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1.
Nat Commun ; 11(1): 3241, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591537

RESUMO

Protein arginine deiminase 4 (PAD4) facilitates the post-translational citrullination of the core histones H3 and H4. While the precise epigenetic function of this modification has not been resolved, it has been shown to associate with general chromatin decompaction and compete with arginine methylation. Recently, we found that histones are subjected to methylglyoxal (MGO)-induced glycation on nucleophilic side chains, particularly arginines, under metabolic stress conditions. These non-enzymatic adducts change chromatin architecture and the epigenetic landscape by competing with enzymatic modifications, as well as changing the overall biophysical properties of the fiber. Here, we report that PAD4 antagonizes histone MGO-glycation by protecting the reactive arginine sites, as well as by converting already-glycated arginine residues into citrulline. Moreover, we show that similar to the deglycase DJ-1, PAD4 is overexpressed and histone citrullination is upregulated in breast cancer tumors, suggesting an additional mechanistic link to PAD4's oncogenic properties.


Assuntos
Histonas/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Aldeído Pirúvico/farmacologia , Animais , Arginina/metabolismo , Neoplasias da Mama/enzimologia , Citrulinação , Citrulina/metabolismo , Feminino , Glicosilação , Humanos , Células MCF-7 , Metilação , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Nucleossomos/metabolismo
2.
Ann Rheum Dis ; 79(9): 1194-1202, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32532752

RESUMO

OBJECTIVES: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. METHODS: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. RESULTS: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). CONCLUSIONS: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Infecções por Bacteroidaceae/imunologia , Epitopos/imunologia , Porphyromonas gingivalis/imunologia , Artrite Reumatoide/microbiologia , Infecções por Bacteroidaceae/microbiologia , Citrulinação/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Peptídeos/imunologia , Desiminases de Arginina em Proteínas/imunologia
3.
Sci Rep ; 10(1): 7596, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371922

RESUMO

Enzymatic and non-enzymatic posttranslational protein modifications by oxidation, glycation and acylation are key regulatory mechanisms in hallmarks of aging like inflammation, altered epigenetics and decline in proteostasis. In this study a mouse cohort was used to monitor changes of posttranslational modifications in the aging process. A protocol for the extraction of histones, cytosolic and mitochondrial proteins from mouse liver was developed and validated. In total, 6 lysine acylation structures, 7 advanced glycation endproducts, 6 oxidative stress markers, and citrullination were quantitated in proteins of subcellular compartments using HPLC-MS/MS. Methionine sulfoxide, acetylation, formylation, and citrullination were the most abundant modifications. Histone proteins were extraordinary high modified and non-enzymatic modifications accumulated in all subcellular compartments during the aging process. Compared to acetylation of histone proteins which gave between 350 and 305 µmol/mol leucine equivalents in young and old animals, modifications like acylation, glycation, and citrullination raised to 43%, 20%, and 18% of acetylation, respectively. On the other hand there was an age related increase of selected oxidative stress markers by up to 150%. The data and patterns measured in this study are mandatory for further studies and will strongly facilitate understanding of the molecular mechanisms in aging.


Assuntos
Senescência Celular , Processamento de Proteína Pós-Traducional , Acetilação , Cromatografia Líquida , Citrulinação , Glicosilação , Espaço Intracelular , Fígado/metabolismo , Organelas/genética , Organelas/metabolismo , Estresse Oxidativo , Frações Subcelulares
4.
Med Sci (Paris) ; 36(5): 465-471, 2020 May.
Artigo em Francês | MEDLINE | ID: mdl-32452368

RESUMO

In the last decade, the association between the periodontitis and rheumatoid arthritis (RA) has been established, suggesting that oral microbiome plays a causal role by initiating this chronic autoimmune inflammatory disease of articulation. Both pathogenesis are similar in term of chronic inflammation, tissue breakdown and bone resorption. Molecular aspects have also revealed that citrullination, a post-translational modification catalyzed by peptidyl-arginine deiminases (PADs), is involved in both diseases. For RA, citrullinated proteins production leads to the synthesis the of anti-citrullinated protein antibodies triggering the loss of immune tolerance. In humans, five PADs have been identified. Recently, studies have found that only Porphyromonas species possess PAD. Thus, a major periodontal pathogen, Porphyromonas gingivalis, is able to generate citrullinated epitopes, and could consequently induce anti-citrullinated protein antibodies. In this review, citrullination process, periodontitis and RA are described to put them in relation with molecular, clinical and epidemiological studies establishing the association between periodontitis and RA.


