Search Results (67)

The peptidylarginine deiminase (PAD) family includes five isozymes (PAD1–4 and PAD6) with unique tissue distributions and substrate specificities. These enzymes facilitate citrullination, a post-translational modification where positively charged arginine residues are converted into neutral citrulline residues in the presence of calcium ions. This process significantly changes protein properties, affecting molecular interactions, structural stability, and biological functions. Over the past six years (2019–2025), there has been significant progress in understanding PAD activity mechanisms and their therapeutic potential. Recent discoveries include the regulated nuclear translocation of PAD2, PAD4’s specific role in forming cancer extracellular chromatin networks (CECNs), and the development of next-generation inhibitors with greatly improved pharmacological profiles. PAD4 is crucial in forming neutrophil extracellular traps (NETs). Citrullination of histones H3 and H4 by PAD4 destabilizes chromatin, helping release DNA-protein networks as an antibacterial defense. However, excessive NET formation can contribute to autoimmune diseases and thrombosis. Similarly, the bacterial peptidylarginine deiminase from Porphyromonas gingivalis (PPAD)—the only known prokaryotic citrullinating enzyme—plays a key role. Working with R-gingipains, PPAD triggers pathological citrullination of host proteins, leading to immune tolerance breakdown and linking periodontal disease with systemic autoimmune disorders such as rheumatoid arthritis, atherosclerosis, and Alzheimer’s disease. Once thought to be a rare post-translational modification, citrullination is now understood as a vital regulatory mechanism in both normal physiology and disease, involving both internal processes of homeostasis and external mechanisms of bacterial pathogenesis. Full article

Although female (XX) and male (XY) placentas generally function the same, it is evident that there are sex-specific postnatal health outcomes following placental dysfunction and pregnancy complications. Although the underlying causes for these sex differences are unclear, it is postulated that differences in XX and XY placental function are involved due to sex chromosomes and/or sex steroids. Studies in breast and prostate cancer cells demonstrated a role for the citrullination enzyme peptidylarginine deiminase 2 (PAD2) in post-translational regulation of estrogen (ESR) and androgen receptor (AR) signaling. The goal of this study is to determine if PAD2 is present in mouse placentas and if XX versus XY differences exist in the relative level of PAD2. Fetuses and placentas were collected from three pregnant mice (C57BL6) at 14 days of gestation. Total RNA and protein were isolated from XX and XY placentas, and relative mRNA and protein were analyzed by real-time PCR and Western blot. AR and PAD2 levels were significantly higher in XY than in XX placentas. This study is the first to demonstrate XX and XY differences in PAD2 and AR in the placenta. It suggests a role for PAD2 regulation of androgen receptor signaling in the XY placenta. Full article

In recent decades, chemotherapy has significantly improved cancer survival, yet its adverse effects on non-cancerous tissues raise increasing concerns. In this context, growing attention has been focused on natural compounds that may be useful in mitigating the undesirable effects of chemotherapeutic agents. Here, we aimed to demonstrate that Cytosporone B (CsnB) is a potent agent for counteracting the cardiovascular effects induced by Imatinib. To this end, 12-week-old male Wistar rats were studied; they were divided into three groups as follows: (1) control, (2) Imatinib-treated (Imatinib: 60 mg/kg/day, per os), (3) Imatinib + CsnB-treated (CsnB: 5 mg/kg/day, i.p.). After the two-week-long experimental period, rats were euthanized. Their hearts were used for the following biochemical measurements: NADPH oxidase (NOX4), high mobility group box 1 (HMGB1), peptidylarginine deiminase 4 (PAD4), inducible nitric oxide synthase (iNOS) expression, tumor necrosis factor-alpha (TNF-α) level, and myeloperoxidase (MPO) activity. Imatinib caused a marked upregulation of key inflammatory and oxidative markers, including HMGB1, TNF-α, MPO, iNOS, PAD4, and NOX4 in cardiac tissue; however, CsnB treatment mitigated these elevations, implying its role in opposing Imatinib-induced inflammatory and oxidative processes in the heart. Our findings suggest that CsnB holds promise as a cardioprotective agent capable of modulating Imatinib-induced adverse cardiac effects. Full article

