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1.
Biochim Biophys Acta Rev Cancer ; 1875(1): 188492, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33321174

RESUMO

Protein arginine deiminases (PADs), is a group of calcium-dependent enzymes, which play crucial roles in citrullination, and can catalyze arginine residues into citrulline. This chemical reaction induces citrullinated proteins formation with altered structure and function, leading to numerous pathological diseases, including inflammation and autoimmune diseases. To date, multiple studies have provided solid evidence that PADs are implicated in cancer progression. Nevertheless, the findings on PADs functions in tumors are too complex to understand due to its involvements in variable signaling pathways. The increasing interest in PADs has heightened the need for a comprehensive description for its role in cancer. The present study aims to identify the gaps in present knowledge, including its structures, biological substrates and tissue distribution. Since several irreversible inhibitors for PADs with good potency and selectivity have been explored, the mechanisms on the dysregulation in tumors remain poorly understood. The present study discusses the relationship between PADs and tumor apoptosis, EMT formation and metastasis as well as the implication of neutrophil extracellular traps (NETs) in tumorigenesis. In addition, the potential uses of citrullinated antigens for immunotherapy were proposed.


Assuntos
Citrulinação/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias/genética , Desiminases de Arginina em Proteínas/genética , Citrulina/genética , Citrulina/metabolismo , Armadilhas Extracelulares/genética , Humanos , Neoplasias/patologia , Transdução de Sinais/genética
2.
Rev. Assoc. Med. Bras. (1992) ; 66(11): 1515-1520, Nov. 2020. tab
Artigo em Inglês | LILACS, Sec. Est. Saúde SP | ID: biblio-1143627

RESUMO

SUMMARY BACKGROUND: The aim of this study is to evaluate the peptidylarginine deiminase 4 (PAD 4) concentration and PADI4 polymorphisms as predictors of acute kidney injury (AKI) development, the need for renal replacement therapy (RRT), and mortality in patients with septic shock. METHODS: We included all individuals aged ≥ 18 years, with a diagnosis of septic shock at ICU admission. Blood samples were taken within the first 24 hours of the patient's admission to determine serum PAD4 concentration and its PADI4 polymorphism (rs11203367) and (rs874881). Patients were monitored during their ICU stay and the development of SAKI was evaluated. Among the patients in whom SAKI developed, mortality and the need for RRT were also evaluated. RESULTS: There were 99 patients, 51.5% of whom developed SAKI and of these, 21.5% needed RRT and 80% died in the ICU. There was no difference between PAD4 concentration (p = 0.116) and its polymorphisms rs11203367 (p = 0.910) and rs874881 (p = 0.769) in patients in whom SAKI did or did not develop. However, PAD4 had a positive correlation with plasma urea concentration (r = 0.269 and p = 0.007) and creatinine (r = 0.284 and p = 0.004). The PAD4 concentration and PADI4 polymorphisms were also not associated with RRT and with mortality in patients with SAKI. CONCLUSION: PAD4 concentration and its polymorphisms were not associated with SAKI development, the need for RRT, or mortality in patients with septic shock. However, PAD4 concentrations were associated with creatinine and urea levels in these patients.


RESUMO OBJETIVO: Avaliar a concentração da peptidilarginina deiminase 4 (PAD4) e os polimorfismos de PADI4, como preditores de desenvolvimento de lesão renal aguda, necessidade de terapia renal substitutiva (TRS) e mortalidade em pacientes com choque séptico. MÉTODOS: Foram incluídos indivíduos com idade ≥18 anos, com diagnóstico de choque séptico na admissão na Unidade de Terapia Intensiva (UTI). Amostras de sangue foram coletadas nas primeiras 24 horas após a admissão do paciente para determinar a concentração sérica de PAD4 e seus polimorfismos PADI4 (rs11203367) e (rs874881). Os pacientes foram acompanhados durante a internação na UTI e tiveram avaliados desenvolvimento da lesão renal aguda séptica (Sepsis-induced acute kidney injury - Saki), necessidade TRS e mortalidade. RESULTADOS: Foram avaliados 99 pacientes; 51,5% desenvolveram Saki e, desses, 21,5% necessitaram de TRS e 80% morreram na UTI. Não houve diferença entre a concentração de PAD4 (p=0,116) e seus polimorfismos rs11203367 (p=0,910) e rs874881 (p=0,769) entre os pacientes. No entanto, o PAD4 apresentou correlação positiva com a concentração plasmática de ureia (r=0,269; p=0,007) e creatinina (r=0,284; p=0,004). A concentração de PAD4 e os polimorfismos da PADI4 também não foram associados à TRS e à mortalidade em pacientes com Saki. CONCLUSÕES: A concentração de PAD4 e seus polimorfismos não foram associados ao desenvolvimento de Saki, à necessidade de TRS ou à mortalidade em pacientes com choque séptico. No entanto, as concentrações de PAD4 foram associadas às concentrações de creatinina e ureia nesses pacientes.