Assuntos
Artrite Reumatoide/etiologia , Bactérias/enzimologia , Citrulinação/fisiologia , Microbiota/fisiologia , Boca/microbiologia , Desiminases de Arginina em Proteínas/fisiologia , Artrite Reumatoide/enzimologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/microbiologia , Bactérias/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Periodontite/complicações , Periodontite/enzimologia , Periodontite/microbiologia , Processamento de Proteína Pós-Traducional
5.
Ann Biol Clin (Paris) ; 78(2): 201-205, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32319950

RESUMO

Anti-citrullinated cyclic peptide antibodies (ACPA) were initially considered very specific for the diagnosis of rheumatoid arthritis (RA), and can predict the prognosis of the disease. However, these antibodies can be detected in other autoimmune diseases, including systemic lupus erythematosus (SLE), the most common manifestation of which is inflammatory arthritis, which is often found in early-stage rheumatoid arthritis. The aim of our study is to evaluate the prevalence of ACPA antibodies and to analyze the profiles of their associations with autoantibodies specific to lupus, in order to look for a possible rhupus overlap syndrome in our patients. This is a retrospective study, carried out at the immunology unit, at Blida University Hospital, Algeria, involving 96 lupus patients, diagnosed according to the criteria of the American college of rheumatology (ACR). ACPA have been identified by the ELISA technique. ACPA was positive in 14,56% of our patients, whereas anti-DNA, anti-Sm and rheumatoid factor (RF) autoantibodies were positive, respectively in 47.09%, 35.41%, and in 26.04% of our patients. In addition, the presence of ACPA with anti DNA was found in 12.5% of patients. Of the 14 with ACPA+, 57.14% had arthritis. Our results confirm that ACPA auto-antibodies do not represent a pathognomonic criterion of RA. This sometimes makes the differential diagnosis with lupus difficult especially at the beginning of the disease.


Assuntos
Artrite Reumatoide/sangue , Biomarcadores/sangue , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/epidemiologia , Fator Reumatoide/sangue , Adolescente , Adulto , Idoso , Argélia/epidemiologia , Anticorpos Antinucleares/análise , Anticorpos Antinucleares/sangue , Artrite Reumatoide/epidemiologia , Autoanticorpos/análise , Autoanticorpos/sangue , Biomarcadores/análise , Criança , Citrulinação , Comorbidade , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/metabolismo , Estudos Retrospectivos , Fator Reumatoide/análise , Adulto Jovem
6.
Nat Rev Rheumatol ; 16(6): 301-315, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32341463

RESUMO

Peptidylarginine deiminases (PADs) have an important role in the pathogenesis of rheumatoid arthritis (RA) owing to their ability to generate citrullinated proteins - the hallmark autoantigens of RA. Of the five PAD enzyme isoforms, PAD2 and PAD4 are the most strongly implicated in RA at both genetic and cellular levels, and PAD inhibitors have shown therapeutic efficacy in mouse models of inflammatory arthritis. PAD2 and PAD4 are additionally targeted by autoantibodies in distinct clinical subsets of patients with RA, suggesting anti-PAD antibodies as possible biomarkers for RA diagnosis and prognosis. This Review weighs the evidence that supports a pathogenic role for PAD enzymes in RA as both promoters and targets of the autoimmune response, as well as discussing the mechanistic and therapeutic implications of these findings in the wider context of RA pathogenesis. Understanding the origin and consequences of dysregulated PAD enzyme activity and immune responses against PAD enzymes will be important to fully comprehend the pathogenic mechanisms involved in this disease and for the development of novel strategies to treat and prevent RA.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Citrulinação , Reações Cruzadas , Predisposição Genética para Doença , Humanos , Doenças Pulmonares Intersticiais/imunologia , Proteína-Arginina Desiminase do Tipo 2/genética , Proteína-Arginina Desiminase do Tipo 2/imunologia , Proteína-Arginina Desiminase do Tipo 3/imunologia , Proteína-Arginina Desiminase do Tipo 4/genética , Proteína-Arginina Desiminase do Tipo 4/imunologia , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Índice de Gravidade de Doença
7.
JCI Insight ; 5(11)2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329756

