Peptidylarginine Deiminase Type Research Articles

Inhibition of PAD4-mediated neutrophil extracellular traps formation attenuates hypoxic–ischemic brain injury in neonatal mice

Neonatal hypoxic–ischemic encephalopathy (HIE) is the primary cause of neonatal mortality and severe neurological sequelae. The interaction of neuroinflammation with the immune system represents a significant pathological mechanism underlying the development of HIE. Neutrophil extracellular traps (NETs) are a recently identified antimicrobial mechanism utilized by neutrophils. NETs can act as damage-associated molecular patterns, thereby amplifying the immune response and exerting proinflammatory effects. However, further research is needed to elucidate their role in the pathogenesis of HIE. In this study, we investigated the role of NETs in a hypoxic–ischemic brain injury (HIBI) model. We first reported that a pharmacological intervention to inhibit peptidylarginine deiminase type IV (PAD4) may constitute an effective strategy for reducing HI insult-induced neuroinflammation, neuronal apoptosis, and brain tissue destruction while also enhancing long-term neurobehavioral function in mice. These results support a pathological role for NETs in HIBI, and targeting PAD4 is a potential direction for the treatment of HIE.

Harnessing marine natural products to inhibit PAD4 triple mutant: A structure-based virtual screening approach for rheumatoid arthritis therapy

Peptidylarginine deiminase type4 (PAD4) is a pivotal pro-inflammatory protein within the human immune system, intricately involved in both inflammatory processes and immune responses. Its role extends to the generation of diverse immune cell types, including T cells, B cells, natural killer cells, and dendritic cells. PAD4 has recently garnered attention due to its association with a spectrum of inflammatory and autoimmune disorders, notably rheumatoid arthritis (RA). Mutations in the PAD4 gene, leading to the conversion of arginine to citrulline, have emerged as significant factors in the pathogenesis of RA and related conditions. As a calcium-dependent enzyme, PAD4 is central to the citrullination process, a crucial post-translational modification implicated in disease pathophysiology. Its critical role in autoimmune disorders and inflammation makes PAD4 a prime candidate for therapeutic intervention in RA. Inhibiting PAD4 presents a promising avenue for mitigating inflammatory responses and curtailing joint degradation and impairment. To explore its therapeutic potential, a structure-based virtual screening (SBVS) approach was employed, harnessing an array of marine natural products (MNPs) sourced from databases such as CMNPD, MNPD, and Seaweed. Notably, MNPD10752, CMNPD12680, and CMNPD2751 emerged as potential hit molecules, exhibiting adherence to essential pharmacokinetic properties and favorable toxicity profiles. Quantum mechanics studies using density functional theory (DFT) calculations revealed the inhibitory potential of these identified natural products. Further structural elucidation through molecular dynamics simulations (MDS) and principal component-based free energy landscape (FEL) analysis shed light on the stability of MNP-bound PAD4 complexes. In conclusion, this computational study serves as a stepping stone for further experimental evaluation, aiming to explore the potential of MNPs in addressing PAD4-related human pathologies.

Neutrophil PAD4 Expression and Its Pivotal Role in Assessment of Alcohol-Related Liver Disease.

Neutrophils release neutrophil extracellular traps (NETs) as a defense strategy in response to broad-spectrum infections and sterile triggers. NETs consist of a DNA scaffold decorated with antimicrobial peptides (AMPs) and enzymatically active proteases, including peptidyl arginine deiminase type 4 (PAD4). Susceptibility to infections and inflammatory dysregulation are hallmarks of alcohol-related liver disease (ALD). Sixty-two patients with ALD were prospectively recruited, and they were followed for 90 days. Twenty-four healthy volunteers served as the control group. PAD4 concentrations were quantified using immunoenzymatic ELISAs. Correlation coefficients between PAD4 blood concentrations and markers of systemic inflammation; liver dysfunction severity scores; and ALD complications were calculated. The receiver operating curves (ROCs) and their areas under the curve (AUCs) were checked in order to assess the accuracy of PAD4 expression in predicting the degree of liver failure and the development of ALD complications. Systemic concentrations of PAD4 were significantly increased in the patients with ALD in comparison with controls. PAD4 levels correlated with the standard markers of inflammation and revealed a good predictive AUC (0.76) for survival in the whole ALD group. PAD4 seems to be an inflammatory mediator and may be potentially applied as a predictor of patient survival in ALD.

