Protein Citrullination by Peptidyl Arginine Deiminase/Arginine Deiminase Homologs in Members of the Human Microbiota and Its Recognition by Anti-Citrullinated Protein Antibodies.

The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100-200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case-control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal-Wallis test and Mann-Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis.

Iguratimod inhibits protein citrullination and inflammation by downregulating NBCe2 in patients with rheumatoid arthritis.

BACKGROUND: Bicarbonate has recently been identified as a crucial factor affecting peptidylarginine deiminase (PAD) activity; however, the mechanism underlying its role in rheumatoid arthritis (RA) remains unclear. Iguratimod (IGU), a small-molecule disease-modifying anti-rheumatic drug, requires further investigation. This study aimed to explore the mechanism by which bicarbonate affects citrullination and inflammation in RA and identify new targets for IGU. METHODS: We enrolled 20 patients with RA in the study. Sodium bicarbonate cotransporter 2 (NBCe2) was detected in the peripheral blood neutrophils and peripheral blood mononuclear cells (PBMCs) of these patients. The effects of varying concentrations of IGU, methotrexate (MTX), dexamethasone (DXM), and S0859 (an NBCe2 inhibitor) on NBCe2, PAD2, PAD4, and citrullinated histone H3 (cit-H3) levels in, migration ability of, and cytokine production from neutrophils and PBMCs were examined. RESULTS: Our findings showed that in patients with RA, citrullinated protein production by peripheral blood neutrophils instead of PBMCs, which showed higher NBCe2 expression levels, increased with an increase in the bicarbonate concentration. In addition, tumor necrosis factor-alpha (TNF-α) promoted NBCe2 expression in neutrophils from patients with RA. Furthermore, we revealed that the inhibitory effects of IGU on neutrophil NBCe2 and cit-H3 levels, degrees of inhibition of neutrophil and PBMC migration, and suppression of interleukin 6, TNF-α, and metalloproteinase-9 secretion from neutrophil-like differentiated HL-60 cells did not substantially differ from those of MTX, DXM, and S0859 at specific doses. CONCLUSIONS: Bicarbonate promotes protein citrullination and inflammation in RA via NBCe2, and IGU can downregulate NBCe2.

Myelin basic proteins charge isomers interact differently with the peptidyl arginine deiminase-2.

The deamination of arginine and its conversion to citrulline is a modification observed in positively charged proteins such as histones or myelin basic protein (MBP). This reaction is catalyzed by peptidyl arginine deiminase (PAD), whose abnormal activation is associated with autoimmune diseases like rheumatoid arthritis and multiple sclerosis. However, the mechanisms that trigger PAD activation and the pathophysiological processes involved in hypercitrullination remain unknown. In this study, we investigated the interaction between PAD and various charged isomers of MBP, each differing in the degree of post-translational modification. Immunoprecipitation experiments were conducted to examine the binding between PAD and the different charge isomers of MBP. Our findings revealed that the phosphorylated forms of MBP (C3 and C4) exhibited a higher affinity for PAD compared to the unmodified (C1) and fully citrullinated forms (C8). Additionally, we observed that only in the presence of the unmodified C1 isomer did PAD undergo autocitrullination, which was inhibited by the endogenous guanidine-containing component, creatine. In the presence of other isomers, PAD did not undergo autocitrullination. Furthermore, we found that the unmodified isomer of MBP-C1 contains methylated arginines, which were not affected by the pre-treatment with PAD. Based on our findings, we propose that the increased phosphorylation of central threonines in the original MBP may trigger PAD activation, leading to increased citrullination of the protein and subsequent disorganization of the myelin sheath. These insights contribute to a better understanding of the underlying mechanisms in autoimmune diseases associated with hypercitrullination, potentially opening new avenues for therapeutic interventions.

PAD2 dysregulation and aberrant protein citrullination feature prominently in reactive astrogliosis and myelin protein aggregation in sporadic ALS.

