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1.
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
2.
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
3.
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
4.
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
5.
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 , Desiminases de Arginina em Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais
6.
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 , 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
7.
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 , Desiminases de Arginina em Proteínas/metabolismo , Couro Cabeludo/patologia , Análise de Sequência de DNA
8.
Mol Carcinog ; 58(1): 66-75, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30242913

RESUMO

An increasing amount of evidence indicates that peptidylarginine deiminase isoform 4 (PADI4) plays an important role in tumorigenesis. However, the effects of PADI4 on tumor-bearing mice are unknown, and no studies have investigated this tumorigenic pathway in an animal model. In the present study, ECA109 cells originating from esophageal squamous cell carcinoma (ESCC) were transfected with PADI4-expressing lentivirus and were injected into BALB/c nude mice. Tumor size and weight were significantly increased in the mouse tumors established with PADI4-overexpressing ECA109 cells. PCR array analysis revealed increased CA9 expression in ECA109 cells transfected with a PADI4-expressing plasmid, while decreased CA9 expression levels were detected in cells transfected with anti-PADI4 siRNA. Furthermore, up-regulation of CA9 expression was detected in mouse tumors established with PADI4-overexpressing cells. Immunohistochemistry detected the increased expression and co-localization of PADI4 and CA9 in ESCC tissues compared with adjacent non-tumor tissues and normal tissue controls. These results were verified using Western blotting. Cell proliferation significantly increased or decreased in ECA109 and EC9706 (another ESCC-originating cell line) cells transfected with a PADI4-expressing plasmid or anti-PADI4 siRNA, respectively. The above findings suggest that increased PADI4 expression in ESCC stimulates tumor growth and up-regulates CA9 expression, which is known to promote metastatic properties in tumor cells.


Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Anidrase Carbônica IX/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Desiminases de Arginina em Proteínas/metabolismo , Animais , Antígenos de Neoplasias/genética , Apoptose , Biomarcadores Tumorais/genética , Anidrase Carbônica IX/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Desiminases de Arginina em Proteínas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Scand J Rheumatol ; 48(2): 133-140, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30269634

RESUMO

OBJECTIVE: Peptidylarginine deiminase-4 (PAD4) is highly expressed by neutrophils and essential for citrullination occurring during the formation of neutrophil extracellular traps, which have been implicated in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). Single-nucleotide polymorphisms (SNPs) in PADI4 influence PAD4 expression and functionality. Here, we investigate whether SNPs in PADI4 influence the risk of SLE or LN. METHOD: Altogether, 234 SLE patients and 484 controls were genotyped for nine PADI4 SNPs known to alter PAD4 functionality and/or expression, or to be associated with other autoimmune diseases, using an in-house multiplex Luminex assay. All analyses were adjusted for age and gender. RESULTS: Heterozygosity for rs1748033, and heterozygosity and homozygosity for rs1635564, were associated with increased occurrence of SLE [odds ratio (OR) 1.55, 95% confidence interval (CI) 1.08-2.23; OR 1.52, 95% CI 1.06-2.19; and OR 2.06, 95% CI 1.08-3.93, respectively]. Homozygosity for rs1635564 was also associated with increased occurrence of LN (OR 3.35, 95% CI 1.2-10.97). Notably, gene dose effects of the rs1635564 variant allele were observed for SLE (p = 0.005) and LN (p = 0.01). Carriage of minor alleles of five other SNPs (rs11203366, rs11203367, rs874881, rs2240340, and rs11203368) was associated with increased occurrence of LN and hypertension. CONCLUSION: The rs1635564 polymorphism of PADI4 is a candidate risk factor for SLE, particularly with renal involvement. Additional PADI4 polymorphisms also conferred increased risk of LN. Overall, these findings support the notion of PAD4 contributing to the pathogenesis of SLE and LN.


Assuntos
Lúpus Eritematoso Sistêmico/genética , Desiminases de Arginina em Proteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Creatinina/metabolismo , Feminino , Humanos , Hipertensão/genética , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/genética , Masculino , Pessoa de Meia-Idade , Síndrome Nefrótica/genética , Polimorfismo de Nucleotídeo Único , Adulto Jovem
10.
Mol Cell ; 73(1): 84-96.e7, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30472187

RESUMO

The post-translational modification of key residues at the C-terminal domain of RNA polymerase II (RNAP2-CTD) coordinates transcription, splicing, and RNA processing by modulating its capacity to act as a landing platform for a variety of protein complexes. Here, we identify a new modification at the CTD, the deimination of arginine and its conversion to citrulline by peptidyl arginine deiminase 2 (PADI2), an enzyme that has been associated with several diseases, including cancer. We show that, among PADI family members, only PADI2 citrullinates R1810 (Cit1810) at repeat 31 of the CTD. Depletion of PADI2 or loss of R1810 results in accumulation of RNAP2 at transcription start sites, reduced gene expression, and inhibition of cell proliferation. Cit1810 is needed for interaction with the P-TEFb (positive transcription elongation factor b) kinase complex and for its recruitment to chromatin. In this way, CTD-Cit1810 favors RNAP2 pause release and efficient transcription in breast cancer cells.


