OBJECTIVE: Posttranslational modifications (PTMs) of proteins are crucial for regulating various biological processes. However, protein alteration via PTMs, and consequently, the creation of new epitopes, can induce abnormal autoimmune responses in predisposed individuals. Immunopathogenesis of several rheumatic diseases, including the most common childhood form, juvenile idiopathic arthritis (JIA), is associated with the generation of autoantibodies against such modified proteins. Dysregulated generation of neutrophil extracellular traps (NETs) can be a source of post-translationally altered proteins. Thus, we investigated the role of PTMs and the presence of NET-associated markers in JIA patients. METHODS: We recruited 30 pediatric patients with JIA (20 with active disease and 10 in remission) and 30 healthy donors. The serum concentrations of citrullinated histone H3 (citH3), peptidyl arginine deiminases (PADs), and NET-related products were detected using ELISA, and the number of citH3+ neutrophils was assessed using flow cytometry. RESULTS: The serum levels of citH3 and PADs were higher in active as well as in remission JIA patients than in healthy donors. Similarly, the number of citH3+ neutrophils was higher in the peripheral blood of patients with JIA, implying an enhanced process of NETosis. This was effectively reflected by elevated serum levels of NET-associated products, such as neutrophil elastase, LL37, and cell-free DNA-histone complexes. Additionally, 16.7% of active JIA patients were seropositive for carbamylated autoantibodies, the levels of which declined sharply after initiation of anti-TNFα therapy. CONCLUSION: Collectively, our data suggest that the accelerated process of NETosis and PTMs in JIA may result in the generation of anti-citrullinated/carbamylated autoantibodies against various epitopes later in life, which could be prevented by effectively regulating inflammation using immune therapy.

• Klíčová slova
• NETosis, carbamylation, citrullination, histone, juvenile idiopathic arthritis, neutrophil, peptidyl arginine deiminases (PAD),
• Publikační typ
• časopisecké články MeSH

LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide's ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.

• MeSH
• autoimunita imunologie   MeSH
• biologický transport MeSH
• buněčné linie MeSH
• citrulin metabolismus   MeSH
• citrulinace fyziologie   MeSH
• dendritické buňky imunologie   MeSH
• DNA imunologie   metabolismus   MeSH
• imunologická tolerance imunologie   MeSH
• kathelicidiny MeSH
• kationické antimikrobiální peptidy metabolismus   MeSH
• lidé MeSH
• myši MeSH
• peptidylarginindeiminasy metabolismus   MeSH
• RAW 264.7 buňky MeSH
• volné cirkulující nukleové kyseliny imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• citrulin MeSH
• DNA MeSH
• kathelicidiny MeSH
• kationické antimikrobiální peptidy MeSH
• peptidylarginindeiminasy MeSH
• volné cirkulující nukleové kyseliny MeSH

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

• MeSH
• autoantigeny krev   imunologie   MeSH
• biologické markery krev   MeSH
• čipová analýza proteinů MeSH
• citrulin metabolismus   MeSH
• citrulinace MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• peptidylarginindeiminasa typu 2 metabolismus   MeSH
• peptidylarginindeiminasa typu 4 metabolismus   MeSH
• protilátky proti citrulinovaným peptidům imunologie   MeSH
• revmatoidní artritida imunologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tandemová hmotnostní spektrometrie MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoantigeny MeSH
• biologické markery MeSH
• citrulin MeSH
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• protilátky proti citrulinovaným peptidům MeSH

OBJECTIVE: Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. METHODS: Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. RESULTS: ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. CONCLUSION: In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

• MeSH
• citrulinace MeSH
• fibrinogen imunologie   MeSH
• fosfopyruváthydratasa imunologie   MeSH
• histony imunologie   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy metabolismus   MeSH
• protilátky proti citrulinovaným peptidům metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• revmatoidní artritida enzymologie   imunologie   MeSH
• sérový albumin imunologie   MeSH
• synoviální tekutina MeSH
• techniky in vitro MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fibrinogen MeSH
• fosfopyruváthydratasa MeSH
• histony MeSH
• PADI2 protein, human MeSH Prohlížeč
• PADI4 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 2 MeSH
• peptidylarginindeiminasa typu 4 MeSH
• peptidylarginindeiminasy MeSH
• protilátky proti citrulinovaným peptidům MeSH
• rekombinantní proteiny MeSH
• sérový albumin MeSH
• vápník MeSH

Rheumatoid factor (RF) is used in human and veterinary medicine in the form of IgM RF traditionally to support the diagnosis of rheumatoid arthritis (RA). In the latest diagnostic criteria, presence of anti - citrullinated protein antibodies (ACPA) was added to the grading system for the diagnosis of RA in humans. A change which is not integrated or routinely used in veterinary medicine. The criteria changed partly because of RF's diagnostic shortcomings, which include its increased titer detection in humans with non-rheumatoid diseases, inability to predict the disease and increased titers over the limit in the older population. Clinical signs similar to human RA were reported in horses in a condition known as idiopathic polysynovitis. Similarities in the clinical presentation to RA raised a question to the usability of RF and ACPA in horses. In our study, sixty clinically healthy horses, ranging from 3 days to 30 years of age, were evaluated for their serum levels of IgM RF. 55 of these horses were tested for ACPA, using methods of ELISA measuring Anti - CFG (Anti citrullinated fibrinogen antibody). The results of the study demonstrated the existence of an age-dependent increase in the level of IgM RF up to the age of about 9 years and ACPA's independence of the horse's age as well as both markers independence of the horse sex.

