Protein Citrullination Research Articles

Impact of Protein Citrullination by Periodontal Pathobionts on Oral and Systemic Health: A Systematic Review of Preclinical and Clinical Studies

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.

Dynamics of the gut microbiome in individuals at risk of rheumatoid arthritis: a cross-sectional and longitudinal observational study

ObjectivesThis work aimed to resolve the conflicting reports on Prevotellaceae abundance in the development of rheumatoid arthritis (RA) and to observe structural, functional and temporal changes in the gut microbiome...

Extracellular vesicles and citrullination signatures are novel biomarkers in sturgeon (Acipenser gueldenstaedtii) during chronic stress due to seasonal temperature challenge

Acipenser gueldenstaedtii is one of the most cultured sturgeon species worldwide and of considerable economic value for caviar production. There are though considerable challenges around chronic stress responses due to increased summer temperatures, impacting sturgeons’ immune responses and their susceptibility to opportunistic infections. The identification of molecular and cellular pathways involved in stress responses may contribute to identifying novel biomarkers reflective of fish health status, crucial for successful sturgeon aquaculture. Protein citrullination is a calcium-catalysed post-translational modification caused by peptidylarginine deiminases (PADs), altering target protein function and affecting protein interactions in physiological and pathobiological processes. PADs can also modulate extracellular vesicle (EVs) profiles, which play critical roles in cellular communication, via transport of their cargoes (proteins, including post-translationally modified proteins, genetic material and micro-RNAs).This study identified differences in EV signatures, and citrullinated proteins in sera from winter and summer farmed sturegeons. EVs were significantly elevated in sera of the summer chronically stressed group. The citrullinated proteins and associated gene ontology (GO) pathways in sera and serum-EVs of chronically heat stressed A. gueldenstaedtii, showed some changes, with specific citrullinated serum protein targets including alpa-2-macroglobulin, alpha globin, calcium-dependent secretion activator, ceruloplasmin, chemokine XC receptor, complement C3 isoforms, complement C9, plectin, selenoprotein and vitellogenin. In serum-EVs, citrullinated protein cargoes identified only in the chronically stressed summer group included alpha-1-antiproteinase, apolipoprotein B-100, microtubule actin crosslinking factor and histone H3. Biological gene ontology (GO) pathways related to citrullinated serum proteins in the chronically stressed group were associated with innate and adaptive immune responses, stress responses and metabolic processes. In serum-EVs of the heat-stressed group the citrullinome associated with various metabolic GO pathways.In addition to modified citrullinated protein content, Serum-EVs from the stressed summer group showed significantly increased levels of the inflammatory associated miR-155 and the hypoxia-associated miR-210, but significantly reduced levels of the growth-associated miR-206.Our findings highlight roles for protein citrullination and EV signatures in response to chronic heat stress in A. gueldenstaedtii, indicating a trade-off in immunity versus growth and may be of value for sturgeon aquaculture.

Brain-Region-Specific Differences in Protein Citrullination/Deimination in a Pre-Motor Parkinson's Disease Rat Model.

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.

Revamped role for approved drug: integrative computational and biophysical analysis of saquinavir's peptidyl arginine deiminase 4 inhibition for rheumatoid arthritis.

The pursuit of novel therapeutics is a complex and resource-intensive endeavor marked by significant challenges, including high costs and low success rates. In response, drug repositioning strategies leverage existing FDA-approved compounds to predict their efficacy across diverse diseases. Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in protein citrullination, a process implicated in the autoimmune pathogenesis of rheumatoid arthritis (RA). Targeting PAD4 has thus emerged as a promising therapeutic approach. This study employs computational and enzyme inhibition strategies to identify potential PAD4-targeting compounds from a library of FDA-approved drugs. In silico docking analyses validated the binding interactions and orientations of screened compounds within PAD4's active site, with key residues such as ASP350, HIS471, ASP473, and CYS645 participating in crucial hydrogen bonding and van der Waals interactions. Molecular dynamics simulations further assessed the stability of top compounds exhibiting high binding affinities. Among these compounds, Saquinavir (SQV) emerged as a potent PAD4 inhibitor, demonstrating competitive inhibition with a low IC50 value of 1.21 ± 0.04 µM. In vitro assays, including enzyme kinetics and biophysical analyses, highlighted significant changes in PAD4 conformation upon SQV binding, as confirmed by circular dichroism spectroscopy. SQV induced localized alterations in PAD4 structure, effectively occupying the catalytic pocket and inhibiting enzymatic activity. These findings underscore SQV's potential as a therapeutic candidate for RA through PAD4 inhibition. Further validation through in vitro and in vivo studies is essential to confirm SQV's therapeutic benefits in autoimmune diseases associated with dysregulated citrullination.