Tenrecs are heterothermic burrowing mammals, which are capable of withstanding extreme environmental stressors, including during hibernation. Their phylogenetic position as reminiscent of an ancestral placental mammal makes tenrecs a unique model for evolutionarily conserved traits, with potential translatability to human physiology and pathobiology, including adaptations to extreme environments. In this study, we compared tenrec plasma for post-translational protein citrullination profiles (citrullinomes) and extracellular vesicle (EV) characteristics, including selected microRNA cargoes (miR-21, miR-155, miR-206, miR-210), between baseline active and hibernating states at low (12 °C) and high (28 °C) ambient temperatures. Our findings show considerable changes in citrullinome plasma profiles and associated Gene Ontology and KEGG pathway analysis linked to physiological and inflammatory processes, comparing hibernating and active states, also differing between the two ambient temperature groups. We furthermore identified modified EV profiles with respect to stress-related (miR-21, miR-155), hypoxia (miR-210) and metabolic/muscle related (miR-206) microRNA cargoes, which showed significant differences between active and hibernating animals, also comparing the two ambient temperature groups. Our findings show novel roles for post-translational protein citrullination in regulating immune and metabolic associated pathways in the tenrec, and highlight EV profiles, based on microRNA cargoes, as indicators for stress and metabolic responses in active versus hibernating states, including at different temperatures. Collectively our data highlights the tenrec as an evolutionary model for regulating pathobiological responses in extreme environments and may have translatable potential for human physiology and pathologies. Full article

Peptidylarginine deiminase 4 (PAD4) catalyzes protein citrullination, a post-translational modification implicated in type 1 diabetes mellitus (T1DM). This study examined PAD4 expression and activity in the pancreas of streptozotocin (STZ)-induced diabetic Wistar rats. Animals were divided into three groups: (A) STZ-induced diabetic rats (60 mg/kg, i.p.), (B) non-diabetic controls, and (C) diabetic rats treated with Cl-amidine (5 mg/kg), a pan-PAD inhibitor, from week six post-induction. Analyses included PAD4 mRNA and protein expression, citrullinated histone H3 (CitH3), calcium concentration, and neutrophil elastase activity. Diabetic rats exhibited increased PAD4 expression, CitH3 levels, and NETosis markers, alongside reduced pancreatic calcium, suggesting calcium consumption during PAD4 activation. Cl-amidine treatment attenuated NETosis. These results implicate PAD4 in T1DM pathogenesis via NETosis and support the utility of STZ-induced diabetic rats as a model for PAD4-targeted studies. Cl-amidine may represent a promising therapeutic approach to reduce pancreatic inflammation in T1DM. Full article

Neutrophils are essential components of innate immunity, executing a range of effector functions including phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs). A key hallmark of NET formation is the presence of citrullinated histone H3 (CitH3), produced by peptidylarginine deiminases (PAD2 and PAD4) to facilitate chromatin decondensation. While NETs play critical antimicrobial roles, excessive or dysregulated NET formation, termed NETosis, can drive tissue injury, chronic inflammation, and organ dysfunction across a wide spectrum of diseases. Beyond its structural role within NETs, CitH3 acts as a damage-associated molecular pattern (DAMP), amplifying immune activation and pathological inflammation. Elevated CitH3 levels have been identified as biomarkers in sepsis, viral infections, ischemia–reperfusion injury, organ transplantation, diabetic wounds, autoimmune diseases, and cancer. Despite increasing recognition of CitH3’s pathogenic contributions, its therapeutic potential remains largely untapped. This review summarizes recent advances in understanding the role of CitH3 in NETosis and immune dysfunction, highlights emerging strategies targeting CitH3 therapeutically, and identifies critical knowledge gaps. Collectively, these insights position CitH3 as a promising druggable biomarker for the diagnosis, prognosis, and treatment of acute and chronic inflammatory diseases. Full article

Neutrophil extracellular traps (NETs) are associated with the extracellular release of nuclear chromatin decorated with cytoplasmic proteins. Excessive release of NETs has been reported in chronic lung diseases, including chronic obstructive pulmonary disease (COPD). However, the role of NETs in the pathogenesis of COPD remains unclear. Peptidylarginine deaminase 4 (PAD4) contributes to NET formation. Therefore, in an elastase (ELS)-induced emphysema mouse model, we examined the role of PAD4 using Padi4 gene knockout (KO) mice. First, we confirmed that ELS induced NET formation in the parenchyma of the lungs. PAD4 deficiency suppressed ELS-induced NET expression and tended to ameliorate the lung tissue injury. The cellular profile of bronchoalveolar lavage fluid (BALF) did not differ between the two groups. Additionally, PAD4 deficiency ameliorated emphysema and apoptosis in lung cells. Finally, we examined the effects of PAD4 on comprehensive gene expression signatures using RNA sequencing. Enrichment analysis of the transcriptomic data revealed that the expression of several genes associated with COPD pathogenesis was altered in the KO mice. Overall, the results suggest that PAD4 deficiency improves NET formation and emphysema in the lungs; this pathway can be a potential therapeutic target for the treatment of COPD. Full article

