Neutrophil-Derived Peptidyl Arginine Deiminase Activity Contributes to Pulmonary Emphysema by Enhancing Elastin Degradation.

In chronic obstructive pulmonary disease (COPD), inflammation gives rise to protease-mediated degradation of the key extracellular matrix protein, elastin, which causes irreversible loss of pulmonary function. Intervention against proteolysis has met with limited success in COPD, due in part to our incomplete understanding of the mechanisms that underlie disease pathogenesis. Peptidyl arginine deiminase (PAD) enzymes are a known modifier of proteolytic susceptibility, but their involvement in COPD in the lungs of affected individuals is underexplored. In this study, we showed that enzyme isotypes PAD2 and PAD4 are present in primary granules of neutrophils and that cells from people with COPD release increased levels of PADs when compared with neutrophils of healthy control subjects. By examining bronchoalveolar lavage and lung tissue samples of patients with COPD or matched smoking and nonsmoking counterparts with normal lung function, we reveal that COPD presents with markedly increased airway concentrations of PADs. Ex vivo, we established citrullinated elastin in the peripheral airways of people with COPD, and in vitro, elastin citrullination significantly enhanced its proteolytic degradation by serine and matrix metalloproteinases, including neutrophil elastase and matrix metalloprotease-12, respectively. These results provide a mechanism by which neutrophil-released PADs affect lung function decline, indicating promise for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

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.

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.

Targeting Peptidylarginine Deiminase 3 to Efficiently Suppress Herpes Simplex Virus Type 2 Infection.

Protein expression is regulated through multiple mechanisms, including post-translational modifications (PTMs), which can alter protein structure, stability, localization, and function. Among these, citrullination stands out due to its ability to convert arginine residues into citrulline, altering protein charge and mass. This modification is catalyzed by calcium-dependent protein arginine deiminases (PADs), enzymes implicated in various inflammatory diseases. We have recently shown that human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) exploit these enzymes to enhance their replication capabilities. Although the role of PADs in HCMV and HSV-1 infections is well documented, their involvement in HSV-2 infection has not yet been thoroughly investigated. Here, we demonstrate that HSV-2 manipulates the overall protein citrullination profile by activating three PAD isoforms: PAD2, PAD3, and PAD4. However, as previously observed during HSV-1 infection, PAD3 is the most significantly upregulated isoform, both at the mRNA and protein levels. Consistently, we demonstrate that inhibiting PAD3, either through the specific inhibitor CAY10727 or via CRISPR/Cas9-mediated gene silencing, markedly reduces HSV-2 replication and viral protein expression. Lastly, we show that CAY10727 displays an IC50 value of 0.3 µM, which is extremely close to what was previously observed for HSV-1. Overall, our findings highlight the crucial role of PAD3 in the life cycle of HSV-2 and suggest that the targeted inhibition of PAD3 may represent a promising approach for treating HSV-2 infections, especially in cases resistant to existing antiviral therapies.

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.

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.

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.

Peptidylarginine Deiminases 2 Mediates Caspase-1-Associated Lethality in Pseudomonas aeruginosa Pneumonia-Induced Sepsis.

BACKGROUND: Pseudomonas aeruginosa (PA) is a pathogenic bacterium that causes severe pneumonia in critically ill and immunocompromised patients. Peptidylarginine deiminase (PAD) 2, PAD4, and caspase-1 are important enzymes in mediating host response to infection. The goal of this study was to determine the interplay between PAD2, PAD4, and caspase-1 in PA pneumonia-induced sepsis. METHODS: Pneumonia was produced in wild-type, Pad2-/-, and Pad4-/- mice by intranasal inoculation of PA (2.5 × 106 colony-forming units per mouse), and survival (n = 15/group) was monitored for 10 days. Bone marrow-derived macrophages (BMDMs) were isolated for in vitro studies. Samples were collected at specific timepoints for Western blot, bacterial load determination, and flow cytometry analysis. RESULTS: Caspase-1-dependent inflammation was diminished in PA-inoculated Pad2-/- mice, contributing to reduced macrophage death and enhanced bacterial clearance. In addition, Pad2-/- mice exhibited improved survival and attenuated acute lung injury after PA infection. In contrast, Pad4-/- mice did not display diminished caspase-1 activation, altered bacterial loads, or improved survival. CONCLUSIONS: Peptidylarginine deiminase 2 plays an essential role in the pathogenesis of pulmonary sepsis by mediating caspase-1 activation. This goes against previous findings of PAD4 in sepsis. Our study suggests that PAD2 is a potential therapeutic target of PA pneumonia-induced sepsis.

