Anti-citrullinated collagen type I antibody is a target of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, but its autoimmune mechanisms are not clearly understood. Recently, anti-citrullinated peptide antibodies have been specifically observed in sera of RA patients. Furthermore, we identified RA-susceptible variant in a gene encoding citrullinating enzyme, peptidylarginine deiminase type 4 (PADI4). Therefore, we hypothesized that proteins which are modified in RA synovium by PADI4 act as autoantigens. Subsequently, we obtained human collagen type I (huCI) as one of the autoantigens using a RA synoviocyte cDNA library by immunoscreening. We also investigated that the levels of anti-citrullinated huCI were significantly higher in RA patient sera than in normal control sera with high specificity (99%) and positively correlated with the levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies. We concluded that huCI is a novel substrate protein of PADIs and that citrullinated huCI is a candidate autoantigen of RA.     

PADI4 Haplotypes Contribute to mRNA Expression,the Enzymatic Activity of Peptidyl Arginine Deaminase and Rheumatoid Arthritis Risk in Patients from Western Mexico

Citrullination is catalyzed by the peptidyl arginine deiminase 4 (PAD4) enzyme, encoded by the PADI4 gene. Increased PAD4 activity promotes the onset and progression of rheumatoid arthritis (RA). This study aimed to evaluate the association of PADI4 haplotypes with RA risk, mRNA expression, and the PAD4 activity in patients with RA from Mexico. Methodology: 100 RA patients and 100 control subjects (CS) were included. Genotyping was performed by PCR-RFLP method, PADI4 mRNA expression was quantified by real-time PCR, the contribution of PADI4 alleles (PADI4_89 G>A, PADI4_90 T>C, and PADI4_92 G>C) to mRNA expression by the ASTQ method, and PAD4 activity by HPLC. Also, the anti-CCP and anti-PADI4 antibodies were quantified by ELISA. Results: The three PADI4 polymorphisms were associated with RA susceptibility (OR = 1.72, p = 0.005; OR = 1.62; p = 0.014; OR = 1.69; p = 0.009; respectively). The 89G, 90T, and 92G alleles have a higher relative contribution to PADI4 mRNA expression from RA patients than 89A, 90C, and 92C alleles in RA patients. Moreover, the GTG/GTG haplotype was associated with RA susceptibility (OR = 2.86; p = 0.024). The GTG haplotype was associated with higher PADI4 mRNA expression (p = 0.04) and higher PAD4 enzymatic activity (p = 0.007) in RA patients. Conclusions: The evaluated polymorphisms contribute to PADI4 mRNA expression and the enzymatic activity of PAD4 in leukocytes. Therefore, the GTG haplotype is a genetic risk factor for RA in western Mexico, and is associated with increased PADI4 mRNA expression and higher PAD4 activity in these patients.     

Overexpression of peptidylarginine deiminase IV features in apoptosis of haematopoietic cells

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via posttranslational modification. One member of the family, PADI4, plays an important role in immune cell differentiation and cell death. To elucidate the participation of PADI4 in haematopoietic cell death, we examine whether inducible overexpression of PADI4 enhances the apoptotic cell death. PADI4 reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells and human acute T leukemia Jurkat cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (Δψ_m), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following PADI4 overexpression, cells arrest in G1 phase significantly before their entrance into apoptotic cell death. PADI4 increases tumor suppressor p53 and its downstream p21 to control cell cycle. In the detections of protein expression and kinase activity, all protein levels of cyclin-dependent kinases (CDKs) and cyclins are not reduced except cyclin D, however, CDK2 (G1 entry S phase) and CDK1 (G2 entry M phase) enzyme activities are inhibited by conditionally inducible PADI4. p53 also expands its other downstream Bax to induce cytochrome c release from mitochondria. According to these data, we suggest that PADI4 induces apoptosis mainly through cell cycle arrest and mitochondria-mediated pathway. Furthermore, p53 features in PADI4-induced apoptosis by increasing intracellular p21 to control cell cycle and by Bax accumulation to decline Bcl-2 function, destroy Δψ_m, release cytochrome c to cytoplasm and activate the caspase cascade.     