Assuntos
Humanos , Sepse , Lesão Renal Aguda/genética , Terapia de Substituição Renal , Desiminases de Arginina em Proteínas/genética , Unidades de Terapia Intensiva
3.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629995

RESUMO

Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Desiminases de Arginina em Proteínas/metabolismo , Betacoronavirus/isolamento & purificação , Estudos de Casos e Controles , Linhagem Celular , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Bases de Dados Factuais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vesículas Extracelulares/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Pandemias , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas , Desiminases de Arginina em Proteínas/genética , RNA Mensageiro/metabolismo
4.
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
5.
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
6.
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
7.
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
8.
J Immunol Res ; 2019: 7592851, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886309

RESUMO

A protein undergoes many types of posttranslation modification. Citrullination is one of these modifications, where an arginine amino acid is converted to a citrulline amino acid. This process depends on catalytic enzymes such as peptidylarginine deiminase enzymes (PADs). This modification leads to a charge shift, which affects the protein structure, protein-protein interactions, and hydrogen bond formation, and it may cause protein denaturation. The irreversible citrullination reaction is not limited to a specific protein, cell, or tissue. It can target a wide range of proteins in the cell membrane, cytoplasm, nucleus, and mitochondria. Citrullination is a normal reaction during cell death. Apoptosis is normally accompanied with a clearance process via scavenger cells. A defect in the clearance system either in terms of efficiency or capacity may occur due to massive cell death, which may result in the accumulation and leakage of PAD enzymes and the citrullinated peptide from the necrotized cell which could be recognized by the immune system, where the immunological tolerance will be avoided and the autoimmune disorders will be subsequently triggered. The induction of autoimmune responses, autoantibody production, and cytokines involved in the major autoimmune diseases will be discussed.


Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Citrulina/metabolismo , Suscetibilidade a Doenças , Desiminases de Arginina em Proteínas/metabolismo , Doenças Autoimunes/diagnóstico , Autoimunidade , Biomarcadores , Citrulinação , Predisposição Genética para Doença , Humanos , Desiminases de Arginina em Proteínas/genética , Fatores de Risco
9.
Int J Mol Sci ; 20(21)2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671738

RESUMO

Naked mole-rats are long-lived animals that show unusual resistance to hypoxia, cancer and ageing. Protein deimination is an irreversible post-translational modification caused by the peptidylarginine deiminase (PAD) family of enzymes, which convert arginine into citrulline in target proteins. Protein deimination can cause structural and functional protein changes, facilitating protein moonlighting, but also leading to neo-epitope generation and effects on gene regulation. Furthermore, PADs have been found to regulate cellular release of extracellular vesicles (EVs), which are lipid-vesicles released from cells as part of cellular communication. EVs carry protein and genetic cargo and are indicative biomarkers that can be isolated from most body fluids. This study was aimed at profiling deiminated proteins in plasma and EVs of naked mole-rat. Key immune and metabolic proteins were identified to be post-translationally deiminated, with 65 proteins specific for plasma, while 42 proteins were identified to be deiminated in EVs only. Using protein-protein interaction network analysis, deiminated plasma proteins were found to belong to KEEG (Kyoto Encyclopedia of Genes and Genomes) pathways of immunity, infection, cholesterol and drug metabolism, while deiminated proteins in EVs were also linked to KEEG pathways of HIF-1 signalling and glycolysis. The mole-rat EV profiles showed a poly-dispersed population of 50-300 nm, similar to observations of human plasma. Furthermore, the EVs were assessed for three key microRNAs involved in cancer, inflammation and hypoxia. The identification of post-translational deimination of critical immunological and metabolic markers contributes to the current understanding of protein moonlighting functions, via post-translational changes, in the longevity and cancer resistance of naked mole-rats.