Inflammation contributes to myocardial injury in ST-elevation myocardial infarction (STEMI). Interleukin-6 receptor (IL-6R) inhibition has been shown to mitigate myocardial injury and reduce levels of the prothrombotic and inflammatory mediator, neutrophil extracellular traps (NETs). The enzyme peptidylarginine deiminase 4 (PAD4) is central in NET formation. We hypothesized that PAD4 links IL-6R activation and NET formation. Methods: We conducted thrombus aspiration and peripheral blood sampling in 33 STEMI patients. In thrombi and leukocytes, we quantified the mRNA of IL-6, IL-6R, and PAD4. In peripheral blood, the protein levels of IL-6, IL-6R, PAD4, dsDNA, H3Cit, MPO-DNA, and troponin T were quantified. Results: In thrombi and circulating leukocytes, PAD4 mRNA was associated with IL-6R mRNA (thrombi: β = 0.34, 95% CI [0.16–0.53], p = 0.001, circulating leukocytes: β = 0.92, 95% CI [0.07–1.77], p = 0.036). There were no correlations between PAD4 and IL-6 in thrombi and leukocytes. The protein levels of IL-6R were associated with the NET marker H3Cit (r_s = 0.40, p = 0.02). In thrombi, PAD4 mRNA was associated with high levels of troponin T (β = 1.15 95% CI [0.27–2.04], p = 0.013). Conclusion: We demonstrate an association between PAD4, IL-6R, and troponin release in STEMI patients. Our findings indicate a PAD4-mediated connection between IL-6R and NET formation and highlight PAD4 as a potential treatment target for mitigating inflammation and myocardial injury in STEMI. Full article

Fetal growth restriction (FGR) is an obstetric condition most frequently caused by placental dysfunction. It is a major cause of perinatal morbidity with limited treatment options, so identifying the underpinning mechanisms is important. Peptidylarginine deiminases (PADs) are calcium-activated enzymes that mediate post-translational citrullination (deimination) of proteins, through conversion of arginine to citrulline. Protein citrullination leads to irreversible changes in protein structure and function and is implicated in many pathobiological processes. Whether placental protein citrullination occurs in FGR is poorly understood. We assessed protein citrullination and PAD isozyme abundance (PAD1, 2, 3, 4 and 6) in human placental samples from pregnancies complicated by early- and late-onset FGR, compared to appropriate-for-gestational-age (AGA) controls. Proteomic mass spectrometry demonstrated that the placental citrullinome profile changed in both early- and late-onset FGR, with 112 and 345 uniquely citrullinated proteins identified in early- and late-onset samples, respectively. Forty-four proteins were citrullinated only in control AGA placentas. The proteins that were uniquely citrullinated in FGR placentas were enriched for gene ontology (GO) terms related to neurological, developmental, immune and metabolic pathways. A greater number of GO and human phenotype pathways were functionally enriched for citrullinated proteins in late- compared with early-onset FGR. Correspondingly, late-onset but not early-onset FGR was associated with significantly increased placental abundance of PAD2 and citrullinated histone H3, determined by Western blotting. PAD3 was downregulated in early-onset FGR while abundance of PAD 1, 4 and 6 was less altered in FGR. Our findings show that placental protein citrullination is altered in FGR placentas, potentially contributing to the pathobiology of placental dysfunction. Full article

Periodontitis (PD), an oral inflammatory disease, is primarily caused by P. gingivalis. Peptidylarginine deiminase (PPAD) is considered an attractive virulence factor because, due to protein citrullination, it may have deleterious effects on host tissues. In this study, the ppad gene sequences from P. gingivalis were analyzed in the context of its impact on bacterial virulence and potential targets for PD therapy. Analyses of ppad sequences from 58 patients with various clinical stages of PD, 20 controls, and 60 sequences from public databases were conducted. Overall, 55 substitutions assigned as polymorphic variants (4), missense mutations (10), or synonymous variants (35) were identified in PD, and 22 synonymous variants were identified in controls. Among them, the G231N, E232T, N235D variant was found in ~25% of P. gingivalis strains from PD samples. It was located close to the catalytic triad and had two-fold higher activity in comparison with reference P. gingivalis, upregulated expression of key inflammatory mediators, and contributed to worsening periodontium conditions in advanced PD, suggesting their unambiguous impact on P. gingivalis virulence. Our results indicate the G231N, E232T, N235D variant of the ppad gene as a potential candidate, opening a path to searching for novel targets for supportive therapy of PD. Further validation of the identified mutations is needed in future studies. Full article