RESUMO

In severe cases of coronavirus disease 2019 (COVID-19), viral pneumonia progresses to respiratory failure. Neutrophil extracellular traps (NETs) are extracellular webs of chromatin, microbicidal proteins, and oxidant enzymes that are released by neutrophils to contain infections. However, when not properly regulated, NETs have the potential to propagate inflammation and microvascular thrombosis - including in the lungs of patients with acute respiratory distress syndrome. We now report that sera from patients with COVID-19 have elevated levels of cell-free DNA, myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (Cit-H3); the latter 2 are specific markers of NETs. Highlighting the potential clinical relevance of these findings, cell-free DNA strongly correlated with acute-phase reactants, including C-reactive protein, D-dimer, and lactate dehydrogenase, as well as absolute neutrophil count. MPO-DNA associated with both cell-free DNA and absolute neutrophil count, while Cit-H3 correlated with platelet levels. Importantly, both cell-free DNA and MPO-DNA were higher in hospitalized patients receiving mechanical ventilation as compared with hospitalized patients breathing room air. Finally, sera from individuals with COVID-19 triggered NET release from control neutrophils in vitro. Future studies should investigate the predictive power of circulating NETs in longitudinal cohorts and determine the extent to which NETs may be novel therapeutic targets in severe COVID-19.


Assuntos
Ácidos Nucleicos Livres/metabolismo , Infecções por Coronavirus/metabolismo , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Neutrófilos/metabolismo , Peroxidase/metabolismo , Pneumonia Viral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Citrulinação , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Pandemias , Contagem de Plaquetas , Pneumonia Viral/sangue , Pneumonia Viral/terapia , Respiração Artificial , Índice de Gravidade de Doença
8.
Arch Oral Biol ; 114: 104695, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32315811

RESUMO

OBJECTIVE: To analyse the citrulline level in the periodontium in association with the presence of or antibody levels against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. DESIGN: Gingival crevicular fluid (GCF), subgingival biofilm and blood serum were sampled from 98 subjects (26 with RA, 72 without RA (NoRA)). GCF was analyzed for the level of citrulline, for interleukin (IL)-1ß, IL-17, IL-10 and monocyte-chemoattractant protein (MCP)-1. Microorganisms were identified in subgingival biofilms. Antibodies againstP. gingivalis, and Aggregatibacter actinomycetemcomitans were quantified in serum. RESULTS: GCF citrulline level was the lowest (by trend) in NoRA group without periodontitis. In NoRA, but not in RA an association between GCF citrulline level and P. gingivalis antibody levels was found and the GCF citrulline levels were higher in P. gingivalis positive samples. Any association of A. actinomycetemcomitans with GCF citrulline level did not exist. A model of univariate variance analysis (p = 0.001) showed a dependence of GCF citrulline level from the number of sites with PD (probing depth) ≥5 mm (p = 0.003) and the GCF MCP-1/CCL2 level (p = 0.019). Compared with NoRA in RA the number of teeth was lower, the number of sites with PD ≥ 5 mm was less, GCF levels of interleukin-17 and MCP-1/CCL2 were higher and those of IL-10 lower. Yeasts were only cultured in 15 RA patients (p < 0.001). CONCLUSION: Citrullination in periodontium might be associated with P. gingivalis supporting the potential role as a trigger in the development of RA. Pathogenesis of periodontal disease in RA patients seems to differ from that in NoRA and should be investigated further.