Assuntos
Proteínas Sanguíneas/metabolismo , Vesículas Extracelulares/metabolismo , Ratos-Toupeira/imunologia , Ratos-Toupeira/metabolismo , Plasma/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Arginina/metabolismo , Biomarcadores , Proteínas Sanguíneas/genética , Citrulina/metabolismo , Regulação da Expressão Gênica , Genoma , Humanos , Imunidade , Longevidade , MicroRNAs/metabolismo , Ratos-Toupeira/genética , Mapas de Interação de Proteínas , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Proteômica
10.
Cell Mol Life Sci ; 76(23): 4635-4662, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31342121

RESUMO

Citrullination is a post-translation modification of proteins, where the proteinaceous arginine residues are converted to non-coded citrulline residues. The immune tolerance to such citrullinated protein can be lost, leading to inflammatory and autoimmune diseases. Citrullination is a chemical reaction mediated by peptidylarginine deiminase enzymes (PADs), which are a family of calcium-dependent cysteine hydrolase enzymes that includes five isotypes: PAD1, PAD2, PAD3, PAD4, and PAD6. Each PAD has specific substrates and tissue distribution, where it modifies the arginine to produce a citrullinated protein with altered structure and function. All mammalian PADs have a sequence similarity of about 70-95%, whereas in humans, they are 50-55% homologous in their structure and amino acid sequences. Being calcium-dependent hydrolases, PADs are inactive under the physiological level of calcium, but could be activated due to distortions in calcium homeostasis, or when the cellular calcium levels are increased. In this article, we analyze some of the currently available data on the structural properties of human PADs, the mechanisms of their calcium-induced activation, and show that these proteins contain functionally important regions of intrinsic disorder. Citrullination represents an important trigger of multiple physiological and pathological processes, and as a result, PADs are recognized to play a number of important roles in autoimmune diseases, cancer, and neurodegeneration. Therefore, we also review the current state of the art in the development of PAD inhibitors with good potency and selectivity.


Assuntos
Autoimunidade , Desiminases de Arginina em Proteínas/metabolismo , Animais , Cálcio/química , Cálcio/metabolismo , Morte Celular , Citrulina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo
11.
Gene ; 713: 143975, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31302167

RESUMO

Hair is one of the defining characteristics of mammals. The hair shaft has a two-layer structure comprising the cortex, which is the inner layer and is composed of cortical cells, and the cuticle, which is the outermost layer. S100 calcium-binding protein A3 (S100A3) is expressed at high levels in the human hair cuticle. Arginine 51 of S100A3 protein is citrullinated specifically by peptidylarginine deiminase 3 (PAD3), and this citrullination is related to maturation of the cuticle. However, the detailed evolutionary processes of S100A3 and PAD3 during mammalian evolution are unknown. Here, we show that nonsynonymous changes in S100A3 accelerated in the common ancestral branch of mammals, probably as a result of positive selection that returned after the acquisition of hair cuticle-specific function in mammals. Later, pseudogenisation or nonfunctionalisation of S100A3 and PAD3 occurred in some species, such as the cetaceans. Our results show that positive selection and relaxation of the functional constraints of genes played important roles in the evolution of mammalian hair.


Assuntos
Evolução Molecular , Cabelo/química , Mamíferos/genética , Desiminases de Arginina em Proteínas/genética , Proteínas S100/genética , Seleção Genética , Sequência de Aminoácidos , Animais , Filogenia , Homologia de Sequência
12.
Fish Shellfish Immunol ; 92: 249-255, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200072

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. Extracellular vesicles 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 and extracellular vesicles (EVs) are described for the first time in shark plasma. We report a poly-dispersed population of shark plasma EVs, positive for phylogenetically conserved EV-specific markers and characterised by TEM. In plasma, 6 deiminated proteins, including complement and immunoglobulin, were identified, whereof 3 proteins were found to be exported in plasma-derived EVs. A PAD homologue was identified in shark plasma by Western blotting and detected an expected 70 kDa size. Deiminated histone H3, a marker of neutrophil extracellular trap formation, was also detected in nurse shark plasma. This is the first report of deiminated proteins in plasma and EVs, highlighting a hitherto unrecognized post-translational modification in key immune proteins of innate and adaptive immunity in shark.


