Evidence of membranolytic targeting and intracellular citrullination in neutrophils isolated from patients with rheumatoid arthritis.

Anti-citrullinated protein autoantibodies (ACPA) are diagnostic for rheumatoid arthritis (RA). The antigens recognized by these autoantibodies are produced by protein arginine deiminases (PADs), particularly PAD4. However, it remains unknown why and how PAD4 causes this aberrant citrullination in RA. Here, we report that poly-perforin pores are present on freshly isolated neutrophils from RA patients, but not on healthy donor neutrophils. Neutrophils with perforin pores also contained intracellular citrullinated proteins in the region adjacent to the pores. This response was replicated in vitro by treating neutrophils with purified perforin, which generated intense dots of anti-perforin immunofluorescence, calcium influx, and intracellular citrullination. Extensive neutrophil killing in Felty's syndrome, an aggressive form of RA, correlated with particularly high ACPA, and PAD4 autoantibodies. In contrast, other forms of death, including NETosis, apoptosis, and pyroptosis, produced minimal citrullination. We conclude that neutrophil targeting by perforin leading to intracellular citrullination takes place in patients with RA.

Levels of asymmetric dimethylarginine in plasma and aqueous humor: a key risk factor for the severity of fibrovascular proliferation in proliferative diabetic retinopathy.

Introduction: Proliferative diabetic retinopathy (PDR) is a common diabetes complication, significantly impacting vision and quality of life. Previous studies have suggested a potential link between arginine pathway metabolites and diabetic retinopathy (DR). Connective tissue growth factor (CTGF) plays a role in the occurrence and development of fibrovascular proliferation (FVP) in PDR patients. However, the relationship between arginine pathway metabolites and FVP in PDR remains undefined. This study aimed to explore the correlation between four arginine pathway metabolites (arginine, asymmetric dimethylarginine[ADMA], ornithine, and citrulline) and the severity of FVP in PDR patients. Methods: In this study, plasma and aqueous humor samples were respectively collected from 30 patients with age-related cataracts without diabetes mellitus (DM) and from 85 PDR patients. The PDR patients were categorized as mild-to-moderate or severe based on the severity of fundal FVP. The study used Kruskal-Wallis test to compare arginine, ADMA, ornithine, and citrulline levels across three groups. Binary logistic regression identified risk factors for severe PDR. Spearman correlation analysis assessed associations between plasma and aqueous humor metabolite levels, and between ADMA and CTGF levels in aqueous humor among PDR patients. Results: ADMA levels in the aqueous humor were significantly greater in patients with severe PDR than in those with mild-to-moderate PDR(P=0.0004). However, the plasma and aqueous humor levels of arginine, ornithine, and citrulline did not significantly differ between mild-to-moderate PDR patients and severe PDR patients (P>0.05). Binary logistic regression analysis indicated that the plasma (P=0.01) and aqueous humor (P=0.006) ADMA levels in PDR patients were risk factors for severe PDR. Furthermore, significant correlations were found between plasma and aqueous humor ADMA levels (r=0.263, P=0.015) and between aqueous humor ADMA and CTGF levels (r=0.837, P<0.001). Conclusion: Elevated ADMA levels in plasma and aqueous humor positively correlate with the severity of FVP in PDR, indicating ADMA as a risk factor for severe PDR.

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.

Mass Spectrometry Reveals Molecular Effects of Citrulline Supplementation during Bone Fracture Healing in a Rat Model.

Bone fracture healing is a complex process in which specific molecular knowledge is still lacking. The citrulline-arginine-nitric oxide metabolism is one of the involved pathways, and its enrichment via citrulline supplementation can enhance fracture healing. This study investigated the molecular effects of citrulline supplementation during the different fracture healing phases in a rat model. Microcomputed tomography (µCT) was applied for the analysis of the fracture callus formation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were used for lipid and protein analyses, respectively. µCT analysis showed no significant differences in the fracture callus volume and volume fraction between the citrulline supplementation and control group. The observed lipid profiles for the citrulline supplementation and control group were distinct for the different fracture healing stages. The main contributing lipid classes were phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs). The changing effect of citrulline supplementation throughout fracture healing was indicated by changes in the differentially expressed proteins between the groups. Pathway analysis showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group via improved angiogenesis and earlier formation of the soft and hard callus. This study showed the molecular effects on lipids, proteins, and pathways associated with citrulline supplementation during bone fracture healing, even though no effect was visible with µCT.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis.