Assuntos
Artrite Reumatoide/complicações , Citrulinação , Citrulina/análise , Periodontite/microbiologia , Periodonto/química , Aggregatibacter actinomycetemcomitans , Infecções por Bacteroidaceae/patologia , Líquido do Sulco Gengival , Humanos , Periodonto/microbiologia , Porphyromonas gingivalis/patogenicidade
9.
PLoS One ; 15(3): e0230719, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218599

RESUMO

INTRODUCTION: Epidemiologically, cigarette smoking is a well-known risk factor for the pathogenesis of rheumatoid arthritis (RA). However, there has been few plausible explanations why cigarette smoking aggravated RA. We investigated the causal effect of smoking in experimental model of arthritis development. METHODS: During induction of experimental arthritis with collagen challenge, mice were exposed to a smoking environment with 3R4F cigarettes. Generated smoke was delivered to mice through a nose-only exposure chamber (ISO standard 3308). Human cartilage pellet was challenged by cigarette smoke extract to identify citrullinating potential in vitro. RESULTS: Cigarette smoke exacerbated arthritis in a collagen-induced arthritis (CIA) model. Exposure to smoke accelerated the onset of arthritis by 2 weeks compared to the conventional model without smoke. Citrullination of lung tissue as well as tarsal joints were revealed in smoke-aggravated CIA mice. Interestingly, tracheal cartilage was a core organ regarding intensity and area size of citrullination. The trachea might be an interesting organ in viewpoint of sharing cartilage with joint and direct smoke exposure. Anti-CCP antibodies were barely detected in the serum of CIA mice, they were significantly elevated in cigarette smoke group. Citrullinated antigens were increased in the serum of smoke-exposed mice. Lastly, a cigarette smoke extract enhanced human cartilage citrullination in vitro. CONCLUSIONS: Missing link of arthritic mechanism between smoke and RA could be partially explained by tracheal citrullination. To control tracheal cartilage citrullination may be beneficial for preventing arthritis development or aggravation if cigarette smoke is becoming a risk factor to pre-arthritic individual.


Assuntos
Artrite Experimental/induzido quimicamente , Fumar Cigarros/efeitos adversos , Animais , Artrite Experimental/patologia , Citrulinação/efeitos dos fármacos , Feminino , Camundongos , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/patologia
10.
Ann Rheum Dis ; 79(4): 472-480, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32041746

RESUMO

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Processamento de Proteína Pós-Traducional/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Acetilação , Idoso , Autoanticorpos/imunologia , Citrulinação/imunologia , Citrulina/análogos & derivados , Citrulina/imunologia , Reações Cruzadas/imunologia , Feminino , Humanos , Imunoglobulina G , Masculino , Pessoa de Meia-Idade , Carbamilação de Proteínas/imunologia
11.
Semin Immunol ; 47: 101393, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932199

RESUMO

Under conditions of cellular stress, proteins can be post-translationally modified causing them to be recognized by the immune system. One such stress-induced post-translational modification (siPTM) is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. PAD enzymes are activated by millimolar concentrations of calcium which can occur during apoptosis, leading to precipitation of proteins, their subsequent uptake by B cells and stimulation of antibody responses. Detection of anti-citrullinated protein antibodies (ACPAs) is a diagnostic of rheumatoid arthritis (RA), where immune complexes stimulate inflammation around the joints. More recently, autophagy has been shown to play a role in the presentation of citrullinated peptides on MHC class II molecules to CD4+ helper T cells, suggesting that citrullination may be a way of alerting immune cells to cellular stress. Additionally, inflammation-induced IFNγ and concomitant MHC class II expression on target cells contributes to immune activation. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a pro-survival mechanism. Cancer cells also over express PAD enzymes and in light of this the hypothesis that citrullinated peptides stimulate CD4+ T cell responses that would recognize these siPTM's produced during autophagy has been investigated. The induction of potent citrullinated peptide-specific CD4 responses has been shown in both humans and HLA transgenic mouse models. Responses in mouse models resulted in potent anti-tumour responses against tumours expressing either constitutive or IFNγ-inducible MHC class II. The anti-tumour effect relied upon direct recognition of tumours by specific CD4 T cells suggesting that citrullinated peptides are attractive targets for cancer vaccines.