• Klíčová slova
• ACPA, Age-dependency, ELISA, Horse, IgM RF, Polysynovitis,
• MeSH
• autoprotilátky krev   MeSH
• biologické markery krev   MeSH
• ELISA MeSH
• imunoglobulin G krev   MeSH
• imunoglobulin M krev   MeSH
• koně imunologie   MeSH
• protilátky proti citrulinovaným peptidům krev   MeSH
• revmatoidní artritida MeSH
• revmatoidní faktor krev   MeSH
• synovitida diagnóza   imunologie   veterinární   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• autoprotilátky MeSH
• biologické markery MeSH
• imunoglobulin G MeSH
• imunoglobulin M MeSH
• protilátky proti citrulinovaným peptidům MeSH
• revmatoidní faktor MeSH

Citrullination is a post-translational protein modification, which is associated with inflammation in general and is thought to play an important pathogenic role in rheumatoid arthritis (RA). Here a mass spectrometry-based proteomics approach was applied to identify citrullination sites in synovial fluid fibrinogen from four RA patients. In general, high disease activity correlated with increased number of identified citrullination sites and higher relative citrulline occupancy. Altogether, 23 sites were identified, of which 9 have not been previously reported to be citrullinated in vivo. Citrullination at site α84, α123, α129, α547, α573, α591, β334 and γ134 was identified in more than one patient, and these positions were therefore regarded as hotspots. Following citrullination of fibrinogen in vitro using human recombinant peptidylarginine deiminase 2 (PAD2), a total of 46 citrullination sites were identified, including 6 hitherto unreported in vitro citrullination sites. Twenty-two out of the 23 citrullination sites identified in vivo were also detected in vitro, supporting the validity of the identifications. SIGNIFICANCE: This work provides information about previously uncharacterized citrullination sites in synovial fluid fibrinogen from rheumatoid arthritis patients. Detection of these novel citrullination sites may prove to have diagnostic or prognostic value in RA and enhance our understanding of the immune pathogenesis.

• Klíčová slova
• Citrullination, Mass spectrometry, Peptidylarginine deiminase, Post-translational modifications, Rheumatoid arthritis, Synovial fluid,
• MeSH
• citrulin metabolismus   MeSH
• fibrinogen metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• peptidylarginindeiminasa typu 2 MeSH
• posttranslační úpravy proteinů * MeSH
• proteomika MeSH
• revmatoidní artritida metabolismus   patologie   MeSH
• senioři MeSH
• synoviální membrána metabolismus   patologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• citrulin MeSH
• fibrinogen MeSH
• PADI2 protein, human MeSH Prohlížeč
• peptidylarginindeiminasa typu 2 MeSH

Fibrinogen is an abundant blood plasma protein that, inter alia, participates in blood coagulation. It polymerizes to form a fibrin clot that is among the major components of the thrombus. Fibrinogen reactions with various reactive metabolites may induce post-translational modifications (PTMs) into the protein structure that affect the architecture and properties of fibrin clots. We reviewed the previous literature to find the positions of PTMs of fibrinogen. For 7 out of 307 reported PTMs, we used molecular dynamics simulations to characterize their effect on the behavior of the fibrinogen coiled-coil domain. Interactions of the γ-coil with adjacent chains give rise to π-helices in Aα and Bβ chains of even unmodified fibrinogen. The examined PTMs suppress fluctuations of the γ-coil, which may affect the fibrinolysis and stiffness of the fibrin fibers. Citrullination of AαR104 and oxidations of γP70 and γP76 to glutamic semialdehyde unfold the α-helical structure of Aα and Bβ chains. Oxidation of γM78 to methionine sulfoxide induces the formation of an α-helix in the γ-coil region. Our findings suggest that certain PTMs alter the protein secondary structure. Thus, the altered protein structure may indicate the presence of PTMs in the molecule and consequently of certain metabolites within the system.