Alteration in protein citrullination (PC), a common posttranslational modification (PTM), contributes to pathogenesis in various inflammatory disorders. We previously reported that PC and protein arginine deiminase 2 (PAD2), the predominant enzyme isoform that catalyzes this PTM in the central nervous system (CNS), are altered in mouse models of amyotrophic lateral sclerosis (ALS). We now demonstrate that PAD2 expression and PC are altered in human postmortem ALS spinal cord and motor cortex compared to controls, increasing in astrocytes while trending lower in neurons. Furthermore, PC is enriched in protein aggregates that contain the myelin proteins PLP and MBP in ALS. These results confirm our findings in ALS mouse models and suggest that altered PAD2 and PC contribute to neurodegeneration in ALS.

Molecular Factors in PAD2 (PADI2) and PAD4 (PADI4) Are Associated with Interstitial Lung Disease Susceptibility in Rheumatoid Arthritis Patients.

Around 50% of rheumatoid arthritis (RA) patients show some extra-articular manifestation, with the lung a usually affected organ; in addition, the presence of anti-citrullinated protein antibodies (ACPA) is a common feature, which is caused by protein citrullination modifications, catalyzed by the peptidyl arginine deiminases (PAD) enzymes. We aimed to identify single nucleotide variants (SNV) in PADI2 and PADI4 genes (PAD2 and PAD4 proteins, respectively) associated with susceptibility to interstitial lung disease (ILD) in RA patients and the PAD2 and PAD4 levels. Material and methods: 867 subjects were included: 118 RA-ILD patients, 133 RA patients, and 616 clinically healthy subjects (CHS). Allelic discrimination was performed in eight SNVs using qPCR, four in PADI2 and four in PADI4. The ELISA technique determined PAD2 and PAD4 levels in serum and bronchoalveolar lavage (BAL) samples, and the population structure was evaluated using 14 informative ancestry markers. Results: The rs1005753-GG (OR = 4.9) in PADI2 and rs11203366-AA (OR = 3.08), rs11203367-GG (OR = 2.4) in PADI4 are associated with genetic susceptibility to RA-ILD as well as the ACTC haplotype (OR = 2.64). In addition, the PAD4 protein is increased in RA-ILD individuals harboring the minor allele homozygous genotype in PADI4 SNVs. Moreover, rs1748033 in PADI4, rs2057094, and rs2076615 in PADI2 are associated with RA susceptibility. In conclusion, in RA patients, single nucleotide variants in PADI4 and PADI2 are associated with ILD susceptibility. The rs1748033 in PADI4 and two different SNVs in PADI2 are associated with RA development but not ILD. PAD4 serum levels are increased in RA-ILD patients.

Delayed puberty, gonadotropin abnormalities and subfertility in male Padi2/Padi4 double knockout mice.

BACKGROUND: Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. METHODS: Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. RESULTS: Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. CONCLUSIONS: The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.

Padi2/3 Deficiency Alters the Epigenomic Landscape and Causes Premature Differentiation of Mouse Trophoblast Stem Cells.

Histone citrullination is a relatively poorly studied epigenetic modification that involves the irreversible conversion of arginine residues into citrulline. It is conferred by a small family of enzymes known as protein arginine deiminases (PADIs). PADI function supports the pluripotent state of embryonic stem cells, but in other contexts, also promotes efficient cellular differentiation. In the current study, we sought to gain deeper insights into the possible roles of PADIs in mouse trophoblast stem cells (TSCs). We show that Padi2 and Padi3 are the most highly expressed PADI family members in TSCs and are rapidly down-regulated upon differentiation. Padi2/3 double knockout (DKO) TSCs express lower levels of stem cell transcription factors CDX2 and SOX2 and are prone to differentiate into extremely large trophoblast giant cells, an effect that may be mediated by centrosome duplication defects. Interestingly, Padi2/3 DKO TSCs display alterations to their epigenomic landscape, with fewer H3K9me3-marked chromocentric foci and globally reduced 5-methylcytosine levels. DNA methylation profiling identifies that this effect is specifically evident at CpG islands of critical trophoblast genes, such as Gata3, Peg3, Socs3 and Hand1. As a consequence of the hypomethylated state, these factors are up-regulated in Padi2/3 DKO TSCs, driving their premature differentiation. Our data uncover a critical epigenetic role for PADI2/3 in safeguarding the stem cell state of TSCs by modulating the DNA methylation landscape to restrict precocious trophoblast differentiation.