The functional haplotype of peptidylarginine deiminase IV (S55G,A82V and A112G) associated with susceptibility to rheumatoid arthritis dominates apoptosis of acute T leukemia Jurkat cells

Peptidylarginine deiminase IV (PADI4) posttranslationally converts peptidylarginine to citrulline. It plays an essential role in immune cell differentiation and apoptosis. A haplotype of single-nucleotide polymorphisms (SNPs) in PADI4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Previously, our study showed that inducible PADI4 causes haematopoietic cell death. Herein, we further investigate whether RA risk PADI4 haplotype (SNP PADI4; S55G, A82V and A112G) and the increase of its enzymatic activity induce apoptosis. In the tetracycline (Tet)-On Jurkat T cells, ionomycin (Ion) only treatment didn't induce apoptosis however it promoted inducible PADI4-decreased cell viability and -enhanced apoptosis. Through in vitro and in vivo PADI enzyme activity assay, we demonstrated that PADI4 enzyme activity of SNP PADI4 was higher than RA non-risk PADI4 haplotype (WT PADI4). The effect of SNP PADI4-induced apoptosis was superior to WT PADI4. In addition, both Ion and SNP PADI4 synergistically provoked apoptosis were compared with both Ion and WT PADI4. Concurrently, in the conditionally inducible SNP PADI4 cells of Ion treatment-induced apoptosis, not only the expression of Bcl-xL was down-regulated and Bax up-regulated, but also cytochrome c was released from mitochondria to cytoplasm in significant amounts. Western blotting data showed the increase in apoptosomal caspase activation during programmed cell death in the inducible SNP PADI4 cells subsequent to Ion treatment. These data demonstrated that both SNP PADI4 increasing their enzyme activity could enhance apoptosis through the mitochondrial pathway and further provide a conceivable explanation in the pathogenesis of RA following the upregulation of PADI4 activity in its SNPs. The authors (H.-C. Hung and C.-Y. Lin) contributed equally to this paper.     

Identification of citrullinated eukaryotic translation initiation factor 4G1 as novel autoantigen in rheumatoid arthritis

Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis. We previously reported that functional variants of the gene encoding peptidylarginine deiminase type 4 were closely associated with RA. The purpose of this study was to investigate the citrullinated autoantigens recognized by serum samples from patients with RA. The human chondrocyte cDNA expression library was citrullinated by PADI4 and was immunoscreened with anti-modified citrulline antibodies and sera from patients with rheumatoid arthritis. One immunoreactive cDNA clone containing a 2480-base pair insert was isolated and sequence analysis revealed that the cDNA included a part of the eukaryotic translation initiation factor 4G1. Immunoreactivity against a recombinant citrullinated eIF4G1 fragment was observed with high specificity in 50.0% of RA patients. The levels of antibodies against citrullinated eIF4G1 were correlated with those of anti-CCP antibodies. Citrullinated eIF4G1 was identified as a candidate citrullinated autoantigen in RA patients. Citrullination of eIF4G1 may thus be involved in the pathogenesis of RA.     

Upregulation of citrullination pathway: From Autoimmune to Idiopathic Lung Fibrosis

Background

Increased protein citrullination and peptidylarginine deiminases (PADIs), which catalyze the citrullination process, are central in Rheumatoid arthritis pathogenesis and probably involved in the initial steps towards autoimmunity. Approximately, 10% of RA patients develop clinically significantly ILD. A possible shared role of protein citrullination in rheumatoid arthritis associated interstitial lung disease (RA-ILD), and idiopathic pulmonary fibrosis (IPF) pathogenesis remains unclear.

Methods

We evaluated PADI2 and PADI4 mRNA expression in bronchoalveolar lavage fluid (BALF) cells of 59 patients with IPF, 27 patients RA-ILD and 10 healthy controls. PADI 2 and 4 expression was analyzed by western blot and immunohistochemistry. Citrullinated protein levels were also quantified.

Results

PADI4 mRNA and protein levels were higher in RA-ILD and IPF than controls. Furthermore, PADI4 mRNA levels showed an increase among smokers in RA-ILD. PADI4 expression was detected in granulocytes and macrophages in all groups, with the strongest cytoplasmic expression observed in granulocytes in RA-ILD and IPF. PADI2 mRNA and immunostaining of BAL cells, were similar in all groups among smokers. Overall, stronger staining was observed in current smokers. Citrullinated peptides were significantly increased in IPF compared to RA-ILD and controls. In RA-ILD, protein citrullination strongly correlated with PADI4 expression and anti-citrullinated protein antibodies (ACPAs).