• Klíčová slova
• citrullination, coiled-coil, fibrinogen, molecular dynamic simulation, oxidation, post-translational modifications,
• Publikační typ
• časopisecké články MeSH

INTRODUCTION: To date, there is not generally accepted and universal indicator of activity, and functional integrity of the small intestine in patients with coeliac disease. The aim of our study was to investigate whether serum concentrations of the non-essential amino acids citrulline and ornithine might have this function. METHODS: We examined serum citrulline and ornithine concentrations in a subgroup of patients with proven coeliac disease and healthy controls (blood donors). RESULTS: A total of 94 patients with coeliac disease (29 men, mean age 53 ± 18 years; 65 women, mean age 44 ± 14 years) and 35 healthy controls (blood donors) in whom coeliac disease was serologically excluded (10 men, mean age 51 ± 14 years; 25 women, mean age 46 ± 12 years) were included in the study. Significantly lower concentrations of serum ornithine were found in patients with coeliac disease (mean 65 ± 3 μmol/L; median 63 μmol/L, IQR 34 μmol/L, p < 0.001). No statistically nor clinically significant differences were found in the citrulline concentrations between the study and control group. CONCLUSIONS: Serum ornithine (but not citrulline) may be useful for assessing the functional status of the small intestine in uncomplicated coeliac disease. Further studies involving more detailed analysis of dietary and metabolic changes in patients will be needed to reach definitive conclusions.

• Klíčová slova
• citrulline, coeliac disease, ornithine,
• MeSH
• celiakie * MeSH
• citrulin * metabolismus   MeSH
• dieta MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ornithin metabolismus   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• citrulin * MeSH
• ornithin MeSH

To observe the protective effects of L-citrulline on the renal I/R injury and elucidate the mechanisms involved, 48 rats were randomized into eight groups: Group 1: sham operated; Group 2: I/R (45 min renal ischaemia and 24 h reperfusion); Group 3: I/R + L-citrulline (300 mg/kg, i.g.); Group 4: I/R + L-citrulline (600 mg/kg, i.g.); Group 5: I/R + L-citrulline (900 mg/kg, i.g.); Group 6: I/R + normal saline (NS, i.g.); Group 7: I/R + N sup ω nitro-L-arginine ester (L-NAME, 20 mg/kg, i.p.); Group 8: I/R + L-citrulline (900 mg/kg, i.g.) + L-NAME (20 mg/ kg, i.p.). At the end of the reperfusion period, serum was collected and the kidneys underwent histological and biochemical examinations. Our results showed that pre-treatment with L-citrulline (300, 600, and 900 mg/kg) significantly ameliorated the renal injury caused by I/R. Moreover, L-citrulline prevented induction of lipid peroxidation and increased the activity of superoxide dismutase and the levels of glutathione and nitric oxide. The I/R-induced decreases in total nitric oxide synthase activity, inducible nitric oxide activity, constitutive nitric oxide activity and endothelial nitric oxide protein expression in the renal cortex were significantly prevented. However, the L-citrulline-mediated protection was significantly antagonized by co-administration of L-NAME. These results suggested that L-citrulline administration exhibited significant protection against renal I/R injury. This protective effect, at least in part, via up-regulation of the endothelial nitric oxide protein expression and constitutive nitric oxide synthase activity, maintained production of nitric oxide at the basal level.

• MeSH
• citrulin farmakologie   terapeutické užití   MeSH
• krysa rodu Rattus MeSH
• oxid dusnatý metabolismus   MeSH
• reperfuzní poškození farmakoterapie   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• citrulin MeSH
• oxid dusnatý MeSH

Citrulline is a non-proteinogenic amino acid that forms as by-product in nitric oxide (NO) synthesis from arginine and may act in concert with NO as an independent signaling molecule that involves in the mechanism of vascular smooth muscle vasodilation. In this study we examined the effects of citrulline on pulmonary artery smooth muscles. Experimental design comprised outward potassium currents measurements in enzymatically isolated rat pulmonary artery smooth muscle (PASMc) cells using whole-cell patch clamp technique, isometric contractile force recordings on rat pulmonary artery rings and method of molecular docking simulation. Citrulline in a concentration 10-9-10-5 M relaxed phenylephrine (PHE)-preactivated SM of rat pulmonary artery in a dose-dependent manner (EC50 0,67 μM). This citrulline-induced relaxation was dependent on an intact endothelium. Bath application of citrulline (10-8-10-5 M) on isolated PASMc induced a significant increase in the amplitude of outward potassium current (Ik). The adenosine antagonist caffeine (10-6 M) effectively blocked both the citrulline-induced relaxation response and Ik increment. Molecular docking modeling suggests that caffeine blocking the potent activity of citrulline results from competitive interactions at the A2 adenosine receptor binding site. In summary, our data suggest that citrulline, released with NO at low concentrations, can effectively interact with adenosine receptors in smooth muscle cells, causing their relaxation, indicating surprising interaction between NO and adenosine pathways.

• Klíčová slova
• Adenosine receptors, Caffeine, Citrulline, Endothelium, Nitric oxide, Vascular smooth muscle,
• MeSH
• arteria pulmonalis účinky léků   metabolismus   MeSH
• citrulin * farmakologie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• potkani Wistar MeSH
• purinergní receptory P1 metabolismus   MeSH
• simulace molekulového dockingu * MeSH
• svaly hladké cévní metabolismus   účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• citrulin * MeSH
• purinergní receptory P1 MeSH