Assuntos
Arginina/metabolismo , Citrulinação/imunologia , Citrulina/metabolismo , Proteínas de Peixes/imunologia , Desiminases de Arginina em Proteínas/imunologia , Tubarões/imunologia , Animais , Citrulina/imunologia , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Proteínas de Peixes/sangue , Proteínas de Peixes/metabolismo , Desiminases de Arginina em Proteínas/genética , Tubarões/genética
13.
Arthritis Res Ther ; 21(1): 108, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039829

RESUMO

OBJECTIVE: To investigate the expression of peptidylarginine deiminases (PADIs) during macrophage differentiation and its role in inflammatory responses. METHODS: The protein expression of PADI2, PADI4, and citrullinated histone 3 in U937 cells, differentiated macrophages, and macrophages stimulated with lipopolysaccharides (LPS) were analyzed by Western blotting. Three PADI inhibitors were used for assessing their effects on the secretion of proinflammatory cytokines in macrophages. The differential expressed citrullinated proteins during macrophage differentiation were probed by self-prepared anti-citrullinated protein antibodies, and the reactive bands were sent for proteomic analyses. Transfection studies were conducted to search for the functions of specific proteins. A specific protein was cloned and citrullinated for its protein binding study. RESULTS: The expression of PADI2 and PADI4 markedly increased during macrophage differentiation, whereas the formation of citrullinated histone 3 increased after stimulated with lipopolysaccharides. Three PADI inhibitors suppressed the LPS mediated proinflammatory cytokines secretion, but did not affect the expression of PADI2 and PADI4. Plasminogen activator inhibitor-2 (PAI-2) was citrullinated during macrophage differentiation. The expression of PAI-2 increased during macrophage differentiation and further increased after stimulated with LPS. Suppressed PAI-2 expression decreased the expression and secretion of proinflammatory cytokines. Decreased PADI2 expression also suppressed the expression of PAI-2 and protein levels of citrullinated PAI-2. The citrullination of PAI-2 inhibited its binding ability to proteasome subunit beta type-1 (PSMB1). CONCLUSION: PADI2 and PADI4 protein levels increased during the macrophage differentiation resulting in protein citrullination, including PAI-2. The increased expression of PAI-2 promoted inflammatory response, and the citrullination of PAI-2 impaired its binding to PSMB1. Therefore, protein citrullination could play a critical role in macrophage differentiation and function.


Assuntos
Diferenciação Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Desiminases de Arginina em Proteínas/biossíntese , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Desiminases de Arginina em Proteínas/genética , Células U937
14.
J Immunol Res ; 2019: 6587570, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30944835

RESUMO

Background: PADI4 has extensive expression in many tumors. This study applied PADI4 as a tumor marker to stimulate DC- (dendritic cell-) CIK (cytokine-induced killer), an immunotherapy approach. Methods: A PADI4 expression plasmid was transfected into EC-originating ECA-109 cells. PADI4 gene was also inserted into a prokaryotic expression vector to produce recombinant protein. Lysate from PADI4-overexpressing cells or the purified recombinant PADI4 protein was used to load DCs, and the cells were then coincubated with CIK cells. DC and CIK cell phenotypes were determined using flow cytometry. The proliferation and viability of CIK cells were analyzed using trypan blue staining. The cytotoxic effect of DC-CIK cells on cultured ECA-109 cells was determined using CCK8 assays. Tumor-bearing mice were prepared by injection of ECA-109 cells. DC-CIK cells stimulated with lysate from PADI4-overexpressing cells or the PADI4 recombinant protein were injected into the tumor-bearing mice. The tumor growth was measured with magnetic resonance imaging (MRI). Results: Following incubation with lysate from PADI4-overexpressing cells, the ratio of CD40+ DCs increased by 17.5%. Induction of CIK cells with PADI4-stimulated DCs elevated the cell proliferation by 53.2% and the ability of CIK cells to kill ECA-109 cells by 12.1%. DC-CIK cells stimulated with lysate from PADI4-overexpressing cells suppressed tumor volume by 18.6% in the tumor-bearing mice. The recombinant PADI4 protein showed a similar effect on CIK cell proliferation and cytotoxicity as that of the lysate from PADI4-overexpressing cells. Furthermore, the recombinant protein elevated the ratio of CD40+ DCs by 111.8%, CD80+ DCs by 6.3%, CD83+ DCs by 30.8%, and CD86+ DCs by 7.8%. Induction of CIK cells with rPADI4-stimulated DCs elevated the cell proliferation by 50.3% and the ability of CIK cells to kill ECA-109 cells by 14.7% and suppressed tumor volume by 35.1% in the animal model. Conclusion: This study demonstrates that stimulation of DC-CIK cells with PADI4 significantly suppressed tumor growth in tumor-bearing mice by promoting DC maturation, CIK cell proliferation, and cytotoxicity. PADI4 may be a potential tumor marker that could be used to improve the therapeutic efficiency of DC-CIK cells.