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.

The roles of PAD2- and PAD4-mediated protein citrullination catalysis in cancers.

Peptidylarginine deiminases (PADs) catalyze the conversion of arginine residues to citrulline residues on target proteins in the presence of calcium ions. This elaborate type of posttranslational modification is termed citrullination. PADs may regulate gene transcriptional activity via histone citrullination. There has been an increasing appreciation for the roles of PADs in a wide variety of biological processes. In this review article, we summarize recent evidence indicating that PADs and citrullinated proteins are involved in several physiological and pathological processes related to cancer. Of particular interest is that PAD2 and PAD4 exhibit characteristic expression levels, activities and specific biological effects in diverse types of cancer. We also list several PAD inhibitors, propose the possible mechanisms underlying the biological actions of PAD-mediated protein citrullination in experimental models and discuss the potential therapeutic value of PADs and their inhibitors for disease diagnosis and treatment.

Angiogenic responses in a 3D micro-engineered environment of primary endothelial cells and pericytes.

Angiogenesis plays a key role in the pathology of diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. Understanding the driving forces of endothelial cell migration and organization, as well as the time frame of these processes, can elucidate mechanisms of action of important pathological pathways. Herein, we have developed an organ-specific microfluidic platform recapitulating the in vivo angiogenic microenvironment by co-culturing mouse primary brain endothelial cells with brain pericytes in a three-dimensional (3D) collagen scaffold. As a proof of concept, we show that this model can be used for studying the angiogenic process and further comparing the angiogenic properties between two different common inbred mouse strains, C57BL/6J and 129S1/SvlmJ. We further show that the newly discovered angiogenesis-regulating gene Padi2 promotes angiogenesis through Dll4/Notch1 signaling by an on-chip mechanistic study. Analysis of the interplay between primary endothelial cells and pericytes in a 3D microfluidic environment assists in the elucidation of the angiogenic response.

Peptidylarginine Deiminase Inhibitor Application, Using Cl-Amidine, PAD2, PAD3 and PAD4 Isozyme-Specific Inhibitors in Pancreatic Cancer Cells, Reveals Roles for PAD2 and PAD3 in Cancer Invasion and Modulation of Extracellular Vesicle Signatures.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with limited survival rate. Roles for peptidylarginine deiminases (PADs) have been studied in relation to a range of cancers with roles in epigenetic regulation (including histone modification and microRNA regulation), cancer invasion, and extracellular vesicle (EV) release. Hitherto though, knowledge on PADs in PDAC is limited. In the current study, two PDAC cell lines (Panc-1 and MiaPaCa-2) were treated with pan-PAD inhibitor Cl-amidine as well as PAD2, PAD3, and PAD4 isozyme-specific inhibitors. Effects were assessed on changes in EV signatures, including EV microRNA cargo (miR-21, miR-126, and miR-221), on changes in cellular protein expression relevant for pancreatic cancer progression and invasion (moesin), for mitochondrial housekeeping (prohibitin, PHB), and gene regulation (deiminated histone H3, citH3). The two pancreatic cancer cell lines were found to predominantly express PAD2 and PAD3, which were furthermore expressed at higher levels in Panc-1, compared with MiaPaCa-2 cells. PAD2 isozyme-specific inhibitor had the strongest effects on reducing Panc-1 cell invasion capability, which was accompanied by an increase in moesin expression, which in pancreatic cancer is found to be reduced and associated with pancreatic cancer aggressiveness. Some reduction, but not significant, was also found on PHB levels while effects on histone H3 deimination were variable. EV signatures were modulated in response to PAD inhibitor treatment, with the strongest effects observed for PAD2 inhibitor, followed by PAD3 inhibitor, showing significant reduction in pro-oncogenic EV microRNA cargo (miR-21, miR-221) and increase in anti-oncogenic microRNA cargo (miR-126). While PAD2 inhibitor, followed by PAD3 inhibitor, had most effects on reducing cancer cell invasion, elevating moesin expression, and modulating EV signatures, PAD4 inhibitor had negligible effects and pan-PAD inhibitor Cl-amidine was also less effective. Compared with MiaPaCa-2 cells, stronger modulatory effects for the PAD inhibitors were observed in Panc-1 cells, which importantly also showed strong response to PAD3 inhibitor, correlating with previous observations that Panc-1 cells display neuronal/stem-like properties. Our findings report novel PAD isozyme regulatory roles in PDAC, highlighting roles for PAD isozyme-specific treatment, depending on cancer type and cancer subtypes, including in PDAC.