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

The Role of Citrullination Modification in CD4+ T Cells in the Pathogenesis of Immune-Related Diseases.

The post-translational modifications (PTMs) of proteins play a crucial role in increasing the functional diversity of proteins and are associated with the pathogenesis of various diseases. This review focuses on a less explored PTM called citrullination, which involves the conversion of arginine to citrulline. This process is catalyzed by peptidyl arginine deiminases (PADs). Different members of the PAD family have distinct tissue distribution patterns and functions. Citrullination is a post-translational modification of native proteins that can alter their structure and convert them into autoantigens; thus, it mediates the occurrence of autoimmune diseases. CD4+ T cells, including Th1, Th2, and Th17 cells, are important immune cells involved in mediating autoimmune diseases, allergic reactions, and tumor immunity. PADs can induce citrullination in CD4+ T cells, suggesting a role for citrullination in CD4+ T cell subset differentiation and function. Understanding the role of citrullination in CD4+ T cells may provide insights into immune-related diseases and inflammatory processes.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

SCOPE: Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinal microbiota imbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. CONCLUSION: L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

Oral post-treatment supplementation with a combination of glutamine, citrulline, and antioxidant vitamins additively mitigates jejunal damage, oxidative stress, and inflammation in rats with intestinal ischemia and reperfusion.

INTRODUCTION: Intestinal ischemia and reperfusion (IIR) injury is closely associated with oxidative stress. Evidence shows that oral supplementation with glutamine and citrulline alleviates IIR-induced jejunal damage. We investigated the effects of a combination of glutamine, citrulline, and antioxidant vitamins on IIR-induced jejunal damage, oxidative stress, and inflammation. METHOD: Male Wistar rats that underwent 60 min of superior mesenteric artery occlusion were orally administered glutamine plus citrulline (GC), vitamin C plus E (CE), or a combination of GC and CE 15 min before and 3, 9, and 21 h after reperfusion. Healthy rats without IIR were used as controls. RESULTS: After reperfusion for 24 h, rats with IIR showed lower levels of red blood cells, hemoglobin, serum glucose, and jejunal DNA and increased white blood cell counts compared to controls (1-way ANOVA with the least significant difference, P < 0.05). The IIR-induced decrease in serum albumin and increase in plasma interleukin-6 and jejunal thiobarbituric acid-reactive substances (TBARS) were significantly reversed by GC and/or CE. The results of the 2-way ANOVA indicated that GC was the main factor that increased jejunal villus height and muscularis DNA, and CE was the main factor that increased jejunal muscularis protein and decreased jejunal proinflammatory cytokine levels and myeloperoxidase activity. In addition, GC and CE are the main factors that decrease plasma proinflammatory cytokine levels and the jejunal apoptotic index. CONCLUSION: Oral post-treatment supplementation with glutamine and citrulline, combined with vitamins C and E, may alleviate IIR-induced oxidative stress, inflammation, and jejunal damage.

A quantitative and site-specific atlas of the citrullinome reveals widespread existence of citrullination and insights into PADI4 substrates.

Despite the importance of citrullination in physiology and disease, global identification of citrullinated proteins, and the precise targeted sites, has remained challenging. Here we employed quantitative-mass-spectrometry-based proteomics to generate a comprehensive atlas of citrullination sites within the HL60 leukemia cell line following differentiation into neutrophil-like cells. We identified 14,056 citrullination sites within 4,008 proteins and quantified their regulation upon inhibition of the citrullinating enzyme PADI4. With this resource, we provide quantitative and site-specific information on thousands of PADI4 substrates, including signature histone marks and transcriptional regulators. Additionally, using peptide microarrays, we demonstrate the potential clinical relevance of certain identified sites, through distinct reactivities of antibodies contained in synovial fluid from anti-CCP-positive and anti-CCP-negative people with rheumatoid arthritis. Collectively, we describe the human citrullinome at a systems-wide level, provide a resource for understanding citrullination at the mechanistic level and link the identified targeted sites to rheumatoid arthritis.