Molecular Mechanism of Protein Arginine Deiminase 2: A Study Involving Multiple Microsecond Long Molecular Dynamics Simulations.

Peptidylarginine deiminase 2 (PAD2) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. This kind of structural modification in histone molecules may affect gene regulation, leading to effects that may trigger several diseases, including breast cancer, which makes PAD2 an attractive target for anticancer drug development. To design new effective inhibitors to control activation of PAD2, improving our understanding of the molecular mechanisms of PAD2 using up-to-date computational techniques is essential. We have designed five different PAD2-substrate complex systems based on varying protonation states of the active site residues. To search the conformational space broadly, multiple independent molecular dynamics simulations of the complexes have been performed. In total, 50 replica simulations have been performed, each of 1 µs, yielding a total simulation time of 50 µs. Our findings identify that the protonation states of Cys647, Asp473, and His471 are critical for the binding and localization of the N-α-benzoyl-l-arginine ethyl ester substrate within the active site. A novel mechanism for enzyme activation is proposed according to near attack conformers. This represents an important step in understanding the mechanism of citrullination and developing PAD2-inhibiting drugs for the treatment of breast cancer.

Peptidylarginine deiminase 2 promotes T helper 17-like T cell activation and activated T cell-autonomous death (ACAD) through an endoplasmic reticulum stress and autophagy coupling mechanism.

Peptididylarginine deiminase type 2 (PADI2) catalyzes the conversion of arginine residues to citrulline residues on proteins. We demonstrate that PADI2 induces T cell activation and investigate how PADI2 promotes activated T cell autonomous death (ACAD). In activated Jurkat T cells, overexpression of PADI2 significantly increases citrullinated proteins and induces endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling, ultimately resulting in the expression of autophagy-related proteins and autophagy. PADI2 promoted autophagy and resulted in the early degradation of p62 and the light chain 3B (LC3B)-II accumulation. In Jurkat T cells, silencing the autophagy-related gene (Atg) 12 protein inhibits PADI2-mediated autophagy and promotes ER stress and apoptosis, whereas overexpression of Atg12 decreased ER stress and prolonged autophagy to promote cell survival. Additionally, PADI2 regulates T cell activation and the production of Th17 cytokines in Jurkat T cells (interleukins 6, IL-17A, IL-17F, IL-21, and IL-22). In Jurkat T cells, silencing IL-6 promotes autophagy mediated by PADI2 and inhibits PADI2-induced apoptosis, whereas silencing Beclin-1 increases the activation and survival of Th17-like T cells while decreasing autophagy and apoptosis. PADI2 silencing alleviates ER stress caused by PADI2 and decreases cytokine expression associated with Th17-like T cell activation and ACAD. We propose that PADI2 was involved in Th17 lymphocyte ACAD via a mechanism involving ER stress and autophagy that was tightly regulated by PADI2-mediated citrullination. These findings suggest that inhibiting Th17 T cell activation and the development of severe autoimmune diseases may be possible through the use of novel antagonists that specifically target PADI2.

ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

PAD-2-mediated citrullination of nucleophosmin provides an effective target for tumor immunotherapy.