Redundant role of PAD2 and PAD4 in the development of cardiovascular lesions in a mouse model of Kawasaki disease vasculitis.

Kawasaki disease (KD) is the leading cause of acquired heart disease in children. While circulating neutrophils are increased and activated during acute KD, it is unclear whether neutrophils and neutrophil extracellular traps (NETs) contribute to the pathogenesis of KD. Peptidylarginine deiminase 4 (PAD4), an enzyme involved in protein citrullination and essential for NETs formation, is implicated in the pathogenesis of various diseases. Here, we used the Lactobacillus casei cell wall extract (LCWE)-induced mouse model of KD vasculitis to determine the contribution of PAD4 in KD vasculitis. We found that the pan-PADs inhibitor, Cl-amidine, significantly reduced LCWE-induced cardiovascular lesions, but neutrophil-specific Padi4 KO mice did not impact the development of KD vasculitis. While in vitro treatment of macrophages, which highly express Padi4, with Cl-amidine inhibited IL-1β secretion, macrophage-specific Padi4 KO mice did not reduce the lesions. Padi4-/- mice also developed KD vasculitis, AFM30a, a PAD2 inhibitor, significantly reduced KD vasculitis in Padi4-/- mice, indicating a compensatory role of PAD2 in PAD4 deficiency. We also identified several citrullinated proteins in macrophages with constitutively active NLRP3 inflammasome that were inhibited by Cl-amidine treatment, suggesting that protein citrullination participates in NLRP3 inflammasome activation. These data indicate a dispensable role for PAD4-dependent NETs formation, and a redundant role of PAD2 and PAD4 in this murine KD vasculitis. The cardioprotective effects of Cl-amidine to reduce the severity of murine KD vasculitis are not limited to PAD4 inhibition and may include decreased citrullination in the inflammasome pathway.

Neutrophil extracellular traps and citrullinated fibrinogen contribute to injury in a porcine model of limb ischemia and reperfusion.

Ischemia/reperfusion injury (IRI) is a complex pathological process, triggered by the restoration of blood flow following an interrupted blood supply. While restoring the blood flow is the only option to salvage the ischemic tissue, reperfusion after a prolonged period of ischemia initiates IRI, triggering a cascade of inflammatory responses ultimately leading to neutrophil recruitment to the inflamed tissue, where they release neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin and neutrophilic proteins, including peptidyl-arginine deiminase 2 and 4 (PAD2, PAD4), that, once outside, can citrullinate plasma proteins, irreversibly changing their conformation and potentially their function. While the involvement of NETs in IRI is known mainly from rodent models, we aimed to determine the effect of NET formation and especially PADs-mediated extracellular protein citrullination in a porcine model of limb IRI. We conducted our study on amputated pig forelimbs exposed to 1h or 9h of ischemia and then reperfused in vivo for 12h. Limb weight, edema formation, compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Fibrin tissue deposition, complement deposition and NETs were investigated by immunofluorescence. Citrullinated plasma proteins were immunoprecipitated and citrullinated fibrinogen was identified in the plasma by Western blot and in the tissue by immunofluorescence and Western blot. Our data consolidate the involvement of NETs in a porcine model of limb IRI, correlating their contribution to damage extension with the duration of the ischemic time. We found a massive infiltration of NETs in the group subjected to 9h ischemia compared to the 1h and citrullinated fibrinogen levels, in plasma and tissue, were higher in 9h ischemia group. We propose fibrinogen citrullination as one of the mechanisms contributing to the worsening of IRI. NETs and protein citrullination represent a potential therapeutic target, but approaches are still a matter of debate. Here we introduce the idea of therapeutic approaches against citrullination to specifically inhibit PADs extracellularly, avoiding the downstream effects of hypercitrullination and keeping PADs' and NETs' intracellular regulatory functions.