Assuntos
Células Matadoras Induzidas por Citocinas/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Neoplasias Esofágicas/terapia , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/farmacologia , Adulto , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Técnicas de Cocultura , Citotoxicidade Imunológica , Modelos Animais de Doenças , Neoplasias Esofágicas/imunologia , Feminino , Humanos , Imunoterapia/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína-Arginina Desiminase do Tipo 4
15.
Nat Commun ; 10(1): 1322, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899022

RESUMO

Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.


Assuntos
Plaquetas/imunologia , Armadilhas Extracelulares/imunologia , Heparina/efeitos adversos , Neutrófilos/imunologia , Receptores de IgG/genética , Trombocitopenia/imunologia , Trombose/imunologia , Animais , Complexo Antígeno-Anticorpo/biossíntese , Plaquetas/efeitos dos fármacos , Citrulinação , Inibidores Enzimáticos/farmacologia , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Histonas/genética , Histonas/imunologia , Humanos , Imunoglobulina G/biossíntese , Camundongos , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/imunologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/imunologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/imunologia , Receptores de IgG/imunologia , Transdução de Sinais , Trombocitopenia/induzido quimicamente , Trombocitopenia/patologia , Trombose/induzido quimicamente , Trombose/patologia , Trombose/prevenção & controle
16.
Mol Med Rep ; 19(4): 3087-3094, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816464

RESUMO

Lung cancer is a complex disease involving multiple genetic and phenotypic alterations. As a histone modification enzyme, protein­arginine deiminase type­4 (PADI4) and its downstream signaling have been studied in the progression of a variety of types of human cancer, but data on PADI4­mediated posttranslational modification in lung cancer are lacking. The aim of present study was to evaluate the expression of PADI4 and its associated molecular signaling in lung cancer metastasis. The results of the present study indicated that PADI4 was overexpressed in lung cancer cells, while knockdown of PADI4 could lead to attenuation of the lung cancer cell invasion and migration phenotype, which was further verified by determining the epithelial­mesenchymal transition (EMT) marker proteins. Additionally, it was demonstrated that stable knockdown of PADI4 in A549 lung cancer cells resulted in a striking reduction of the EMT­associated Snail1/mothers against decapentaplegic homolog 3/4 transcriptional complex, which was consistent with alterations in migratory and invasive phenotypes of A549 lung cancer cells. Therefore, PADI4­mediated EMT transition is proposed to represent a novel mechanism underlying the epigenetic and phenotypic alterations in lung cancer cells, and the PADI4 associated signaling pathway may be a therapeutic target for treating lung cancer in a clinical setting.


Assuntos
Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Desiminases de Arginina em Proteínas/genética , Células A549 , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais
17.
Acc Chem Res ; 52(3): 818-832, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30844238