Crystal structure of human peptidylarginine deiminase type VI (PAD6) provides insights into its inactivity.

Human peptidylarginine deiminase isoform VI (PAD6), which is predominantly limited to cytoplasmic lattices in the mammalian oocytes in ovarian tissue, is essential for female fertility. It belongs to the peptidylarginine deiminase (PAD) enzyme family that catalyzes the conversion of arginine residues to citrulline in proteins. In contrast to other members of the family, recombinant PAD6 was previously found to be catalytically inactive. We sought to provide structural insight into the human homologue to shed light on this observation. We report here the first crystal structure of PAD6, determined at 1.7 Å resolution. PAD6 follows the same domain organization as other structurally known PAD isoenzymes. Further structural analysis and size-exclusion chromatography show that PAD6 behaves as a homodimer similar to PAD4. Differential scanning fluorimetry suggests that PAD6 does not coordinate Ca2+ which agrees with acidic residues found to coordinate Ca2+ in other PAD homologs not being conserved in PAD6. The crystal structure of PAD6 shows similarities with the inactive state of apo PAD2, in which the active site conformation is unsuitable for catalytic citrullination. The putative active site of PAD6 adopts a non-productive conformation that would not allow protein-substrate binding due to steric hindrance with rigid secondary structure elements. This observation is further supported by the lack of activity on the histone H3 and cytokeratin 5 substrates. These findings suggest a different mechanism for enzymatic activation compared with other PADs; alternatively, PAD6 may exert a non-enzymatic function in the cytoplasmic lattice of oocytes and early embryos.

Genetic factors underlying host resistance to Rhipicephalus microplus tick infestation in Braford cattle: a systems biology perspective.

Approximately 80% of the world's cattle are raised in regions with a high risk of tick-borne diseases, resulting in significant economic losses due to parasitism by Rhipicephalus (Boophilus) microplus. However, the lack of a systemic biology approach hampers a comprehensive understanding of tick-host interactions that mediate tick resistance phenotypes. Here, we conducted a genome-wide association study (GWAS) of 2933 Braford cattle and found 340 single-nucleotide polymorphisms (SNPs) associated with tick counts. Gene expression analyses were performed on skin samples obtained from previously tick-exposed heifers with extremely high or low estimated breeding values for R. microplus counts. Evaluations were performed both before and after artificial infestation with ticks. Differentially expressed genes were found within 1-Mb windows centered at significant SNPs from GWAS. A total of 330 genes were related to the breakdown of homeostasis that was induced by larval attachment to bovine skin. Enrichment analysis pointed to a key role of proteolysis and signal transduction via JAK/STAT, NFKB and WNT/beta catenin signaling pathways. Integrative analysis on matrixEQTL revealed two cis-eQTLs and four significant SNPs in the genes peptidyl arginine deiminase type IV (PADI4) and LOC11449251. The integration of genomic data from QTL maps and transcriptome analyses has identified a set of twelve key genes that show significant associations with tick loads. These genes could be key candidates to improve the accuracy of genomic predictions for tick resistance in Braford cattle.

4-(Phenylselanyl)-2H-chromen-2-one-Loaded Nanocapsule Suspension-A Promising Breakthrough in Pain Management: Comprehensive Molecular Docking, Formulation Design, and Toxicological and Pharmacological Assessments in Mice.