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.

Helicobacter pylori upregulates PAD4 expression via stabilising HIF-1α to exacerbate rheumatoid arthritis

ObjectiveHelicobacter pylori infection has been reported to aggravate rheumatoid arthritis (RA), but the relevant mechanism remains unclear. This study aimed to investigate the underlying pathogenic mechanism of H. pylori infection...

Anti-carbamylated Protein Antibodies Positivity in Rheumatoid Arthritis and Its Association With Rheumatoid Factor and Anti-cyclic Citrullinated Protein Antibodies.

Background Rheumatoid arthritis (RA) is a widespread autoimmune disease affecting millions of people worldwide. The current markers include anti-cyclic citrullinated peptide (anti-CCP) antibodiesand rheumatoid factor (RF), which are nonspecific and elevated in variousconditions and do not have a prognostic value. They are also elevated in the later stages of the disease. Anti-carbamylated protein (anti-CarP) antibodies have been reported to be associated with joint damage in RA. Therefore, this study aimed to evaluate the sensitivity and specificity of anti-CarP antibodies in individuals with RA and their relationship with inflammatory markers such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Methods This was a cross-sectional case-control study conducted from April 2020 to March 2021 at the Saveetha Medical College, Chennai, India. The age makeup of the three groups was evaluated: Group 1 comprised anti-CCP and RF-positive patients; Group 2 comprised anti-CCP and RF-negative patients; and Group 3 was the control group, which comprised healthy volunteers. Patient samples, including blood and serum, have been utilized to conduct various assessments aimed at evaluating biomarkers such as CRP, ESR, RF, and autoantibodies like anti-CCP and anti-CarP. Results This study examined the role of various autoantibodies and biomarkers in RA across three distinct groups. Group 1 predominantly consisted of middle-aged individuals, and women constituted the majority in both Group 1 and Group 2, consistent with higher RA prevalence among females. In Group 1, 65.7% of patients tested positive for anti-CarP, while in Group 2, 48.6% tested positiveeven when RF and anti-CCP antibodies were absent. This suggests a potential diagnostic role for anti-CarP antibodies in identifying RA patients early. CRP and ESR levels were significantly elevated in RA patients (Groups 1 and 2) compared to healthy controls (Group 3), indicating higher inflammatory activity in affected individuals. We also observed that anti-CarPantibodies had a specificity of 69.1% and a sensitivity of 78.2%. Positive correlations between the diagnosis of RA and anti-CarP antibody positivity were observed across the groups and correlated well with the inflammatory markers and signs such as joint damage. The data were found to be statistically significant. Conclusions Our study showed a significant correlation between joint damage and CRP levels and a positive correlation between anti-CarP antibodies and ESR and CRP values. These findings suggest that anti-CarP antibodies can offer certain advantages over RF and anti-CCP antibodies in RA diagnosis due to their early detection potential, higher specificity, complementary diagnostic role, and predictive value for disease severity.

Determination of the derivatization reactivity between α/β-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two β-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the β-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the β-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