Conclusions

These results suggest that the citrullination pathway is upregulated in IPF and in RA-ILD.

     

Role for PADI6 in securing the mRNA-MSY2 complex to the oocyte cytoplasmic lattices

The oocyte cytoplasmic lattices (CPLs) have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo. Our previous studies have demonstrated that ribosomal component S6 is stored in the oocyte CPLs and peptidylarginine deiminase 6 (PADI6) is critical for CPLs formation. Additionally, we found that depletion of PADI6 reduced de novo protein synthesis prior to the maternal-to-embryonic transition, therefore causing embryos to arrest at the 2-cell stage. Here, we present evidence further supporting the association of ribosomes with the CPLs by demonstrating that rRNAs are dramatically decreased in Padi6 KO oocytes. We also show that the abundance and localization of mRNAs is affected upon PADI6 depletion, suggesting that mRNAs are very possibly associated with CPLs. Consistent with this observation, the amount of the major RNA binding protein, MSY2, that is associated with the insoluble fraction of the oocytes after Triton X-100 extraction is also markedly decreased in the Padi6 KO oocytes. Furthermore, treatment of the oocytes with RNase A followed by Triton X-100 extraction severely impairs the localization of PADI6 and MSY2 in oocytes. These results indicate that mRNAs, possibly in a complex with MSY2 and PADI6, are bound in the CPLs and may play a role in securing the mRNA-MSY2 complex to the CPLs.     

肽基精氨酸脱亚胺酶4(PADI4)对RA的影响

类风湿关节炎(RA)是一种病因和发病机制尚不清楚的自身免疫疾病,一般认为是由多种遗传因素和环境因素共同作用的结果。遗传因素中以组织相容性白细胞抗原HLA最为重要,另外作为非HLA的肽基精氨酸脱亚胺酶4(PADI4)也参与了RA的发病。PADI4是一种翻译后修饰酶,可在钙离子存在的情况下将精氨酸残基转化为瓜氨酸残基,瓜氨酸化后的蛋白质往往改变其分子构象,从而导致其生化活性亦发生改变。在不用种族的人群中,PADI4基因多态性与RA的易感性不尽相同。PADI4在RA患者血清中含量明显升高,在机体内产生自身抗PADI4抗体,并且PADI4瓜氨酸化多种蛋白质引起机体自身的免疫反应参与RA的发生与发展。近些年来的其他研究表明PAID4也参与了肿瘤、溃疡性结肠炎、多发性硬化症的发病。尽管针对PADI4的研究已经取得了很多重大进展,但是仍然存在很多悬而未决的问题等待科研工作者进一步的研究和证实。     

Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case–control study

Peptidylarginine deiminase type 4 (PADI4) genotypes were shown to influence susceptibility to rheumatoid arthritis (RA) in the Japanese population. Such an association could not previously be confirmed in different European populations. In the present study, we analysed exons 2–4 of PADI4 in 102 German RA patients and 102 healthy individuals to study the influence of PADI4 variability on RA susceptibility by means of haplotype-specific DNA sequencing. Analyses of the influence of PADI4 and HLA-DRB1 genotypes on disease activity and on levels of anti-cyclic citrullinated peptide antibodies were performed.     

Association of single nucleotide polymorphisms (SNPs) of PADI4 gene with rheumatoid arthritis (RA) in Indian population

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting ~ 1% of the population worldwide. The genome wide association studies on RA patients revealed linkage with 1p36 locus containing peptidyl arginine deiminase 4 (PADI4) genes. Case-control association studies and mRNA stability assays reported the association of PADI4 gene with RA in Korean and Japanese populations. However, such association was not found in Spanish population. Differences in the association of PADI4 with RA in different populations prompted the present study in Indian population. Anti-CCP antibodies, RF antibody, disease activity scores at 28 joints (DAS28) and mutations in three exons of PADI4 were investigated in RA patients and control group. Among the patients anti-CCP antibody levels were found to be associated with high DAS28 values (r = 0.4526, P < 0.0001). Polymorphism in exon-4 (padi4_104, [rs1748033]) of PADI4 showed significant association of 'C' allele with RA in the study population (P = 0.0008). Polymorphism in exon-3 (padi4_92, [rs874881]) also exhibited moderate association with the disease (P = 0.075). However, no association of the disease was found with the SNPs padi4_89 [rs11203366] and padi4_90 [rs11203367] in exon-2 of PADI4.     

