti-PAD4 antibodies in 40Egyptian subjects with RA and their first degree relatives in order to determine whether these autoantibodies play a role inearly disease evolution. In addition, we aimed to describe the Anti-PAD4 relation to anti-CCP autoantibodies, and potentialassociations with a more severe RA phenotype. The study was carried out on total of 100 subjects divided into; 40 RApatients, 40 of first degree relatives (sister or brother) of RA patients and 20 matched controls. Results: The resultsindicated that the serum level of Anti-PAD 4 is increased in Egyptian RA patients and their relatives and has a positiveassociation with disease activity. Also its level together with Anti-CCP provides a good diagnostic tool of RA. Conclusion:The serum level of Anti-PAD4 may provide diagnostic information of RA and may be considered as an early marker of thedisease among the first degree relatives of RA patients. [Egypt J Rheumatology & Clinical Immunology, 2016; 4(1): 59-66]

Characterization of the porcine peptidylarginine deiminase type VI gene (PADI6) promoter: Sp1 regulates basal transcription of the porcine PADI6

It is a general consensus that oocyte quality is the key to embryo survival in pig reproduction. Thus, study on regulation of the ovary-associated gene is of great significance in pig breeding. Peptidylarginine deiminases (PADs) are a family of enzymes which catalyze the conversion of arginine to citrulline in proteins. The peptidylarginine deiminases type VI gene (PADI6) is mainly expressed in the ovary, and plays an important role in oocyte growth, fertilization and early embryo development. However, until now, little is known about its transcriptional regulation mechanism. Here, we firstly isolated and characterized the 5′-flanking region of porcine PADI6 gene. We determined the transcription start site using 5′-rapid amplification of cDNA ends (RACE) analysis, and identified the minimal promoter (−85/+68) that drove the basal expression of PADI6 by constructing various progressive deletions. Mutational analysis and electrophoretic mobility shift assays demonstrated Sp1 bound to the −56/−47 region of the PADI6 promoter. Furthermore, overexpression of Sp1 significantly increased the promoter activity and promoted PADI6 gene expression, and accordingly, inhibition of Sp1 expression with specific siRNA significantly reduced the promoter activity and suppressed the PADI6 expression. In addition, inhibition of Sp1 binding by Mithramycin A treatment reduced the transcriptional activity of PADI6 in a dose-dependent manner. Taken together, these data indicate that Sp1 is essential for the transcriptional regulation of PADI6.

The W620 Polymorphism in PTPN22 Disrupts Its Interaction With Peptidylarginine Deiminase Type 4 and Enhances Citrullination and NETosis.

A C-to-T single-nucleotide polymorphism (SNP) located at position 1858 of human protein tyrosine phosphatase PTPN22 complementary DNA carries the highest risk of rheumatoid arthritis (RA) among all non-HLA genetic variants. This C1858T SNP converts an arginine (R620) to a tryptophan (W620), but it is unclear why it has such a strong impact on RA, a disease characterized by anti-citrullinated protein antibodies. The aim of this study was to test the hypothesis that PTPN22 regulates protein citrullination. The level of citrullinated proteins in immune cells was quantified by Western blotting. The physical interaction between PTPN22 and peptidyl arginine deiminase type 4 (PAD-4), which is one of the enzymes that catalyzes protein citrullination, was examined by coimmunoprecipitation. Neutrophils were collected from healthy donors carrying the C1858T SNP and healthy donors not carrying this SNP. The formation of neutrophil extracellular traps (NETs) was examined by immunocytochemistry. PTPN22 physically interacted with PAD-4, and a deficiency in PTPN22 enhanced protein citrullination. This abnormality was reversed by exogenous wild-type PTPN22 or catalytically dead mutant PTPN22. The R-to-W conversion rendered PTPN22 unable to interact with PAD-4 and suppress citrullination. The C1858T SNP was associated with hypercitrullination in peripheral blood mononuclear cells and a heightened propensity for spontaneous formation of NETs, which is a PAD-4-dependent process. PTPN22 is an inhibitor of PAD-4 and protein citrullination. This function of PTPN22 is independent of its phosphatase activity but requires R620. Our data not only establish a molecular link between PTPN22 and PAD-4, but also suggest that the C1858T SNP increases the risk of RA by enhancing protein citrullination and spontaneous formation of NETs.

Peptidylarginine deiminase type 4 deficiency reduced arthritis severity in a glucose-6-phosphate isomerase-induced arthritis model.

Peptidyl arginine deiminase 4 (PAD4) is an enzyme that is involved in protein citrullination, and is a target for anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Genetic polymorphisms in the PADI4 gene encoding PAD4 are associated with RA susceptibility. We herein analyzed the roles of PADI4 in inflammatory arthritis using a glucose-6-phosphate isomerase (GPI)-induced arthritis (GIA) model in Padi4 knockout (KO) mice. Arthritis severity, serum anti-GPI antibody titers, and IL-6 concentrations were significantly reduced in Padi4 KO mice. The frequency of Th17 cells was decreased in GPI-immunized Padi4 KO mice, whereas WT and Padi4-deficient naïve CD4+ T cells displayed the same efficiencies for Th17 cell differentiation in vitro. In addition, the numbers of myeloid lineage cells were reduced with the increased expression of pro-apoptotic genes in GPI-immunized Padi4 KO mice. Furthermore, the survival of Padi4-deficient neutrophils was impaired in vitro. Our results suggest that PADI4 exacerbates arthritis with diverse immunological modifications.

Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis.

IntroductionSmoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4).MethodsLung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test.ResultsWithin the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase.ConclusionsWe have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0520-x) contains supplementary material, which is available to authorized users.

Association of susceptible genetic markers and autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder of unknown aetiology resulting in inflammation of the synovium, cartilage and bone. The disease has a heterogeneous character, consisting of clinical subsets of anti-citrullinated protein antibody (ACPA)-positive and APCA-negative disease. Although, the pathogenesis of RA is incompletely understood, genetic factors play a vital role in susceptibility to RA as the heritability of RA is between 50 and 60%, with the human leukocyte antigen (HLA) locus accounting for at least 30% of overall genetic risk. Non-HLA genes, i.e. tumour necrosis factor-α (TNF-α) within the MHC (major histocompatibility complex) have also been investigated for association with RA. Although, some contradictory results have originated from several studies on TNF-α gene, the data published so far indicate the possible existence of TNF-α gene promoter variants that act as markers for disease severity and response to treatment in RA. The correlation of HLA and non-HLA genes within MHC region is apparently interpreted. A considerable number of confirmed associations with RA and other autoimmune disease susceptibility loci including peptidylarginine deiminase type 4 (PADI4), protein tyrosine phosphatase non-receptor type 22 (PTPN22), signal transducer and activator of transcription (STAT4), cluster of differentiation 244 (CD244) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4), located outside the MHC have been reported recently. In this review, we aim to give an update on recent progress in RA genetics, the importance of the combination of HLA-DRB1 alleles, non-HLA gene polymorphism, its detection and autoantibodies as susceptibility markers for early RA disease.

Vimentin is involved in peptidylarginine deiminase 2-induced apoptosis of activated Jurkat cells.

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca2+-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.