Therapies for the treatment of pain and inflammation continue to pose a global challenge, emphasizing the significant impact of pain on patients' quality of life. Therefore, this study aimed to investigate the effects of 4-(Phenylselanyl)-2H-chromen-2-one (4-PSCO) on pain-associated proteins through computational molecular docking tests. A new pharmaceutical formulation based on polymeric nanocapsules was developed and characterized. The potential toxicity of 4-PSCO was assessed using Caenorhabditis elegans and Swiss mice, and its pharmacological actions through acute nociception and inflammation tests were also assessed. Our results demonstrated that 4-PSCO, in its free form, exhibited high affinity for the selected receptors, including p38 MAP kinase, peptidyl arginine deiminase type 4, phosphoinositide 3-kinase, Janus kinase 2, toll-like receptor 4, and nuclear factor-kappa β. Both free and nanoencapsulated 4-PSCO showed no toxicity in nematodes and mice. Parameters related to oxidative stress and plasma markers showed no significant change. Both treatments demonstrated antinociceptive and anti-edematogenic effects in the glutamate and hot plate tests. The nanoencapsulated form exhibited a more prolonged effect, reducing mechanical hypersensitivity in an inflammatory pain model. These findings underscore the promising potential of 4-PSCO as an alternative for the development of more effective and safer drugs for the treatment of pain and inflammation.

Structure-based drug design for protein arginine deiminase Type IV (PAD4) receptor: Chemoinformatics approach

Rheumatoid Arthritis (RA) is the second most common type of arthritis with symptoms first appearing in patients between 40 and 60 years of age. The number of older persons is projected to double to 1.5 billion in 2050, globally. Peptidylarginine deiminase Type 4 (PAD4), which catalyzes the conversion of peptidyl-arginine to peptidyl-citrulline, is widely believed to play a causative role in RA disease. Nonsteroidal anti-inflammatories (NSAIDs) and corticosteroids encompass a large group of clinically effective compounds whose mode of action is well established, these compounds relieve pain and reduce inflammation by preventing prostaglandin synthesis through inhibition of cyclooxygenase 2 and the production of arachidonic acid, respectively. We developed a computational protocol/pipeline, using virtual screening approaches to search for new chemical agents (NCA), capable of inhibiting the action of PAD4, in potential, in view of the treatment of RA. Our results allowed the selection of structures with suitable indices of pharmacokinetic properties and estimated low toxicological, in potential, important evidence for selection of more promising molecular candidates against the studied disease. Molecules ZINC20452582 and ZINC67673633 presented better results, as well as the drug-receptor interactions were similar to those observed between the crystallographic compound (GSK147) here used. They presented excellent results for predictions of metabolites, and they can be indicated as promising molecules, resulting from virtual screening approaches, as new PAD4 receptor inhibitors with activity against RA, in potential.

Peptidylarginine Deiminase Type 2 Predicts Tumor Progression and Poor Prognosis in Patients with Curatively Resected Biliary Tract Cancer.

(1) Background: PADI2 is a post-translational modification (PTM) enzyme that catalyzes citrullination, which then triggers autoimmune disease and cancer. This study aimed to evaluate the prognostic value of peptidylarginine deiminase 2 (PADI2) protein expression in biliary tract cancer (BTC) patients. (2) Methods: Using immunohistochemistry, the PADI2 protein expression in BTC tissues was analyzed. The correlations between PADI2 protein expression and clinicopathologic characteristics were analyzed using Chi-square tests. The Kaplan-Meier procedure was used for comparing survival distributions. We used Cox proportional hazards regression for univariate and multivariate analyses. From 2014 to 2020, 30 resected BTC patients were enrolled in this study. (3) Results: Patients with high PADI2 protein expression were associated with shorter progress-free survival (PFS; p = 0.041), disease-specific survival (DSS; p = 0.025), and overall survival (OS; p = 0.017) than patients with low PADI2 protein expression. (4) Conclusions: The results indicated that PADI2 protein expression was an independent poor prognostic factor for BTC patients regarding PFS, DSS, and OS.