Detailed analysis of the variability of peptidylarginine deiminase type 4 in German patients with rheumatoid arthritis: a case-control study

Peptidylarginine deiminase type 4 (PADI4) genotypes were shown to influence susceptibility to rheumatoid arthritis (RA) in the Japanese population. Such an association could not previously be confirmed in different European populations. In the present study, we analysed exons 2-4 of PADI4 in 102 German RA patients and 102 healthy individuals to study the influence of PADI4 variability on RA susceptibility by means of haplotype-specific DNA sequencing. Analyses of the influence of PADI4 and HLA-DRB1 genotypes on disease activity and on levels of anti-cyclic citrullinated peptide antibodies were performed. Comparing the frequencies of PADI4 haplotype 4 (padi4_89*G, padi4_90*T, padi4_92*G, padi4_94*T, padi4_104*C, padi4_95*G, padi4_96*T) (patients, 14.7%; controls, 7.8%; odds ratio = 2.0, 95% confidence interval = 1.1-3.8) and carriers of this haplotype (patients, 27.5%; controls, 13.7%; odds ratio = 2.4, 95% confidence interval = 1.2-4.8), a significant positive association of PADI4 haplotype 4 with RA could be demonstrated. Other PADI4 haplotypes did not differ significantly between patients and controls. Regarding the individual PADI4 variants, padi4_89 (A-->G), padi4_90 (C-->T), and padi4_94 (C-->T) were significantly associated with RA (patients, 49.5%; controls, 38.7%; odds ratio = 1.6, 95% confidence interval = 1.1-2.3). Considering novel PADI4 variants located in or near to exons 2, 3, and 4, no quantitative or qualitative differences between RA patients (8.8%) and healthy controls (10.8%) could be demonstrated. While the PADI4 genotype did not influence disease activity and the anti-cyclic citrullinated peptide antibody level, the presence of the HLA-DRB1 shared epitope was significantly associated with higher anti-cyclic citrullinated peptide antibody levels (P = 0.033). The results of this small case-control study support the hypothesis that variability of the PADI4 gene may influence susceptibility to RA in the German population. Quantitative or qualitative differences in previously undefined PADI4 variants between patients and controls could not be demonstrated.     

Novel functional polymorphism on PADI-4 gene and its association with arthritis onset

BackgroundCitrullinated proteins formed by peptidyl arginine deiminases (PADIs) deimination of arginine residues in proteins are of particular interest in arthritis pathogenesis. Polymorphisms on the PADI-4 gene lead to the malfunctioning of PADIs leading to the onset of arthritis.ObjectiveThe present study was conducted to determine the polymorphisms on the PADI-4 gene and their association with rheumatoid arthritis (RA) as well as Osteoarthritis (OA).MethodologyTo achieve the above-mentioned objective a case-control study was conducted. Blood samples were collected from RA, OA, and control subjects. DNA was extracted from each blood sample by modified organic method and was quantified as well as qualified by DNA gel electrophoresis and Nanodrop. Patients were tested for rs874881, rs11203366, rs11203367, rs2240336, rs2240337, rs2240339, rs1748033 and rs2240340 polymorphic sites by amplifying targeted regions through PCR with site-specific primers. Genotyping was performed by Restriction Fragment Length Polymorphism and direct sequencing method. Mutations were identified by analyzing sequences on BioEdit software. Allelic, genetic, and multiple site analysis were performed by SHEsis and PLINK software. Change in the amino acid sequence was identified by MEGA 6.0 software.ResultsPolymorphisms were identified on all targeted polymorphic sites except rs2240337 in both RA and OA individuals. In addition, two novel mutations were also identified in exon 4 identified i-e SCV000804840: c.218T > C and SCV000807675: c.241G > T. All the SNPs except rs11203366 were found to be significantly associated with RA at an allelic level whereas all SNP’s have been significant risk factors in the onset of OA. At genotypic level rs874881, rs11203366, rs2240339, SCV000804840 and SCV000807675 were significantly associated to RA development whereas rs874881, rs11203366, rs11203367, rs2240339, SCV000804840 and SCV000807675 were genetic risk factors in OA onset. Haplotype analysis indicated that GACCACGCC and GACCACGCT were highly significant in disease development. Polymorphisms identified altered the functioning of PADIs by altering their amino acid sequence.ConclusionIn conclusion, it was found that PADI-4 gene polymorphism was not only involved in the onset of RA but was also found to be a significant risk factor in OA onset.     