BACKGROUND: The enzymatic conversion of arginine to citrulline is involved in gene and protein regulation and in alerting the immune system to stressed cells, including tumor cells. Nucleophosmin (NPM) is a nuclear protein that plays key roles in cellular metabolism including ribosome biogenesis, mRNA processing and chromatin remodeling and is regulated by citrullination. In this study, we explored if the same citrullinated arginines within NPM are involved in gene regulation and immune activation. METHODS: HLA-DP4 and HLA-DR4 transgenic mice were immunized with 22 citrullinated NPM overlapping peptides and immune responses to the peptides were assessed by ex vivo ELISpot assays. Antitumor immunity of NPM targeted vaccination was assessed by challenging transgenic mice with B16F1 HHDII/iDP4, B16F1 HHDII/PAD2KOcDP4, B16F1 HHDII and Lewis lung carcinoma cells/cDP4 cells subcutaneously. Peripheral blood mononuclear cells isolated from healthy donors were stimulated with NPM266-285cit peptides with/without CD45RO+memory cell depletion to assess if the responses in human were naïve or memory. RESULTS: In contrast to NPM regulation, which is mediated by peptidylarginine deiminase (PAD4) citrullination of arginine at position 197, only citrullinated NPM266-285 peptide induced a citrulline-specific CD4 T cell response in transgenic mice models expressing human HLA-DP4 or HLA-DR4. Vaccinations with the NPM266-285cit peptide stimulated antitumor responses that resulted in dramatic tumor therapy, greatly improved survival, and protected against rechallenge without further vaccination. The antitumor response was lost if MHCII expression on the tumor cells was knocked out demonstrating direct presentation of the NPM266-285cit epitope in tumors. This antitumor response was lost in B16 tumors lacking PAD2 enzyme indicating NPM266cit is citrullinated by PAD2 in this model. Assessment of the T cell repertoire in healthy individuals and patients with lung cancer also showed CD4 T cells that respond to NPM266-285cit. The proliferative CD4 responses displayed a Th1 profile as they were accompanied with increased IFNγ and granzyme B expression. Depletion of CD45RO+ memory cells prior to stimulation suggested that responses originated from a naïve population in healthy donors. CONCLUSION: This study indicates PAD2 can citrullinate the nuclear antigen NPM at position 277 which can be targeted by CD4 T cells for antitumor therapy. This is distinct from PAD4 citrullination of arginine 197 within NPM which results in its transport from the nucleoli to the nucleoplasm.

Syndecan-2 regulates PAD2 to exert antifibrotic effects on RA-ILD fibroblasts.

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Peptidylarginine Deiminase 2 in Murine Antiviral and Autoimmune Antibody Responses.

The peptidylarginine deiminases (PADs) and the citrullinated proteins that they generate have key roles in innate immunity and rheumatoid arthritis, an inflammatory arthritis with antibodies that target citrullinated proteins. However, the importance of PADs, particularly PAD2, in the adaptive immune response, both normal and pathogenic, is newly emerging. In this study, we evaluated a requirement for PAD2 in the antibody response in collagen-induced arthritis (CIA), a T and B cell-driven murine model of rheumatoid arthritis, and in the protective antibody response to murine influenza infection. Using PAD2-/- and PAD2+/+ mice on the DBA/1J background, we found that PAD2 is required for maximal anti-collagen antibody levels, but not collagen-specific plasma cell numbers, T cell activation or polarization, or arthritis severity in CIA. Also, we found that PAD2 is required not just for normal levels of persistent hemagglutination inhibiting antibodies but also for full protection from lethal influenza rechallenge. Together, these data provide evidence for a novel modest requirement for PAD2 in a normal antiviral antibody response and in an abnormal autoantibody response in inflammatory arthritis.

The Clinical and Prognostic Significance of Protein Arginine Deiminases 2 and 4 in Colorectal Cancer.