Histopathological evaluation of docetaxel effects in treatment of rheumatoid arthritis induced in rat model

Rheumatoid arthritis is an immune-mediated condition that affects synovial joints. Synovial tissue, cartilage, bone, and less frequently extra-articular structures which in turn experience
 inflammatory changes. Paclitaxel's semi-synthetic equivalent, docetaxel, is an anti-neoplastic drug. Methotrexate is a treatment for early RA and may have a mildly negative impact on peptidyl arginine deiminase type 4 fluorescence test. However, 30% of patients fail to complete treatment within the first year due to resistance or side effects. The synovial membrane of Rheumatoid arthritis patient infiltrated with macrophages and neutrophils that express peptidyl arginine deiminase type 4 which their effect in rheumatoid arthritis pathogenesis lies in the generation of citrullinated neoepitopes that are Anti cyclic citrullinated peptide antibodies-targeted.
 The purpose of this study: was to assess the anti-inflammatory effects of docetaxel and methotrexate on the joint structure.
 Methods: Five groups of eight rats were formed from the 40 male Wister rats. Complete Freund’s adjuvant was injected subcutaneously into rats to induce the disease. The first group is control group which was the only group consists of (healthy untreated) rats. Second group was received complete Freund’s adjuvant. 0.5ml of ordinary saline was intraperitoneally administered to both the control and induction groups. Based on a preliminary experiment, the third group was given intraperitoneally 1 mg/kg/on alternative day docetaxel. The fourth group was given intraperitoneally 1 mg/kg/week of Methotrexate. Fifth group was given a half dose of both Methotrexate and docetaxel concurrently. Arthritis index was measured and Knee joint was histopathological examined.
 Results: significant Arthritis Index decrease in docetaxel group (p≤0.05). Significant lowering Histometric scoring (p≤0.05) in docetaxel, and Methotrexate group (cellular hyperplasia, formation of granulation tissue, infiltration of leukocytes, destroying of cartilage and intensity of erosion & Articular cartilage thickness) level in rats induced arthritis. Conclusion: This study showed that docetaxel may have anti-arthritic effects through their significant lowering Histometric scoring(p≤0.05).

PADI4 and IL-33 gene polymorphisms with susceptibility to systemic lupus erythematosus and juvenile idiopathic arthritis, a systematic review and meta-analysis.

This study evaluated the association between peptidyl arginine deiminase type IV (PADI4) and interleukin 33 (IL-33) with systemic lupus erythematosus (SLE) and juvenile idiopathic arthritis (JIA). We searched the PubMed, Web of Science, Embase and Cochrane Library databases to retrieve articles published up to January 20, 2023. Stata/SE 17.0 (College Station, TX) software was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs). The cohort study, case-control study focusing on the PADI4, IL-33 polymorphism, and SLE, JIA were retrieved. The data included basic information of each study and the genotypes and allele frequencies. Studies in PADI4 rs2240340 = 2 and 3 IL-33(rs1891385 = 3, rs10975498 = 2, rs1929992 = 4) were found in 6 articles. Overall, only the IL-33 rs1891385 show significant association between SLE in all 5 models. The results were OR (95% CI) = 1.528 (1.312, 1.778), P = .000 in Allele model (C vs A), OR (95% CI) =1.473 (1.092, 1.988), P = .000 in Dominant model (CC + CA vs AA), 2.302 (1.583, 3.349), P = .000 in Recessive model (CC vs CA + AA), 2.711 (1.845, 3.983), P = .000 in Homozygote model (CC vs AA), 5.568 (3.943, 7.863), P = .000 in Heterozygote model (CA vs AA). PADI4 rs2240340, IL-33 rs10975498, IL-33 rs1929992 were not found to be association with the risk of SLE and JIA. In gene model, statistically significant association was found between IL-33 rs1891385 and SLE in sensitivity analysis. Egger's publication bias plot showed there was no publication bias (P = .165). Only in recessive model the heterogeneity test was significant (I2 = 57.9%, P ≤ .093) of IL-33 rs1891385. The current study suggests that in all 5 model, IL-33 rs1891385 polymorphism may be associated with genetic susceptibility to SLE. There was unclear association found between PADI4 rs2240340, IL-33 rs10975498, and IL-33 rs1929992 polymorphisms and SLE and JIA. Due to the limitations of included studies and the risk of heterogeneity, additional research is required to confirm our findings. CRD42023391268.