PADI2 Is Significantly Associated with Rheumatoid Arthritis

Citrullination, a posttranslational modification of peptidyl arginine to citrulline, plays an essential role in rheumatoid arthritis (RA). Citrullination is catalyzed by a group of peptidylarginine deiminases (PADs) including PADI 1, 2, 3, 4 and 6. Many studies have indicated that the gene encoding PADI4 is a factor in susceptibility to RA. Some studies have detected PADI2 expression in RA synovial tissues, suggesting that PADI2 also plays an important role in the disease. This study evaluated the possible association between the PADI2-encoding gene and RA. Seventeen tag SNPs across the PAD locus were genotyped using a custom-designed Illumina 96-SNP VeraCode microarray. Peripheral blood samples were collected from patients with RA (n = 267), ankylosing spondylitis (AS, n = 51) and healthy controls (n = 160). The results of genotyping were verified using Sequenom MassARRAY in an independent cohort of 307 patients with RA, 324 patients with AS and 509 healthy controls. A western blot analysis was performed using synovial tissue from patients with RA (n = 7), osteoarthritis (OA, n = 7) and AS (n = 5) to determine the levels of expression of PADI2. A microarray analysis revealed a significant association between three selected PADI2 SNPs (rs2235926, rs2057094, rs2076616) and the presence of RA. The increased susceptibility to RA associated with rs2235926 (OR = 1.706733, 95% CI = [1.576366–1.866587], p = 0.000839) and rs2057094 (OR = 1.360432, 95% CI = [1.065483–1.869482], p = 0.003291) was further confirmed by the Sequenom MassARRAY. No tag SNPs in the PADI2 locus showed a significant association with AS. Increased expression of PADI2 was detected in RA synovial tissues compared with samples from patients with OA and AS. PADI2 is significantly associated with RA and may be involved in the pathogenesis of the disease.     

PADI4 and HLA-DRB1 Are Genetic Risks for Radiographic Progression in RA Patients,Independent of ACPA Status: Results from the IORRA Cohort Study

Introduction

Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease influenced by both genetic and environmental factors, leading to joint destruction and functional impairment. Recently, a large-scaled GWAS meta-analysis using more than 37,000 Japanese samples were conducted and 13 RA susceptibility loci were identified. However, it is not clear whether these loci have significant impact on joint destruction or not. This is the first study focused on the 13 loci to investigate independent genetic risk factors for radiographic progression in the first five years from onset of RA.

Methods

Sharp/van der Heijde score of hands at 5-year disease duration, which represents joint damage, were measured retrospectively and used as an outcome variable in 865 Japanese RA patients. Genetic factors regarded as putative risk factors were RA-susceptible polymorphisms identified by the Japanese GWAS meta-analysis, including HLA-DRB1 (shared epitope, SE), rs2240340 (PADI4), rs2230926 (TNFAIP3), rs3093024 (CCR6), rs11900673 (B3GNT2), rs2867461 (ANXA3), rs657075 (CSF2), rs12529514 (CD83), rs2233434 (NFKBIE), rs10821944 (ARID5B), rs3781913 (PDE2A-ARAP1), rs2841277 (PLD4) and rs2847297 (PTPN2). These putative genetic risk factors were assessed by a stepwise multiple regression analysis adjusted for possible non-genetic risk factors: autoantibody positivity (anti-citrullinated peptide antibody [ACPA] and rheumatoid factor), history of smoking, gender and age at disease onset.