INTRODUCTION: Protein arginine deiminases (PADIs) are a family of enzymes that catalyse the post-translational modification of proteins. Association between PADI expression and clinicopathology, protein expression, and outcome was determined. METHODS: PADI2 and PADI4 expression was assessed immunohistochemically in a cohort of colorectal cancer (CRC) patients. RESULTS: CRC tissues expressed variable levels of PADI2 which was mainly localised in the cytoplasm and correlated with patient survival (p = 0.005); high expression increased survival time from 43.5 to 67.6 months. Expression of cytoplasmic PADI2 correlated with the expression of nuclear ß catenin, PADI4, and alpha-enolase. In contrast, expression of nuclear PADI2 correlated with a decrease in survival (p = 0.010), with high expression decreasing survival from 76.4 to 42.9 months. CRC tissues expressed variable levels of PADI4 in both the nucleus and cytoplasm. Expression of cytoplasmic PADI4 correlated with survival (p = 0.001) with high expression increasing survival time from 48.1 to 71.8 months. Expression of cytoplasmic PADI4 correlated with expression of nuclear ß catenin, alpha-enolase (p ≤ 0.0001, p = 0.002), and the apoptotic related protein, Bcl-2. Expression of nuclear PADI4 also correlated with survival (p = 0.011), with high expression of nuclear PADI4 increasing survival time from 55.4 to 74 months. Expression of nuclear PADI4 correlated with p53, alpha-enolase, and Bcl-2. Multivariate analysis showed that TNM stage, cytoplasmic PADI2, and PADI4 remained independent prognostic factors in CRC. Both PADI2 and PADI4 are good prognostic factors in CRC. CONCLUSION: High expression of cytoplasmic PADI2, PADI4, and nuclear PADI4 were associated with an increase in overall survival.

Circulating CitH3 Is a Reliable Diagnostic and Prognostic Biomarker of Septic Patients in Acute Pancreatitis.

Purpose: Acute pancreatitis (AP) is an inflammatory disease. AP starts with sterile inflammation and is often complicated with critical local or systemic infection or sepsis in severe cases. Septic AP activates peptidyl arginine deiminase (PAD) and citrullinates histone H3 (CitH3), leading to neutrophil extracellular trap (NET) formation. Investigating the role of NETs and underlying mechanisms in septic AP may facilitate developing diagnostic and therapeutic approaches. In this study, we sought to identify the expression of CitH3 in septic AP patients and to analyze the correlation of CitH3 concentration with NET components as well as clinical outcomes. Methods: Seventy AP patients with or without sepsis (40 septic cases, 30 nonseptic cases) and 30 healthy volunteers were recruited in this study. Concentration of NET components (CitH3 and double-strain DNA) and key enzymes (PAD2/4) were measured. Clinical and laboratory characteristics of patients were recorded and analyzed. Results: Levels of CitH3 were elevated significantly in septic AP patients compared with those in nonseptic AP and healthy volunteers. The area under the curve (AUC, 95% confidence interval) for diagnosing septic AP was 0.93 (0.86-1.003), and the cutoff was 43.05 pg/ml. Among septic AP cases (n = 40), the concentration of CitH3 was significantly increased in those who did not survive or were admitted to the intensive care unit, when compared with that in those who survived or did not require intensive care unit. Association analysis revealed that CitH3 concentration was positively correlated with PAD2, PAD4, dsDNA concentration, and Sequential Organ Failure Assessment scores. Conclusion: CitH3 concentration increased in septic AP patients and was closely correlated with disease severity and clinical outcomes. CitH3 may potentially be a diagnostic and prognostic biomarker of septic AP.

Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4.

Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4+ T cells, CD8+ T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 µM, while BB-Cl-amidine was cytotoxic at concentrations above 1 µM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential.

Proximity-Dependent Labeling of Cysteines.