Background: This review synthesizes the role of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in modulating immune responses through citrullination and assesses its impact on periodontitis and systemic conditions. Methods: A systematic review was conducted on preclinical and clinical studies focusing on P. gingivalis- and A. actinomycetemcomitans-induced citrullination and its effects on immune responses, particularly inflammatory pathways, and systemic diseases. The search included PubMed, Scopus, Google Scholar, Web of Science, and gray literature. Quality and risk of bias were assessed using OHAT Rob Toll and QUIN-Tool. The review is registered in PROSPERO (ID: CRD42024579352). Results: 18 articles published up to August 2024 were included. Findings show that P. gingivalis and A. actinomycetemcomitans citrullination modulates immune responses, leading to neutrophil dysfunction and chronic inflammation. Key mechanisms include citrullination of antimicrobial peptides, CXCL10, histone H3, α-enolase, and C5a, impairing neutrophil activation and promoting NET formation. Conclusions: This review suggests that P. gingivalis and A. actinomycetemcomitans citrullination modulates immune responses and may influence periodontitis and systemic conditions like RA. Beyond ACPA production, these pathogens affect key proteins such as H3, C5a, and CXCL10, as well as antimicrobial peptides, NET formation, and phagocytosis. These interactions lead to neutrophil dysfunction and potentially affect other cells, subsequently disrupting local and systemic inflammatory responses. Full article

The detection of early molecular mechanisms and potential biomarkers in Parkinson’s disease (PD) remains a challenge. Recent research has pointed to novel roles for post-translational citrullination/deimination caused by peptidylarginine deiminases (PADs), a family of calcium-activated enzymes, in the early stages of the disease. The current study assessed brain-region-specific citrullinated protein targets and their associated protein–protein interaction networks alongside PAD isozymes in the 6-hydroxydopamine (6-OHDA) induced rat model of pre-motor PD. Six brain regions (cortex, hippocampus, striatum, midbrain, cerebellum and olfactory bulb) were compared between controls/shams and the pre-motor PD model. For all brain regions, there was a significant difference in citrullinated protein IDs between the PD model and the controls. Citrullinated protein hits were most abundant in cortex and hippocampus, followed by cerebellum, midbrain, olfactory bulb and striatum. Citrullinome-associated pathway enrichment analysis showed correspondingly considerable differences between the six brain regions; some were overlapping for controls and PD, some were identified for the PD model only, and some were identified in control brains only. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways identified in PD brains only were associated with neurological, metabolic, immune and hormonal functions and included the following: “Axon guidance”; “Spinocerebellar ataxia”; “Hippo signalling pathway”; “NOD-like receptor signalling pathway”; “Phosphatidylinositol signalling system”; “Rap1 signalling pathway”; “Platelet activation”; “Yersinia infection”; “Fc gamma R-mediated phagocytosis”; “Human cytomegalovirus infection”; “Inositol phosphate metabolism”; “Thyroid hormone signalling pathway”; “Progesterone-mediated oocyte maturation”; “Oocyte meiosis”; and “Choline metabolism in cancer”. Some brain-region-specific differences were furthermore observed for the five PAD isozymes (PADs 1, 2, 3, 4 and 6), with most changes in PAD 2, 3 and 4 when comparing control and PD brain regions. Our findings indicate that PAD-mediated protein citrullination plays roles in metabolic, immune, cell signalling and neurodegenerative disease-related pathways across brain regions in early pre-motor stages of PD, highlighting PADs as targets for future therapeutic avenues. Full article

Background: Immune cells from rheumatoid arthritis (RA) patients display a reduced in vitro response to Porphyromonas gingivalis (P. gingivalis), which may have functional immune consequences. The aim of this study was to characterize, by flow cytometry, the frequency/activity of monocytes and naturally occurring myeloid dendritic cells (mDCs) in peripheral blood samples from patients with periodontitis and patients with periodontitis and RA. Methods: The relative frequency of monocytes and mDCs in the whole blood, the frequency of these cells producing TNFα or IL-6 and the protein expression levels for each cytokine, before and after stimulation with lipopolysaccharide (LPS) from Escherichia coli plus interferon-γ (IFN-γ), were assessed by flow cytometry, in peripheral blood samples from 10 healthy individuals (HEALTHY), 10 patients with periodontitis (PERIO) and 17 patients with periodontitis and RA (PERIO+RA). Results: The frequency of monocytes and mDCs producing IL-6 or TNF-α and the expression of IL-6 and TNF-α in the PERIO group were generally higher. Within the PERIO+RA group, P. gingivalis and related antibodies were negatively correlated with the monocyte and mDC expression of IL-6. A subgroup of the PERIO+RA patients that displayed statistically significantly lower frequencies of monocytes producing IL-6 after activation presented statistically significantly higher peptidylarginine deiminase (PAD)2/4 activity, anti-arg-gingipain (RgpB) IgG levels, mean probing depth (PD), periodontal inflamed surface area (PISA) and bleeding on probing (BoP). Conclusions: In the patients with PERIO+RA, innate immune cells seemed to produce lower amounts of pro-inflammatory cytokines, which are correlated with worse periodontitis-related clinical and microbiological parameters. Full article

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. Full article

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. Full article