Potential peptidyl arginine deiminase type 4 inhibitors from Morinda citrifolia: a structure-based drug design approach.

The online version contains supplementary material available at 10.1007/s40203-023-00147-3.

Serum antibodies to peptidylarginine deiminase-4 in rheumatoid arthritis associated-interstitial lung disease are associated with decreased lung fibrosis and improved survival

ObjectiveInterstitial lung disease (ILD) is a leading cause of mortality in rheumatoid arthritis (RA), particularly in those with the usual interstitial pneumonia subtype (RA-UIP). Serum antibodies to peptidylarginine deiminase type 4 (anti-PAD4), particularly a subset that cross-react with PAD3 (PAD3/4XR), have been associated with imaging evidence of ILD. We aimed to determine the specificity of anti-PAD4 antibodies in RA-ILD and to examine associations with markers of ILD severity. Methods48 RA-ILD and 31 idiopathic pulmonary fibrosis (IPF) patients were identified from the National Jewish Health Biobank. RA-ILD subtype was defined by imaging pattern on high-resolution chest computed tomography (CT), and serum was tested for anti-PAD4 and anti-PAD3/4XR antibodies. Antibody prevalence, measures of ILD severity (% predicted forced vital capacity, FVC; % predicted diffusion capacity carbon monoxide, DLCO; quantitative CT fibrosis) and mortality were compared between groups. ResultsAnti-PAD4 antibodies were present in 9/48 (19%) subjects with RA-ILD and no subjects with IPF. Within RA-ILD, anti-PAD4 antibodies were found almost exclusively in RA-UIP (89%). Within RA-UIP subjects, % predicted FVC was higher in anti-PAD4+ subjects, and this finding was most strongly associated with anti-PAD3/4XR antibodies. In addition, quantitative CT fibrosis score was lower in anti-PAD4+ RA-UIP subjects, including those with mono-reactive anti-PAD4 antibodies and anti-PAD3/4XR antibodies. Anti-PAD4+ RA-UIP subjects also exhibited decreased mortality. ConclusionsWe demonstrate the presence of serum anti-PAD4 antibodies in a subset of patients with RA-UIP that were notably associated with better lung function, less fibrosis and decreased mortality.

PADI4 and IL-33 gene polymorphisms associated with differential susceptibility to juvenile-onset systemic lupus erythematosus and juvenile idiopathic arthritis in Chinese children

Juvenile systemic lupus erythematosus (JSLE) and juvenile idiopathic arthritis (JIA) are two common types of autoimmune diseases in children with unclear pathogenesis. Both peptidyl arginine deiminase type IV (PADI4) and interleukin 33 (IL-33) are the key molecular involved in immune responses in autoimmune diseases. Usually, it may share the same risk genetic alleles for autoimmune diseases. So measurement of PADI4 and IL-33 polymorphisms was conducted with 303 healthy controls, 144 JSLE patients and 160 JIA patients in this study. It demonstrated that there was a significant association between PADI4 genotypes (rs2240340: CT, CT + CC), IL-33 genotype (rs1929992: TT) and JSLE susceptibility in Southwest China population. While no significant association with the risk of JIA were observed no matter at allelic or genotypic levels. Our study reveals the importance of PADI4 and IL-33 polymorphisms with JSLE risk and their roles in the development of the diseases need more further researches.

Selinexor, a selective inhibitor of nuclear export, inhibits human neutrophil extracellular trap formation in vitro.