DIPG-16. THE BIG AND SMALL OF PROTEIN POST-TRANSLATIONAL MODIFICATIONS: TARGETING SUMOYLATION AND CITRULLINATION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Protein post-translational modifications (PTMs) alter the properties of protein through proteolytic cleavage, or the addition of modifying groups to one or more amino acids. Histones are major targets of PTMs, including through SUMOylation of lysine residues and citrullination of arginine residues, with both processes able to regulate gene transcription. We aimed to delineate the potential role of SUMOylation and citrullination in the epigenetic dysregulation that drives Diffuse Midline Glioma (DMG) tumorigenicity. METHODS AND RESULTS DMG tumours demonstrated an upregulation of SUMOylation machinery pathway components and increased expression of peptidylarginine deiminases (PADs), the enzymes responsible for citrullination. Nanomolar doses of the SUMOylation inhibitor TAK-981 blocks DMG cell cycle G2/M transition and results in potent decreases in cell viability and clonogenic potential, triggering ‘viral mimicry’ in DMG cells, resulting in increased expression of interferon stimulated genes and inflammatory mediators including IL-1β, IL-6 and TNFα, whilst also causing a pro-inflammatory M1 macrophage response. These data suggest that SUMOylation inhibition may lead to a double-punch effect against DMG by inhibiting tumorgenicity and stimulating an immune response. In further studies, we have shown that pharmacological inhibition of PAD activity in DMG decreases histone citrullination and restores histone methylation – considered the Achilles heel of DMG. This was accompanied by induction of reactive oxygen species and an unfolded protein response, resulting in DMG cell death in a dose dependent manner. In addition to fully characterising the epigenetic and global changes within DMG initiated through SUMOylation or PAD inhibition, we are currently performing preliminary drug efficacy studies in orthotopic DMG models, providing the necessary pre-clinical research pipeline towards an effective treatment for DMG. CONCLUSIONS Protein SUMOylation and citrullination are previously unrecognised therapeutic targets in DMG and potentially play key roles in tumour initiation and pathogenesis. Our data suggests a novel therapeutic approach for children with DMG.

Early predictive factors in routine clinical practice for rituximab therapy response in patients with rheumatoid arthritis.

Identifying early predictive factors of how rheumatoid arthritis (RA) patients respond to rituximab (RTX) treatment is crucial for both individual treatment outcome and the improvement of clinical practice overall. This study aimed to identify early predictive factors available in standard clinical practice for predicting RTX treatment outcomes in RA patients. Data on seventy patients diagnosed with RA treated with RTX (two 1,000 mg doses 2 weeks apart or two 500 mg doses 2 weeks apart) were retrospectively collected. Baseline information collected at the initiation of RTX treatment included patient characteristics such as age, sex, disease duration, disease activity, Health Assessment Questionnaire score, erythrocyte sedimentation rate, C-reactive protein, and serological status regarding rheumatoid factor (RF) and anti-cyclic citrullinated protein antibodies (ACPA). Clinical responses were analyzed 6 months after RTX initiation using the European Alliance of Associations for Rheumatology criteria. Potential predictors associated with positive RTX response at 6 months were identified using a multivariate ordinal logistic regression model. The analysis showed that persistently active RA disease, Disease Activity Score with 28-joint count (DAS28) values at the treatment onset and after 3 months, along with erythrocyte sedimentation rate at treatment initiation, were negatively correlated with the response to RTX therapy (p < 0.05). All these correlations were statistically significant at the 99% confidence interval. The correlation and logistic regression analyses indicate that there are no significant association between RF and ACPA concerning therapy response, despite a higher number of RTX responders in the seropositive groups. Additionally, the study emphasizes the prognostic significance of the DAS28 value at treatment initiation in predicting therapy response at 6 months. The optimal model for predicting RTX response at 6 months involves the interaction of all clinical factors examined in this study, as revealed by the analysis of multiple variables.

Investigating the Precise Identification of Citrullination Sites with High- Performance Score Metrics Using a Powerful Computation Predicting Tool.

To elucidate the detailed mechanisms of citrullination at the molecular level and design drugs applicable to major human diseases, predicting protein citrullination sites (PCSs) is essential. Using experimental approaches to predict PCSs is time-consuming and costly. However, there is a limited scope of the current PCS predictors. In particular, most predictors are commonly used for PCS prediction and have limited performance scores. This work aims to provide an improved sophisticated predictor of citrullination sites using a benchmark dataset in a machine learning platform. This study presents a reliable citrullination site predictor based on a benchmark dataset containing a 1:1 ratio of positive and negative samples. We classified citrullination sites using the Composition of the K-Spaced Amino Acid Pairs (CKSAAP) and Support Vector Machine (SVM). We developed PCS predictors using integrated machine-learning methods that produced the highest average scores. Using 10-fold cross-validation on test datasets, the True Positive Rate (TPR) was 98.34%, the True Negative Rate (TNR) was 99.44%, the accuracy was 98.89%, the Mathew Correlation Coefficient (MCC) was 98.21%, the Area Under the ROC Curve (AUC) was 0.999, and the partial Area Under the ROC Curve (pAUC) was 0.1968. According to overall performance, our developed predictor has a significantly higher implementation in comparison with the current tools on the same benchmark dataset. Moreover, it showed better performance metrics on both test and training datasets. Our developed predictor is promising and can be implemented as a complementary technique for identifying fast and precise citrullination sites.