Mapping protein-protein interactions is crucial for understanding various signaling pathways in living cells, and developing new techniques for this purpose has attracted significant interest. Classic methods (e.g., the yeast two-hybrid) have been supplanted by more sophisticated chemical approaches that label proximal proteins (e.g., BioID, APEX). Herein we describe a proximity-based approach that uniquely labels cysteines. Our approach exploits the nicotinamide N-methyltransferase (NNMT)-catalyzed methylation of an alkyne-substituted 4-chloropyridine (SS6). Upon methylation of the pyridinium nitrogen, this latent electrophile diffuses out of the active site and labels proximal proteins on short time scales (≤5 min). We validated this approach by identifying known (and novel) interacting partners of protein arginine deiminase 2 (PAD2) and pyruvate dehydrogenase kinase 1 (PDK1). To our knowledge, this technology uniquely exploits a suicide substrate to label proximal cysteines in live cells.

Peptidylarginine Deiminase 2 in Host Immunity: Current Insights and Perspectives.

Peptidylarginine deiminases (PADs) are a group of enzymes that catalyze post-translational modifications of proteins by converting arginine residues into citrullines. Among the five members of the PAD family, PAD2 and PAD4 are the most frequently studied because of their abundant expression in immune cells. An increasing number of studies have identified PAD2 as an essential factor in the pathogenesis of many diseases. The successes of preclinical research targeting PAD2 highlights the therapeutic potential of PAD2 inhibition, particularly in sepsis and autoimmune diseases. However, the underlying mechanisms by which PAD2 mediates host immunity remain largely unknown. In this review, we will discuss the role of PAD2 in different types of cell death signaling pathways and the related immune disorders contrasted with functions of PAD4, providing novel therapeutic strategies for PAD2-associated pathology.

PADI2 Polymorphisms Are Significantly Associated With Rheumatoid Arthritis, Autoantibodies Serologic Status and Joint Damage in Women from Southern Mexico.

The enzymes of the family peptidylarginine deiminases (PADs) have an important role in the pathogenesis of rheumatoid arthritis (RA) due to their association with the anti-citrullinated protein antibodies (ACPA) production. To evaluate the association between single-nucleotide polymorphisms (SNPs) in the PADI2 gene and RA susceptibility, related clinical parameters, and the serologic status of autoantibodies in a women population with RA from southern Mexico, a case-control study was conducted (case n=229; control n=333). Sociodemographic characteristics were evaluated, along with clinical parameters, inflammation markers, the levels of ACPAs as anti-cyclic citrullinated peptides (anti-CCPs), anti-modified citrullinated vimentin (anti-MCV), and rheumatoid factor (RF). Genomic DNA was extracted from peripheral blood, and three SNPs of the PADI2 gene (rs1005753, rs2057094, and rs2235926) were performed by qPCR using TaqMan probes. The data analysis reveals that the carriers of the T allele for rs2057094 and rs2235926 presented an earlier onset of the disease (ß= -3.26; p = 0.03 and ß = -4.13; p = 0.015, respectively) while the carriers of the T allele for rs1005753 presented higher levels of anti-CCPs (ß= 68.3; p = 0.015). Additionally, the T allele of rs2235926 was associated with a positive RF (OR = 2.90; p = 0.04), anti-MCV (OR = 2.92; p = 0.05), and with the serologic status anti-CCP+/anti-MCV+ (OR = 3.02; p = 0.03), and anti-CCP+/anti-MCV+/RF+ (OR = 3.79; p = 0.004). The haplotypes GTT (OR =1.52; p = 0.027) and TTT (OR = 1.32; p = 0.025) were associated with the presence of RA. In addition, in this study the haplotype TTT is linked to the presence of radiographic joint damage defined by a Sharp-van der Heijde score (SHS) ≥2 (OR = 1.97; p = 0.0021) and SHS ≥3 (OR = 1.94; p = 0.011). The haplotype TTT of SNPs rs1005753, rs2057094, and rs2235926 of the PADI2 gene confers genetic susceptibility to RA and radiographic joint damage in women from southern Mexico. The evidence reveals that SNPs of the PADI2 gene favors the presence of a positive serologic status in multiple autoantibodies and the clinical manifestations of RA at an early onset age.

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays.

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.