RESUMO

Proteins are well-known to undergo a variety of post-translational modifications (PTMs). One such PTM is citrullination, an arginine modification that is catalyzed by a group of hydrolases called protein arginine deiminases (PADs). Hundreds of proteins are known to be citrullinated and hypercitrullination is associated with autoimmune diseases including rheumatoid arthritis (RA), lupus, ulcerative colitis (UC), Alzheimer's disease, multiple sclerosis (MS), and certain cancers. In this Account, we summarize our efforts to understand the structure and mechanism of the PADs and to develop small molecule chemical probes of protein citrullination. PAD activity is highly regulated by calcium. Structural studies with PAD2 revealed that calcium-binding occurs in a stepwise fashion and induces a series of dramatic conformational changes to form a catalytically competent active site. These studies also identified the presence of a calcium-switch that controls the overall calcium-dependence and a gatekeeper residue that shields the active site in the absence of calcium. Using biochemical and site-directed mutagenesis studies, we identified the key residues (two aspartates, a cysteine, and a histidine) responsible for catalysis and proposed a general mechanism of citrullination. Although all PADs follow this mechanism, substrate binding to the thiolate or thiol form of the enzyme varies for different isozymes. Substrate-specificity studies revealed that PADs 1-4 prefer peptidyl-arginine over free arginine and certain citrullination sites on a peptide substrate. Using high-throughput screening and activity-based protein profiling (ABPP), we identified several reversible (streptomycin, minocycline, and chlorotetracycline) and irreversible (streptonigrin, NSC 95397) PAD-inhibitors. Screening of a DNA-encoded library and lead-optimization led to the development of GSK199 and GSK484 as highly potent PAD4-selective inhibitors. Furthermore, use of an electrophilic, cysteine-targeted haloacetamidine warhead to mimic the guanidinium group in arginine afforded several mechanism-based pan-PAD-inhibitors including Cl-amidine and BB-Cl-amidine. These compounds are highly efficacious in various animal models, including those mimicking RA, UC, and lupus. Structure-activity relationships identified numerous covalent PAD-inhibitors with different bioavailability, in vivo stability, and isozyme-selectivity (PAD1-selective: D-Cl-amidine; PAD2-selective: compounds 16-20; PAD3-selective: Cl4-amidine; and PAD4-selective: TDFA). Finally, this Account describes the development of PAD-targeted and citrulline-specific chemical probes. While PAD-targeted probes were utilized for identifying off-targets and developing high-throughput inhibitor screening platforms, citrulline-specific probes enabled the proteomic identification of novel diagnostic biomarkers of hypercitrullination-related autoimmune diseases.


Assuntos
Citrulinação , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas/metabolismo , Proteínas/metabolismo , Animais , Ácido Aspártico/química , Catálise , Domínio Catalítico/genética , Cisteína/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Histidina/química , Humanos , Camundongos , Modelos Químicos , Mutação , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/química , Desiminases de Arginina em Proteínas/genética
18.
N Engl J Med ; 380(9): 833-841, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30763140

RESUMO

BACKGROUND: Central centrifugal cicatricial alopecia (CCCA) is the most common form of scarring alopecia among women of African ancestry. The disease is occasionally observed to affect women in families in a manner that suggests an autosomal dominant trait and usually manifests clinically after intense hair grooming. We sought to determine whether there exists a genetic basis of CCCA and, if so, what it is. METHODS: We used exome sequencing in a group of women with alopecia (discovery set), compared the results with those in a public repository, and applied other filtering criteria to identify candidate genes. We then performed direct sequencing to identify disease-associated DNA variations and RNA sequencing, protein modeling, immunofluorescence staining, immunoblotting, and an enzymatic assay to evaluate the consequences of potential etiologic mutations. We used a replication set that consisted of women with CCCA to confirm the data obtained with the discovery set. RESULTS: In the discovery set, which included 16 patients, we identified one splice site and three heterozygous missense mutations in PADI3 in 5 patients (31%). (The approximate prevalence of the disease is up to 5.6%.) PADI3 encodes peptidyl arginine deiminase, type III (PADI3), an enzyme that post-translationally modifies other proteins that are essential to hair-shaft formation. All three CCCA-associated missense mutations in PADI3 affect highly conserved residues and are predicted to be pathogenic; protein modeling suggests that they result in protein misfolding. These mutations were found to result in reduced PADI3 expression, abnormal intracellular localization of the protein, and decreased enzymatic activity - findings that support their pathogenicity. Immunofluorescence staining showed decreased expression of PADI3 in biopsy samples of scalp skin obtained from patients with CCCA. We then directly sequenced PADI3 in an additional 42 patients (replication set) and observed genetic variants in 9 of them. A post hoc analysis of the combined data sets showed that the prevalence of PADI3 mutation was higher among patients with CCCA than in a control cohort of women of African ancestry (P = 0.002 by the chi-square test; P = 0.006 by Fisher's exact test; and after adjustment for relatedness of persons, P = 0.03 and P = 0.04, respectively). CONCLUSIONS: Mutations in PADI3, which encodes a protein that is essential to proper hair-shaft formation, were associated with CCCA. (Funded by the Ram Family Foundation and others.).