Neutrophils are central players in the innate immune system. To protect against invading pathogens, neutrophils can externalize chromatin to create neutrophil extracellular traps (NETs). While NETs are critical to host defense, they also have deleterious effects, and dysregulation of NETs formation has been implicated in autoimmune diseases, atherosclerosis and thrombotic conditions, cancer progression and dissemination, and acute respiratory distress syndrome. Here, we report that selinexor, a first-in-class selective inhibitor of nuclear export approved for the treatment of multiple myeloma and diffuse large B-cell lymphoma, markedly suppressed the release of NETs in vitro. Furthermore, we demonstrate a significant inhibitory effect of selinexor on NETs formation, but not on oxidative burst or enzymatic activities central to NETs release such as neutrophil elastase, myeloperoxidase or peptidyl arginine deiminase type IV. The inhibitory effect of selinexor was demonstrated in neutrophils activated by a variety of NETs-inducers, including PMA, TGF-β, TNF-α and IL-8. Maximal inhibition of NETs formation was observed using TGF-β, for which selinexor inhibited NETs release by 61.6%. These findings pave the way to the potential use of selinexor in an effort to reduce disease burden by inhibition of NETs.

Tale of Two Alopecias: Alopecia Areata and Central Centrifugal Cicatricial Alopecia Occurring in the Same Patient

Abstract Introduction: Scarring and non-scarring alopecias have rarely been described to occur together in the same patient. Distinguishing these two different types of alopecia is important as treatment and prognosis can be different. Case presentation: Here, we report the first case of simultaneous alopecia areata (AA) and central centrifugal cicatricial alopecia (CCCA) in a 35-year-old woman. New alopecic patches were noted on her frontal and vertex scalp. Biopsy of the frontal scalp revealed miniaturized hair follicles and dense lymphocytic infiltrate surrounding the hair bulbs, consistent with AA; while biopsy of the vertex scalp revealed decreased hair follicles, perifollicular fibroplasia with eccentric atrophy of the follicular epithelium, and premature desquamation of the inner root sheath at the level of the lower isthmus, consistent with CCCA. Discussion: Proposed mechanisms of these two alopecia types occurring together include loss of immune privilege, genetic predisposition, as well as unknown external factors that trigger an autoimmune lymphocytic response. Most recently, the peptidylarginine deiminase type III gene has been implicated in both diseases. Although treatment options can overlap between the two diseases, treatment response can differ and CCCA tends to have a worse prognosis. Conclusion: Awareness of this concomitant presentation of two alopecic types is important for appropriate treatment and prognostication.

Characterizing Enzyme Cooperativity with Imaging SAMDI-MS.

This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca2+ . Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K0.5 ) for the Ca2+ - dependent activation of PAD2. This work establishes a high-throughput and label-free method for studying enzyme-ligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.

Single Nucleotide Polymorphism of Padi4 Gene )Rs11203367( in A Sample of Rheumatoid Arthritis Iraqi Patients

Rheumatoid arthritis (RA) is an inflamed chronic autoimmune disease in which genetics and environment are the most common causative factors. Peptidyl arginine deiminase type IV (PADI4) is an enzyme responsible for the posttranslational conversion of arginine residues into citrulline. Real-time polymerase chain reaction (RT-PCR) is a specific technique was used to determine gene polymorphism. One hundred twenty three patients molecularly confirmed with RA and sixty healthy control subjects were recruited. By applying the logistic regression analysis, some alleles and genotypes were associated with susceptibility to RA. Under the allelic model, C allele frequency was significantly increased in RA patients compared to control (58.5 vs. 46.7%; p = 0.034). The odds ratio (OR) of such difference was 1.61 (95% CI: 1.04 – 2.50). Under the dominant model, TC+CC genotype frequency was higher in patients than in control (82.1vs. 65.5%; OR = 2.47; 95% CI: 1.23 – 4.97; p = 0.015). Under the codominant model, frequency of the heterozygous genotype TC was significantly increased in patients compared to control (47.1 vs. 36.7%; OR = 2.52; 95% CI: 1.17 – 5.41; p = 0.028). The results revealed the role of the rs11203367 variant of padi4 gene in susceptibility to RA in Iraqi patients.