Assuntos
Biomarcadores Tumorais , Citrulinação , Neoplasias/etiologia , Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunoterapia , Terapia de Alvo Molecular , Neoplasias/terapia , Peptídeos/imunologia , Peptídeos/metabolismo , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo
12.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952341

RESUMO

Deimination, also known as citrullination, corresponds to the conversion of the amino acid arginine, within a peptide sequence, into the non-standard amino acid citrulline. This post-translational modification is catalyzed by a family of calcium-dependent enzymes called peptidylarginine deiminases (PADs). Deimination is implicated in a growing number of physiological processes (innate and adaptive immunity, gene regulation, embryonic development, etc.) and concerns several human diseases (rheumatoid arthritis, neurodegenerative diseases, female infertility, cancer, etc.). Here, we update the involvement of PADs in both the homeostasis of skin and skin diseases. We particularly focus on keratinocyte differentiation and the epidermal barrier function, and on hair follicles. Indeed, alteration of PAD activity in the hair shaft is responsible for two hair disorders, the uncombable hair syndrome and a particular form of inflammatory scarring alopecia, mainly affecting women of African ancestry.


Assuntos
Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas/genética , Dermatopatias/genética , Fenômenos Fisiológicos da Pele/genética , Citrulinação , Homeostase/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Queratinócitos/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Pele/metabolismo , Pele/patologia , Dermatopatias/metabolismo
13.
J Leukoc Biol ; 107(1): 69-83, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478251

RESUMO

Aspergillus fumigatus (A. fumigatus) is an environmental fungus and a human pathogen. Neutrophils are critical effector cells during the fungal infections, and neutropenia is a risk factor for the development of pulmonary aspergillosis. Neutrophil extracellular traps (NETs) are released by neutrophils in response to A. fumigatus and inhibit the conidial germination. In this work, we observed that the receptors TLR2, TLR4, and Dectin-1 were dispensable for the A. fumigatus induced NET release. In contrast CD11b/CD18 was critical for the NET release in response to A. fumigatus conidia, and this required the CD11b I-domain-mediated recognition, whereas the blockade of the CD11b lectin domain did not affect the A. fumigatus induced NET release. A. fumigatus induced NET release relied on the activity of spleen tyrosine kinase (Syk), Src family kinase(s), and class IA PI3 kinase δ. Although A. fumigatus promoted histone citrullination, this process was dispensable for the NET release in response to A. fumigatus conidia. The A. fumigatus induced NET release required the reactive oxygen species generation by the NOX2 complex, in a downstream pathway requiring CD11b/CD18, Src kinase family activity, Syk and PI3K class IA δ. Our findings thus reveal the signaling pathways involved in the formation of NETs in response to A. fumigatus.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , DNA/imunologia , Armadilhas Extracelulares/imunologia , Histonas/química , Antígeno de Macrófago 1/metabolismo , Neutrófilos/imunologia , Proteína-Arginina Desiminase do Tipo 4/química , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/metabolismo , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Citrulinação , DNA/metabolismo , Armadilhas Extracelulares/microbiologia , Humanos , Antígeno de Macrófago 1/genética , Neutrófilos/microbiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Quinase Syk/metabolismo , Quinases da Família src/metabolismo
14.
Mol Cell Proteomics ; 19(1): 167-180, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31754044

RESUMO

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.