Vitamin B12 inhibits peptidylarginine deiminases and ameliorates rheumatoid arthritis in CAIA mice.

Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.

Amino acid kinetics in the critically ill.

PURPOSE OF REVIEW: Stable isotope methods have been used for many years to assess whole body protein and amino acid kinetics in critically ill patients. In recent years, new isotope approaches and tracer insights have been developed. The tracer pulse approach has some advantages above the established primed-continuous tracer infusion approach because of the high amount of metabolic information obtained, easy applicability, and low tracer costs. Effects of disease severity and sex on amino acid kinetics in ICU patients will also be addressed. RECENT FINDINGS: Current knowledge was synthesized on specific perturbations in amino acid metabolism in critically ill patients, employing novel methodologies such as the pulse tracer approach and computational modeling. Variations were evaluated in amino acid production and linked to severity of critical illness, as measured by SOFA score, and sex. Production of the branched-chain amino acids (BCAAs), glutamine, tau-methylhistidine and hydroxyproline were elevated in critical illness, likely related to increased transamination of the individual BCAAs or increased breakdown of proteins. Citrulline production was reduced, indicative of impaired gut mucosa function. Sex and disease severity independently influenced amino acid kinetics in ICU patients. SUMMARY: Novel tracer and computational approaches have been developed to simultaneously measure postabsorptive kinetics of multiple amino acids that can be used in critical illness. The collective findings lay the groundwork for targeted individualized nutritional strategies in ICU settings aimed at enhancing patient outcomes taking into account disease severity and sex.

From Fruit Waste to Medical Insight: The Comprehensive Role of Watermelon Rind Extract on Renal Adenocarcinoma Cellular and Transcriptomic Dynamics.

Cancer researchers are fascinated by the chemistry of diverse natural products that show exciting potential as anticancer agents. In this study, we aimed to investigate the anticancer properties of watermelon rind extract (WRE) by examining its effects on cell proliferation, apoptosis, senescence, and global gene expression in human renal cell adenocarcinoma cells (HRAC-769-P) in vitro. Our metabolome data analysis of WRE exhibited untargeted phyto-constituents and targeted citrulline (22.29 µg/mg). HRAC-769-P cells were cultured in RPMI-1640 media and treated with 22.4, 44.8, 67.2, 88.6, 112, 134.4, and 156.8 mg·mL-1 for 24, 48, and 72 h. At 24 h after treatment, (88.6 mg·mL-1 of WRE) cell proliferation significantly reduced, more than 34% compared with the control. Cell viability decreased 48 and 72 h after treatment to 45% and 37%, respectively. We also examined poly caspase, SA-beta-galactosidase (SA-beta-gal), and wound healing activities using WRE. All treatments induced an early poly caspase response and a significant reduction in cell migration. Further, we analyzed the transcript profile of the cells grown at 44.8 mg·mL-1 of WRE after 6 h using RNA sequencing (RNAseq) analysis. We identified 186 differentially expressed genes (DEGs), including 149 upregulated genes and 37 downregulated genes, in cells treated with WRE compared with the control. The differentially expressed genes were associated with NF-Kappa B signaling and TNF pathways. Crucial apoptosis-related genes such as BMF, NPTX1, NFKBIA, NFKBIE, and NFKBID might induce intrinsic and extrinsic apoptosis. Another possible mechanism is a high quantity of citrulline may lead to induction of apoptosis by the production of increased nitric oxide. Hence, our study suggests the potential anticancer properties of WRE and provides insights into its effects on cellular processes and gene expression in HRAC-769-P cells.

Circulating arginine metabolites in Alzheimer's disease and vascular dementia: A systematic review and meta-analysis.