Neutrophil Extracellular Traps Caused by Gut Leakage Trigger the Autoimmune Response in Nonobese Diabetic Mice.

Increased formation of neutrophil extracellular traps (NETs) is associated with gut leakage in type 1 diabetes (T1D). To explore the mechanism of how enteropathy exacerbated by NETs triggers pancreatic autoimmunity in T1D, we carried out a correlation analysis for NET formation with gut barrier functions and autoimmunity in nonobese diabetic (NOD) mice. Inducing chronic colitis or knocking out of peptidyl arginine deiminase type 4 (PAD4) in NOD mice were used to further study the effect of NET formation on the progression of T1D. Microbial alterations in Deferribacteres and Proteobacteria, along with the loss of gut barrier function, were found to be associated with increased endotoxin and abnormal formation of NETs in NOD mice. Both DSS-induced colitis and knockout of PAD4 in NOD mice indicated that PAD4-dependent NET formation was involved in the aggravation of gut barrier dysfunction, the production of autoantibodies, and the activation of enteric autoimmune T cells, which then migrated to pancreatic lymph nodes (PLNs) and caused self-damage. The current study thus provides evidence that PAD4-dependent NET formation is engaged in leaky gut triggering pancreatic autoimmunity and suggests that either degradation of NETs or inhibition of NET formation may be helpful for innovative therapeutic interventions in T1D.

Identification of a Novel Serological Marker in Seronegative Rheumatoid Arthritis Using the Peptide Library Approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation mainly affecting the joints leading to cartilage and bone destruction. The definition of seropositive or seronegative RA is based on the presence or absence of rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs). Other autoantibodies have been identified in the last decade such as antibodies directed against carbamylated antigens, peptidyl-arginine deiminase type 4 and v-Raf murine sarcoma viral oncogene homologue B. In order to identify relevant autoantigens, we screened a random peptide library (RPL) with pooled IgGs obtained from 50 patients with seronegative RA. Patients’ sera were then used in an ELISA test to identify the most frequently recognized peptide among those obtained by screening the RPL. Sera from age- and sex-matched healthy subjects were used as controls. We identified a specific peptide (RA-peptide) recognized by RA patients’ sera, but not by healthy subjects or by patients with other immune-mediated diseases. The majority of sera from seronegative and seropositive RA patients (73.8% and 63.6% respectively) contained IgG antibodies directed against the RA-peptide. Interestingly, this peptide shares homology with some self-antigens, such as Protein-tyrosine kinase 2 beta, B cell scaffold protein, Liprin-alfa1 and Cytotoxic T lymphocyte protein 4. Affinity purified anti-RA-peptide antibodies were able to cross react with these autoantigens. In conclusion, we identified a peptide that is recognized by seropositive and, most importantly, by seronegative RA patients’ sera, but not by healthy subjects, conferring to this epitope a high degree of specificity. This peptide shares also homology with other autoantigens which can be recognized by autoantibodies present in seronegative RA sera. These newly identified autoantibodies, although present also in a percentage of seropositive RA patients, may be considered as novel serum biomarkers for seronegative RA, which lacks the presence of RF and/or ACPAs.

Structural characterization of human peptidyl-arginine deiminase type III by X-ray crystallography.

The Ca2+-dependent enzyme peptidyl-arginine deiminase type III (PAD3) catalyses the deimination of arginine residues to form citrulline residues in proteins such as keratin, filaggrin and trichohyalin. This is an important post-translation modification that is required for normal hair and skin formation in follicles and keratocytes. The structure of apo human PAD3 was determined by X-ray crystallography to a resolution of 2.8 Å. The structure of PAD3 revealed a similar overall architecture to other PAD isoforms: the N-terminal and middle domains of PAD3 show sequence and structural variety, whereas the sequence and structure of the C-terminal catalytic domain is highly conserved. Structural analysis indicates that PAD3 is a dimer in solution, as is also the case for the PAD2 and PAD4 isoforms but not the PAD1 isoform.