Assuntos
Membrana Externa Bacteriana/metabolismo , Citrulinação , Vesículas Extracelulares/metabolismo , Peptídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Alanina/metabolismo , Artrite Reumatoide/microbiologia , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cromatografia Líquida , Técnicas de Inativação de Genes , Humanos , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Proteômica/métodos
15.
Clin Chim Acta ; 501: 6-11, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31730822

RESUMO

BACKGROUND: Citrullination is the post-translational conversion of arginine into citrulline in proteins. The reaction is catalyzed by peptidylarginine deiminase (PAD), of which five isoforms exist. Fibrinogen is a substrate for PAD2 and PAD4, and citrullinated fibrinogen (cFBG) has been detected in patients with inflammatory diseases. In purified systems, cFBG is known to inhibit the release of fibrinopeptide A (FPA) and B (FPB) and impairs fibrin polymerization. However, the effect of cFBG on fibrin structure and fibrinolysis in a plasma environment remains unclear. We hypothesized that citrullination of fibrinogen impairs fibrin properties. METHODS: Fibrinogen was citrullinated by recombinant PAD2 and PAD4. The impact of cFBG on fibrin structure was investigated by turbidity measurements in fibrinogen-deficient plasma spiked with cFBG or native fibrinogen. RESULTS: Citrullination of fibrinogen by PAD2 dose-dependently reduced the rate of fibrin polymerization, as well as the overall hemostasis potential of fibrin, the maximum velocity of fibrin formation, the fibrin mass/length ratio, and the lysis of fibrin clots. CONCLUSION: Citrullination of fibrinogen by PAD2 affects not only fibrin polymerization but also fibrin fiber properties, indicating that the fibrin network formed in the presence of cFBG may influence hemostasis. Our results suggest that citrullination of fibrinogen alters the composition of fibrin fibers which may lead to a looser fibrin network that is more susceptible to fibrinolysis and thereby affecting the hemostatic balance.


Assuntos
Citrulinação , Fibrina/metabolismo , Fibrinogênio/metabolismo , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Fibrina/química , Fibrinogênio/química , Humanos , Conformação Proteica , Proteína-Arginina Desiminase do Tipo 2/química
16.
Amino Acids ; 52(1): 103-110, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31832896

RESUMO

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.


Assuntos
Anticorpos/farmacologia , Arginina/genética , Diabetes Mellitus Tipo 1/tratamento farmacológico , Fator de Necrose Tumoral alfa/genética , Animais , Anticorpos/imunologia , Glicemia/genética , Citrulinação/efeitos dos fármacos , Citrulinação/genética , Metilação de DNA/genética , Metilação de DNA/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Humanos , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T alfa-beta/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Baço/efeitos dos fármacos , Baço/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores
17.
Am J Respir Cell Mol Biol ; 62(3): 364-372, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31647878

RESUMO

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.


Assuntos
Isquemia Fria/efeitos adversos , DNA Mitocondrial/farmacologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/efeitos dos fármacos , Disfunção Primária do Enxerto/imunologia , Receptor Toll-Like 9/fisiologia , Lesão Pulmonar Aguda/etiologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Citrulinação , DNA Mitocondrial/administração & dosagem , Desoxirribonuclease I/metabolismo , Humanos , Transplante de Pulmão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Disfunção Primária do Enxerto/metabolismo , Proteína-Arginina Desiminase do Tipo 4/deficiência , Proteína-Arginina Desiminase do Tipo 4/fisiologia , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Organismos Livres de Patógenos Específicos , Receptor Toll-Like 9/deficiência , Isquemia Quente/efeitos adversos
18.
Mol Immunol ; 117: 37-53, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733447