Neuroinflammation and histone H3 citrullination are increased in X-linked Dystonia Parkinsonism post-mortem prefrontal cortex.

Neuroinflammation plays a pathogenic role in neurodegenerative diseases and recent findings suggest that it may also be involved in X-linked Dystonia-Parkinsonism (XDP) pathogenesis. Previously, fibroblasts and neuronal stem cells derived from XDP patients demonstrated hypersensitivity to TNF-α, dysregulation in NFκB signaling, and an increase in several pro-inflammatory markers. However, the role of inflammatory processes in XDP patient brain remains unknown. Here we demonstrate that there is a significant increase in astrogliosis and microgliosis in human post-mortem XDP prefrontal cortex (PFC) compared to control. Furthermore, there is a significant increase in histone H3 citrullination (H3R2R8R17cit3) with a concomitant increase in peptidylarginine deaminase 2 (PAD2) and 4 (PAD4), the enzymes catalyzing citrullination, in XDP post-mortem PFC. While there is a significant increase in myeloperoxidase (MPO) levels in XDP PFC, neutrophil elastase (NE) levels are not altered, suggesting that MPO may be released by activated microglia or reactive astrocytes in the brain. Similarly, there was an increase in H3R2R8R17cit3, PAD2 and PAD4 levels in XDP-derived fibroblasts. Importantly, treatment of fibroblasts with Cl-amidine, a pan inhibitor of PAD enzymes, reduced histone H3 citrullination and pro-inflammatory chemokine expression, without affecting cell survival. Taken together, our results demonstrate that inflammation is increased in XDP post-mortem brain and fibroblasts and unveil a new epigenetic potential therapeutic target.

Peptidyl Arginine Deiminase 2 (PADI2)-Mediated Arginine Citrullination Modulates Transcription in Cancer.

Protein arginine deimination leading to the non-coded amino acid citrulline remains a key question in the field of post-translational modifications ever since its discovery by Rogers and Simmonds in 1958. Citrullination is catalyzed by a family of enzymes called peptidyl arginine deiminases (PADIs). Initially, increased citrullination was associated with autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, as well as other neurological disorders and multiple types of cancer. During the last decade, research efforts have focused on how citrullination contributes to disease pathogenesis by modulating epigenetic events, pluripotency, immunity and transcriptional regulation. However, our knowledge regarding the functional implications of citrullination remains quite limited, so we still do not completely understand its role in physiological and pathological conditions. Here, we review the recently discovered functions of PADI2-mediated citrullination of the C-terminal domain of RNA polymerase II in transcriptional regulation in breast cancer cells and the proposed mechanisms to reshape the transcription regulatory network that promotes cancer progression.

The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage.

OBJECTIVE: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. METHODS: The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate. RESULTS: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1ß and TNF-α in the U937 cells and IL-1ß and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1ß, IL-6, and TNF-α were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. CONCLUSION: This study showed that PADI2 could promote IL-1ß, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines.

Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.

Calcium Regulates the Nuclear Localization of Protein Arginine Deiminase 2.

Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the post-translational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor target genes. However, PAD2 lacks a canonical nuclear localization sequence, and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Binding of calcium to PAD2 weakens this cytoplasmic interaction, which generates a pool of calcium-bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into the nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus.

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.

Peptidylarginine deiminase (PAD): A promising target for chronic diseases treatment.

In 1958, the presence of citrulline in the structure of the proteins was discovered for the first time. Several years later they found that Arginine converted to citrulline during a post-translational modification process by PAD enzyme. Each PAD is expressed in a certain tissue developing a series of diseases such as inflammation and cancers. Among these, PAD2 and PAD4 play a role in the development of rheumatoid arthritis (RA) by producing citrullinated autoantigens and increasing the production of inflammatory cytokines. PAD4 is also associated with the formation of NET structures and thrombosis. In the crystallographic structure, PAD has several calcium binding sites, and the active site of the enzyme consists of different amino acids. Various PAD inhibitors have been developed divided into pan-PAD and selective PAD inhibitors. F-amidine, Cl-amidine, and BB-Cl-amidine are some of pan-PAD inhibitors. AFM-30a and JBI589 are selective for PAD2 and PAD4, respectively. There is a need to evaluate the effectiveness of existing inhibitors more accurately in the coming years, as well as design and production of novel inhibitors targeting highly specific isoforms.

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.