DCM-Spheroid Morphs Express PADs and Citrullinated Cytoskeletal Proteins.

During investigating the role of peptidylarginine deiminase (PAD) enzymes in dilated cardiomyopathy (DCM), we observed unique spheroid formation in DCM-myofibroblasts that distinguished them from normal cardiac myofibroblasts. The present study aimed to assess the presence of PADs, the extracellular matrix (ECM), and citrullination in DCM spheroids using immunofluorescence staining and imaging techniques. The results revealed that spheroids derived from DCM-myofibroblasts displayed a more distinctive, tightly packed structure compared with those derived from human cardiac fibroblasts. DCM spheroids showed abundant protein expression of the PAD 2, 3, and 4 enzymes. Notably, increased Ki67 protein expression was associated with increased proliferation in DCM spheroids. Cytoskeletal proteins such as Col-1A, vimentin, α-SMA, and F-actin were highly abundant in DCM spheroids. Furthermore, DCM spheroids contained citrullinated cytoskeletal proteins, mainly citrullinated vimentin and citrullinated fibronectin. These observations supported the occurrence of PAD-mediated citrullination of ECM proteins in DCM spheroids. Collectively, these findings describe the distinctive features of DCM spheroids, representing the cellular characteristics of DCM myofibroblasts. Therefore, DCM spheroids can serve as an in vitro model for further investigations of disease morphology and therapeutic efficacy.

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.

Abstract PS07-06: A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention

Abstract Purpose: To evaluate the merits of a novel citrullinated-enolase 1 (ENO1) peptide-based vaccine for inducing an effective response against triple negative breast cancer (TNBC). Background: Targets for cancer vaccines have included mutated proteins and peptides, as well as proteins that are overexpressed in cancer but otherwise ubiquitous in their structure. However, protein modifications other than mutation may also induce an immune response. Several lines of evidence have demonstrated a role for the posttranslational modification (PTM) citrullination as inducing immunogenicity. Dysregulated protein citrullination by peptidyl arginine deiminases (PADI) has been associated with autoimmune diseases and is currently being explored for its relevance to cancer. Previously, our group established PADI2, which catalyzes the PTM from arginine to citrulline, to be highly expressed in TNBC compared to normal mammary and other tissues. We observed that PADI2-associated protein citrullination promotes antigenicity. Importantly, we demonstrated that protein citrullination, non-occurring in DNA and RNA-based vaccines, is highly cancer specific. On the basis of these findings, we developed a novel vaccine based on cancer-selective citrullinated-enolase 1 peptides (cit-ENO1) that were identified from mass spectrometry surfaceome and immunopeptidome data from breast cancer cells. We assessed the extent to which a citENO1 peptide based vaccine elicits a potent host response and attenuates tumor development in a syngeneic mouse model of TNBC. Design: To confirm that the cit-ENO1 peptide vaccine elicited an immune response, we immunized female B6129SF1/J mice with 10nmol cit-ENO1 peptides or 10nmol of corresponding unmodified peptides as the antigen, plus the TLR3 agonist poly I:C as an adjuvant (10mg), subcutaneously once a week, for three weeks. Saline or the adjuvant alone were used as controls. One week post the last round of immunization, draining lymph nodes (LNs) and immunized skin were harvested, and T cell phenotypes evaluated using flow-cytometry. To assess anti-cancer efficacy, female B6129SF1/J mice were immunized with cit-ENO1 peptides or unmodified peptides with poly I:C as an adjuvant followed by orthotopic implantation with murine BRCA1co/co; MMTV-Cre; p53+/- TNBC cells (1.5 x 106) and tumor growth as well as overall survival were assessed. Results: We observed that the cit-ENO1 peptide vaccination induced a statistically significantly greater percentage of activated CD8+ PD-1+ effector T cells as well as CD8+ CD44+ CD62L- effector memory T cells in the LNs compared with either the control groups or the unmodified ENO1 peptide. CD8+ PD-1+ T-cells were also found to be significantly elevated in immunized skin of cit-ENO1 vaccinated mice. Both cit-ENO1 and unmodified ENO1 vaccine increased CD4+ PD-1+ T cells from the LNs and immunized skin. Remarkably, the cit-ENO1 vaccine but not the unmodified-ENO1 vaccine significantly delayed tumor growth and markedly improved overall survival compared to respective controls (Logrank test for trend 2-sided p&amp;lt; 0.05). Conclusion: We have developed a novel vaccine based on cancer-selective cit-ENO1 peptides that stimulates a potent anti-cancer immune response and significantly delays tumor initiation and progression in a preclinical, immune competent mouse model of TNBC. Importantly, the anti-cancer effects were not observed with corresponding unmodified ENO1 peptides. Citation Format: Ricardo Leon Letelier, Hiroyuki Katayama, Yihui Chen, Yinning Cai, Fuchung Hsiao, Banu Arun, Samir Hanash. A novel citrullinated-ENO1 peptide-based vaccine for triple-negative breast cancer prevention [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-06.