Results

The number of SE alleles (P = 0.002) and risk alleles of peptidyl arginine deiminase type IV gene (PADI4, P = 0.04) had significant impact on progressive joint destruction, as well as following non-genetic factors: ACPA positive (P = 0.0006), female sex (P = 0.006) and younger age of onset (P = 0.02).

Conclusions

In the present study, we found that PADI4 risk allele and HLA-DRB1 shared epitope are independent genetic risks for radiographic progression in Japanese rheumatoid arthritis patients. The results of this study give important knowledge of the risks on progressive joint damage in RA patients.     

Genome-wide pathway analysis of genome-wide association studies on systemic lupus erythematosus and rheumatoid arthritis

The aim of this study was to explore candidate single nucleotide polymorphisms (SNPs) and candidate mechanisms of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two SLE genome-wide association studies (GWASs) datasets were included in this study. Meta-analysis was conducted using 737,984 SNPs in 1,527 SLE cases and 3,421 controls of European ancestry, and 4,429 SNPs that met a threshold of p?<?0.01 in a Korean RA GWAS dataset was used. ICSNPathway (identify candidate causal SNPs and pathways) analysis was applied to the meta-analysis results of the SLE GWAS datasets, and a RA GWAS dataset. The most significant result of SLE GWAS meta-analysis concerned rs2051549 in the human leukocyte antigen (HLA) region (p?=?3.36E?22). In the non-HLA region, meta-analysis identified 6 SNPs associated with SLE with genome-wide significance (STAT4, TNPO3, BLK, FAM167A, and IRF5). ICSNPathway identified five candidate causal SNPs and 13 candidate causal pathways. This pathway-based analysis provides three hypotheses of the biological mechanism involved. First, rs8084 and rs7192?→?HLA-DRA?→?bystander B cell activation. Second, rs1800629?→?TNF?→?cytokine network. Third, rs1150752 and rs185819?→?TNXB?→?collagen metabolic process. ICSNPathway analysis identified three candidate causal non-HLA SNPs and four candidate causal pathways involving the PADI4, MTR, PADI2, and TPH2 genes of RA. We identified five candidate SNPs and thirteen pathways, involving bystander B cell activation, cytokine network, and collagen metabolic processing, which may contribute to SLE susceptibility, and we revealed candidate causal non-HLA SNPs, genes, and pathways of RA.     

Citrullination Was Introduced into Animals by Horizontal Gene Transfer from Cyanobacteria

Protein posttranslational modifications add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms, and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologs. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than 1 billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared with products of likely endosymbiont gene transfer, mitochondrial proteins, and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.     

Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4

Candidate-gene association studies in rheumatoid arthritis (RA) have lead to encouraging yet apparently inconsistent results. One explanation for the inconsistency is insufficient power to detect modest effects in the context of a low prior probability of a true effect. To overcome this limitation, we selected alleles with an increased probability of a disease association, on the basis of a review of the literature on RA and other autoimmune diseases, and tested them for association with RA susceptibility in a sample collection powered to detect modest genetic effects. We tested 17 alleles from 14 genes in 2,370 RA cases and 1,757 controls from the North American Rheumatoid Arthritis Consortium (NARAC) and the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) collections. We found strong evidence of an association of PTPN22 with the development of anti-citrulline antibody-positive RA (odds ratio [OR] 1.49; P=.00002), using previously untested EIRA samples. We provide support for an association of CTLA4 (CT60 allele, OR 1.23; P=.001) and PADI4 (PADI4_94, OR 1.24; P=.001) with the development of RA, but only in the NARAC cohort. The CTLA4 association is stronger in patients with RA from both cohorts who are seropositive for anti-citrulline antibodies (P=.0006). Exploration of our data set with clinically relevant subsets of RA reveals that PTPN22 is associated with an earlier age at disease onset (P=.004) and that PTPN22 has a stronger effect in males than in females (P=.03). A meta-analysis failed to demonstrate an association of the remaining alleles with RA susceptibility, suggesting that the previously published associations may represent false-positive results. Given the strong statistical power to replicate a true-positive association in this study, our results provide support for PTPN22, CTLA4, and PADI4 as RA susceptibility genes and demonstrate novel associations with clinically relevant subsets of RA.     

Regulation of mouse oocyte microtubule and organelle dynamics by PADI6 and the cytoplasmic lattices

Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.