RESUMO

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, causing functional and structural changes in target proteins. Protein deimination causes generation of neo-epitopes, affects gene regulation and also allows for protein moonlighting. Furthermore, PADs have been found to be a phylogenetically conserved regulator for extracellular vesicle (EVs) release. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins in serum and serum-EVs are described for the first time in camelids, using the llama (Lama glama L. 1758) as a model animal. We report a poly-dispersed population of llama serum EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In serum, 103 deiminated proteins were overall identified, including key immune and metabolic mediators including complement components, immunoglobulin-based nanobodies, adiponectin and heat shock proteins. In serum, 60 deiminated proteins were identified that were not in EVs, and 25 deiminated proteins were found to be unique to EVs, with 43 shared deiminated protein hits between both serum and EVs. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in llama serum. PAD homologues were identified in llama serum by Western blotting, via cross reaction with human PAD antibodies, and detected at an expected 70 kDa size. This is the first report of deiminated proteins in serum and EVs of a camelid species, highlighting a hitherto unrecognized post-translational modification in key immune and metabolic proteins in camelids, which may be translatable to and inform a range of human metabolic and inflammatory pathologies.


Assuntos
Camelídeos Americanos/imunologia , Citrulinação/imunologia , Vesículas Extracelulares/imunologia , Animais , Camelídeos Americanos/sangue , Camelídeos Americanos/metabolismo , Vesículas Extracelulares/metabolismo , Masculino
19.
Arthritis Rheumatol ; 72(6): 912-918, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31876120

RESUMO

OBJECTIVE: To address the independent roles of peptidylarginine deiminase type 2 (PAD2) and PAD4 in generating rheumatoid arthritis (RA) autoantigens by using a system that mimics intracellular citrullination in the RA joint. METHODS: PAD2- or PAD4-expressing 293T cells and mock-transfected cells were used as targets in cytotoxic assays using lymphokine-activated killer cells, cytotoxic YT cell granule contents, or purified human perforin. Protein citrullination and autoantigen production were determined by immunoblotting using the anti-modified citrulline-Senshu method and RA sera (n = 30), respectively. RESULTS: RA sera recognized at least 3 categories of autoantigens in PAD-expressing target cells killed by the cytotoxic lymphocyte granule-induced death pathway. These included: 1) autoantigens targeted in their native form, 2) citrullinated antigens, and 3) antigens cleaved by cytotoxic proteases (e.g., granzymes). Interestingly, although target cells expressing PAD2 or PAD4 showed prominent hypercitrullination of a broad range of proteins during cytotoxic granule-induced cell damage, autoantibodies in RA sera targeted only a very limited number of antigens in hypercitrullinated cells. Furthermore, RA sera showed distinct reactivities to autoantigens generated by PAD2 or PAD4. CONCLUSION: The cytotoxic granule-induced death pathway has the capacity to modify antigens by inducing hypercitrullination and antigen cleavage in target cells. Interestingly, among a large number of citrullinated proteins generated by PAD2 and PAD4 in cells, only a few are likely involved in the production of autoantibodies in RA.


Assuntos
Artrite Reumatoide/imunologia , Autoantígenos/imunologia , Citrulinação/imunologia , Proteína-Arginina Desiminase do Tipo 2/imunologia , Proteína-Arginina Desiminase do Tipo 4/imunologia , Artrite Reumatoide/sangue , Citrulina/metabolismo , Células HEK293 , Humanos , Perforina/metabolismo
20.
Adv Exp Med Biol ; 1185: 175-179, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884608

RESUMO

We present evidence that protein citrullination, a proinflammatory and immune system-activating posttranslational modification (PTM) of arginine residues mediated by peptidyl arginine deiminases (PADs), is elevated in mouse models of retinal degenerations. Together with the fact that the animal models that we investigated (and their human counterparts) exhibit also anti-retinal autoantibodies, we propose that retinal citrullination is an immunogenic trigger that activates the immune system both locally and systemically, contributing to disease pathogenesis. Consistent with this possibility, we show that PAD compromise reduces the severity of Mertk-related retinal degeneration. Thus, PAD inhibition may be as a potential treatment strategy for retinal degenerations.


Assuntos
Autoimunidade , Citrulinação , Sistema Imunitário , Inflamação/patologia , Desiminases de Arginina em Proteínas/fisiologia , Degeneração Retiniana/patologia , Animais , Citrulina , Humanos , Camundongos
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