BACKGROUND: Alterations in nitric oxide (NO) synthesis have been reported in Alzheimer's disease and vascular dementia. However, as the measurement of NO in biological samples is analytically challenging, alternative, stable circulatory biomarkers of NO synthesis may be useful to unravel new pathophysiological mechanisms and treatment targets in dementia. METHODS: We conducted a systematic review and meta-analysis of the circulating concentrations of arginine metabolites linked to NO synthesis, arginine, citrulline, asymmetric (ADMA) and symmetric (SDMA) dimethylarginine, and ornithine, in Alzheimer's disease and vascular dementia. We searched for relevant studies in PubMed, Scopus, and Web of Science from inception to the 31st of May 2023. The JBI checklist and GRADE were used to assess the risk of bias and the certainty of evidence, respectively. RESULTS: In 14 selected studies, there were no significant between-group differences in arginine and ornithine concentrations. By contrast, compared to controls, patients with dementia had significantly higher ADMA (standard mean difference, SMD=0.62, 95% CI 0.06-1.19, p = 0.029), SDMA (SMD=0.70, 95% CI 0.34-1.35, p<0.001), and citrulline concentrations (SMD=0.50, 95% CI 0.08-0.91, p = 0.018). In subgroup analysis, the effect size was significantly associated with treatment with cholinesterase inhibitors and/or antipsychotics for ADMA, and underlying disorder (Alzheimer's disease), study continent, and analytical method for citrulline. CONCLUSION: Alterations in ADMA, SDMA, and citrulline, biomarkers of NO synthesis, may be useful to investigate the pathophysiology of different forms of dementia and identify novel therapeutic strategies. (PROSPERO registration number: CRD42023439528).

Protein citrullination: inhibition, identification and insertion.

Protein citrullination is a post-translational modification (PTM) that is catalysed by the protein arginine deiminase (PAD) family of enzymes. This PTM involves the transformation of an arginine residue into citrulline. Protein citrullination is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation and DNA damage-induced apoptosis. Aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis (RA), lupus, atherosclerosis and ulcerative colitis. In RA, anti-citrullinated protein antibodies can be detected prior to disease onset and are thus a valuable diagnostic tool for RA. Notably, citrullinated proteins may serve more generally as biomarkers of specific disease states; however, the identification of citrullinated protein residues remains challenging owing to the small 1 Da mass change that occurs upon citrullination. Herein, we highlight the progress made so far in the development of pan-PAD and isozyme selective inhibitors as well as the identification of citrullinated proteins and the site-specific incorporation of citrulline into proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

An unbiased proteomic analysis of PAD4 in human monocytes: novel substrates, binding partners and subcellular localizations.

Peptidylarginine deiminase IV (PAD4) post-translationally converts arginine residues in proteins to citrullines and is implicated in playing a central role in the pathogenesis of several diseases. Although PAD4 was historically thought to be a nuclear enzyme, recent evidence has revealed a more complex localization of PAD4 with evidence of additional cytosolic and cell surface localization and activity. However, the mechanisms by which PAD4, which lacks conventional secretory signal sequences, traffics to extranuclear localizations are unknown. In this study, we show that PAD4 was enriched in the organelle fraction of monocytes with evidence of citrullination of organelle proteins. We also demonstrated that PAD4 can bind to several cytosolic, nuclear and organelle proteins that may serve as binding partners for PAD4 to traffic intracellularly. Additionally, cell surface expression of PAD4 increased with monocyte differentiation into monocyte-derived dendritic cells and co-localized with several endocytic/autophagic and conventional secretory pathway markers, implicating the use of these pathways by PAD4 to traffic within the cell. Our results suggest that PAD4 is expressed in multiple subcellular localizations and may play previously unappreciated roles in physiological and pathological conditions. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Citrulline is not a major determinant of autoantibody reactivity to neutrophil extracellular traps.