Estrogen Regulation of PAD2 and Citrullination in the Aging Female Heart

Advanced age is the primary risk factor for heart failure, an enormous public health burden. With aging, men tend to develop systolic dysfunction whereas women are predisposed to develop diastolic dysfunction. Despite these known phenotype differences, underlying molecular mechanisms of sex differences in cardiac aging are unknown. Citrullination, a post-translational modification catalyzed by the peptidylarginine deiminases (PAD) enzyme family, is positively regulated by the sex hormone estrogen (E2) suggesting a potential novel mechanism of age-related cardiac functional changes in women. We hypothesized that PAD expression and citrullination are sexually dimorphic with aging such that as E2 levels decline, so too does citrullination, thus contributing to diastolic dysfunction. We quantified expression of PAD2, the primary cardiac isoform, in female young reproductively competent adult (3 months) and aged (21 months) C57Bl6 mice. Consistent with clinical reports, in our model of aging, female mice developed diastolic dysfunction, with a prolonged linear phase relaxation (54.0 ± 1.7 msec, adult and 66.0 ± 3.3 msec aged, p&lt;0.05). PAD2 expression decreased with age in the female heart, concomitant with decreases in myocardial protein citrullination. To identify potential mechanisms of differential citrullination by age, we performed cit-mass spectrometry. Adult female mice had higher overall citrullination patterns than aged females with enrichment of citrullinated proteins in metabolic, mitochondrial, and sarcomeric pathways. In reproductively competent adult females, cardiac PAD2 expression was higher during estrus compared to diestrus. To confirm direct regulation of PAD2 by E2, a cohort of adult mice underwent ovariectomy (OVX) with or without E2 replacement, provided orally in the water (0.5μM E2) for three weeks. Exogenous E2 supplementation post-OVX increased uterine weight and PAD2 expression, further confirming positive regulation by E2. Together, we suggest that hormonally regulated changes in PAD2 and citrullination with age contribute to sex-specific cardiac aging. Ongoing experiments will directly test the role of PAD2 in the heart by generation of a novel myocyte specific PAD2 deleted mouse across the female lifespan. This abstract was made possible by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under 2P20GM103432. This work was supported by the National Institutes of Health [K01 AG058810 DRB, [K01 AG066845 KCW]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures

BackgroundEnvironmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear.MethodsCCR2 RFP+ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 μg), or gram-positive peptidoglycan (PGN, 100 μg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey’smultiple comparison) and nonparametric statistical (Kruskal–Wallis, Dunn’s multiple comparison) tests were used following Shapiro–Wilk testing for normality.ResultsIntratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2+ CD11cintCD11bhi monocytes/macrophages and both CCR2+ and CCR2− CD11c−CD11bhi monocytes at 48 h. There were also increases in CCR2+ CD4+ and CD8+ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11cintCD11bhi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11cintCD11bhi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome.ConclusionHighly concentrated environmental/occupational exposures induced the recruitment of CCR2+ and CCR2− transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.