Assuntos
Afro-Americanos/genética , Alopecia/genética , Predisposição Genética para Doença , Cabelo/crescimento & desenvolvimento , Mutação , Desiminases de Arginina em Proteínas/genética , Adolescente , Adulto , Idade de Início , Alopecia/etnologia , Distribuição de Qui-Quadrado , Cicatriz/genética , Exoma , Feminino , Heterozigoto , Humanos , Pessoa de Meia-Idade , Mutagênese , Linhagem , Proteína-Arginina Desiminase do Tipo 3 , Desiminases de Arginina em Proteínas/metabolismo , Couro Cabeludo/patologia , Análise de Sequência de DNA
19.
Protein Sci ; 28(3): 478-486, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30638292

RESUMO

Citrullination is an essential post-translational modification in which the guanidinium group of protein and peptide arginines is deiminated by peptidylarginine deiminases (PADs). When deregulated, excessive citrullination leads to inflammation as in severe periodontal disease (PD) and rheumatoid arthritis (RA). Porphyromonas gingivalis is the major periodontopathogenic causative agent of PD and also an etiological agent of RA. It secretes a PAD, termed Porphyromonas PAD (PPAD), which is a virulence factor that causes aberrant citrullination. Analysis of P. gingivalis genomes of laboratory strains and clinical isolates unveiled a PPAD variant (PPAD-T2), which showed three amino-acid substitutions directly preceding catalytic Residue H236 (G231 N/E232 T/N235 D) when compared with PPAD from the reference strain (PPAD-T1). Mutation of these positions in the reference strain resulted in twofold higher cell-associated citrullinating activity. Similar to PPAD-T1, recombinant PPAD-T2 citrullinated arginines at the C-termini of general peptidic substrates but not within peptides. Catalytically, PPAD-T2 showed weaker substrate binding but higher turnover rates than PPAD-T1. In contrast, no differences were found in thermal stability. The 1.6 Å-resolution X-ray crystal structure of PPAD-T2 in complex with the general human PAD inhibitor, Cl-amidine, revealed that the inhibitor moiety is tightly bound and that mutations localize to a loop engaged in substrate/inhibitor binding. In particular, mutation G231 N caused a slight structural rearrangement, which probably originated the higher substrate turnover observed. The present data compare two natural PPAD variants and will set the pace for the design of specific inhibitors against P. gingivalis-caused PD.


Assuntos
Inibidores Enzimáticos/farmacologia , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/genética , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Substituição de Aminoácidos , Infecções por Bacteroidaceae/tratamento farmacológico , Infecções por Bacteroidaceae/microbiologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Porphyromonas gingivalis/química , Conformação Proteica , Desiminases de Arginina em Proteínas/química , Desiminases de Arginina em Proteínas/metabolismo
20.
NPJ Biofilms Microbiomes ; 5(1): 7, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32029738

RESUMO

Porphyromonas gingivalis is the only known human-associated prokaryote that produces a peptidylarginine deiminase (PPAD), a protein-modifying enzyme that is secreted along with a number of virulence factors via a type IX secretion system (T9SS). While the function of PPAD in P. gingivalis physiology is not clear, human peptidylarginine deiminases are known to convert positively charged arginine residues within proteins to neutral citrulline and, thereby, impact protein conformation and function. Here, we report that the lack of citrullination in a PPAD deletion mutant (Δ8820) enhances biofilm formation. More Δ8820 cells attached to the surface than the parent strain during the early stages of biofilm development and, ultimately, mature Δ8820 biofilms were comprised of significantly more cell-cell aggregates and extracellular matrix. Imaging by electron microscopy discovered that Δ8820 biofilm cells secrete copious amounts of protein aggregates. Furthermore, gingipain-derived adhesin proteins, which are also secreted by the T9SS were predicted by mass spectrometry to be citrullinated and citrullination of these targets by wild-type strain 381 in vitro was confirmed. Lastly, Δ8820 biofilms contained more gingipain-derived adhesin proteins and more gingipain activity than 381 biofilms. Overall, our findings support the model that citrullination of T9SS cargo proteins known to play a key role in colonization, such as gingipain-derived adhesin proteins, is an underlying mechanism that modulates P. gingivalis biofilm development.


Assuntos
Biofilmes/crescimento & desenvolvimento , Citrulinação , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/fisiologia , Adesinas Bacterianas/metabolismo , Arginina/metabolismo , Citrulina/metabolismo , Deleção de Genes , Porphyromonas gingivalis/enzimologia , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo
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