One of the main strategies of neutrophils in responding to microbial infections is the formation of neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin associated with antimicrobial proteins. Citrullination plays an important role during NET formation and a substantial fraction of NET-associated proteins appeared to be citrullinated. The release of citrullinated intracellular proteins from netting neutrophils led to the hypothesis that the production of anti-citrullinated protein autoantibodies by autoimmune patients, in particular patients with rheumatoid arthritis, might be initiated when citrullinated NET components are not properly cleared and are exposed to the immune system. Here, we discuss the processes that lead to NET formation, including the role of peptidylarginine deiminase activation and our current knowledge on citrullinated NET-associated proteins. Citrulline-dependent epitopes do not appear to play a major role in the recognition of NETs by autoantibodies from rheumatoid arthritis and systemic lupus erythematosus patients, even though anti-NET autoantibodies are frequently observed in sera from these patients. The neutrophil proteases associated with NETs have a major impact on the integrity of NET-associated proteins when NET formation is induced by activating isolated human neutrophils. Cleavage/degradation of these proteins also resulted in a strong reduction of the reactivity with autoantibodies. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Pentachlorophenol exposure induced neurotoxicity by disrupting citrulline metabolism in larvae and adult zebrafish.

Pentachlorophenol (PCP) is a ubiquitous environmental toxicant with various adverse effects. Although its neurotoxicity has been reported, the underlying mechanism and subsequent detoxification remain unclear. In this study, embryos and adult zebrafish were exposed to PCP to determine its potential neurotoxic mechanism and protective indicators. The survival rate, heart rate, mobility time, active status and moving distance were significantly decreased in larvae after 30 µg/L PCP exposure. Likewise, the mobile time, latency to the first movement, velocity and moving distance of adult zebrafish were significantly reduced by PCP exposure. Untargeted metabolomics analysis of larvae revealed that arginine and proline metabolism was the primary pathway affected by PCP exposure, reflected by increased proline and decreased citrulline (CIT) contents, which were confirmed by quantitative data. PCP exposure suppressed the conversion from arginine to CIT in larvae by downregulating the expression of nos1 and nos2a. Ornithine content was increased in the brains and intestines of adult zebrafish after PCP exposure, which inhibited ornithine catabolism to CIT by downregulating otc, resulting in reduced CIT. Intriguingly, CIT supplementation significantly restored the neurobehavioral defects induced by PCP in larvae and adult zebrafish. CIT supplementation upregulated the expression of ef1α and tuba1 in larvae and inhibited the downregulation of ef1α in the brains of adult zebrafish. Taken together, these results indicated that CIT supplementation could protect against PCP-induced neurotoxicity by upregulating the expression of genes involved in neuronal development and function.

The interaction of ammonia and manganese in abnormal metabolism of minimal hepatic encephalopathy: A comparison metabolomics study.

This study was to investigate the effects of ammonia and manganese in the metabolism of minimal hepatic encephalopathy (MHE). A total of 32 Sprague-Dawley rats were divided into four subgroups: chronic hyperammonemia (CHA), chronic hypermanganese (CHM), MHE and control group (CON). 1H-NMR-based metabolomics was used to detect the metabolic changes. Sparse projection to latent structures discriminant analysis was used for identifying and comparing the key metabolites. Significant elevated blood ammonia were shown in the CHA, CHM, and MHE rats. Significant elevated brain manganese (Mn) were shown in the CHM, and MHE rats, but not in the CHA rats. The concentrations of γ-amino butyric acid (GABA), lactate, alanine, glutamate, glutamine, threonine, and phosphocholine were significantly increased, and that of myo-inositol, taurine, leucine, isoleucine, arginine, and citrulline were significantly decreased in the MHE rats. Of all these 13 key metabolites, 10 of them were affected by ammonia (including lactate, alanine, glutamate, glutamine, myo-inositol, taurine, leucine, isoleucine, arginine, and citrulline) and 5 of them were affected by manganese (including GABA, lactate, myo-inositol, taurine, and leucine). Enrichment analysis indicated that abnormal metabolism of glutamine and TCA circle in MHE might be affected by the ammonia, and abnormal metabolism of GABA might be affected by the Mn, and abnormal metabolism of glycolysis and branched chain amino acids metabolism might be affected by both ammonia and Mn. Both ammonia and Mn play roles in the abnormal metabolism of MHE. Chronic hypermanganese could lead to elevated blood ammonia. However, chronic hyperammonemia could not lead to brain Mn deposition.