Assuntos
Neoplasias da Mama/enzimologia , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Transcrição Genética , Arginina , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Citrulinação , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , RNA Polimerase II/química , RNA Polimerase II/genética , Transdução de Sinais
11.
Dev Comp Immunol ; 92: 1-19, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30395876

RESUMO

Post-translational protein deimination is mediated by peptidylarginine deiminases (PADs), which are calcium dependent enzymes conserved throughout phylogeny with physiological and pathophysiological roles. Protein deimination occurs via the conversion of protein arginine into citrulline, leading to structural and functional changes in target proteins. In a continuous series of early halibut development from 37 to 1050° d, PAD, total deiminated proteins and deiminated histone H3 showed variation in temporal and spatial detection in various organs including yolksac, muscle, skin, liver, brain, eye, spinal cord, chondrocytes, heart, intestines, kidney and pancreas throughout early ontogeny. For the first time in any species, deimination of complement components C3 and C4 is shown in halibut serum, indicating a novel mechanism of complement regulation in immune responses and homeostasis. Proteomic analysis of deiminated target proteins in halibut serum further identified complement components C5, C7, C8 C9 and C1 inhibitor, as well as various other immunogenic, metabolic, cytoskeletal and nuclear proteins. Post-translational deimination may facilitate protein moonlighting, an evolutionary conserved phenomenon, allowing one polypeptide chain to carry out various functions to meet functional requirements for diverse roles in immune defences and tissue remodelling.


Assuntos
Citrulinação , Complemento C3/metabolismo , Complemento C4/metabolismo , Proteínas de Peixes/metabolismo , Linguado/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Animais , Evolução Biológica , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Homeostase , Imunidade , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas/genética , Proteômica , Transcriptoma
12.
JCI Insight ; 3(23)2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30518690

RESUMO

The peptidylarginine deiminases PAD2 and PAD4 are implicated in the pathogenesis of several autoimmune diseases. PAD4 may be pathogenic in systemic lupus erythematosus (SLE) through its role in neutrophil extracellular trap (NET) formation that promotes autoantigen externalization, immune dysregulation, and organ damage. The role of this enzyme in mouse models of autoimmunity remains unclear, as pan-PAD chemical inhibitors improve clinical phenotype, whereas PAD4-KO models have given conflicting results. The role of PAD2 in SLE has not been investigated. The differential roles of PAD2 and PAD4 in TLR-7-dependent lupus autoimmunity were examined. Padi4-/- displayed decreased autoantibodies, type I IFN responses, immune cell activation, vascular dysfunction, and NET immunogenicity. Padi2-/- mice showed abrogation of Th subset polarization, with some disease manifestations reduced compared with WT but to a lesser extent than Padi4-/- mice. RNA sequencing analysis revealed distinct modulation of immune-related pathways in PAD-KO lymphoid organs. Human T cells express both PADs and, when exposed to either PAD2 or PAD4 inhibitors, displayed abrogation of Th1 polarization. These results suggest that targeting PAD2 and/or PAD4 activity modulates dysregulated TLR-7-dependent immune responses in lupus through differential effects of innate and adaptive immunity. Compounds that target PADs may have potential therapeutic roles in T cell-mediated diseases.


Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Desiminases de Arginina em Proteínas/imunologia , Desiminases de Arginina em Proteínas/metabolismo , Receptor 7 Toll-Like/imunologia , Animais , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Armadilhas Extracelulares , Feminino , Regulação da Expressão Gênica , Histonas , Humanos , Hidrolases/genética , Hidrolases/imunologia , Hidrolases/metabolismo , Inflamação , Interferon Tipo I , Camundongos , Camundongos Knockout , Desiminases de Arginina em Proteínas/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th1 , Células Th17 , Transcriptoma
13.
Nat Commun ; 9(1): 4783, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30429478

RESUMO

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.


Assuntos
Citrulinação/genética , Neoplasias Colorretais/genética , Matriz Extracelular/metabolismo , Neoplasias Hepáticas/genética , Desiminases de Arginina em Proteínas/genética , Animais , Adesão Celular , Movimento Celular , Colágeno Tipo I/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Células HCT116 , Células HT29 , Humanos , Hidrolases/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos , Metástase Neoplásica
14.
Sci Rep ; 8(1): 15228, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323221

RESUMO

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.


Assuntos
Artrite Reumatoide/genética , NADPH Oxidases/genética , Desiminases de Arginina em Proteínas/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Membrana Celular/genética , Citrulinação/genética , Citosol/metabolismo , Humanos , Neutrófilos/enzimologia , Neutrófilos/patologia , Fagócitos/metabolismo , Fagocitose/genética , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade
15.
JCI Insight ; 3(18)2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30232279

RESUMO

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.


Assuntos
Armadilhas Extracelulares/imunologia , Neutrófilos/imunologia , Trombocitopenia/imunologia , Trombose/imunologia , Animais , Doenças Autoimunes/imunologia , Movimento Celular , Células Endoteliais , Humanos , Leucócitos , Vasos Linfáticos , Masculino , Camundongos , Camundongos Knockout , Ativação de Neutrófilo , Fator Plaquetário 4/genética , Desiminases de Arginina em Proteínas/genética , Receptores de Interleucina-8B/metabolismo
16.
Sci Signal ; 11(546)2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30181240

RESUMO

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain-like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase-independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8-dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8-dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.


Assuntos
Apoptose , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Armadilhas Extracelulares/genética , Histonas/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Neutrófilos/microbiologia , Neutrófilos/ultraestrutura , Proteínas Quinases/genética , Desiminases de Arginina em Proteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
17.
Sci Rep ; 8(1): 13263, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-30185873

RESUMO

Peptidyl arginine deiminases (PADIs) are enzymes that change the charge of proteins through citrullination. We recently found Padi2 was expressed exclusively in fetal Sertoli cells. In this study, we analyzed the transcriptional regulation of Padi2 and the role of PADI2 in testicular development. We showed SOX9 positively regulated Padi2 transcription and FOXL2 antagonized it in TM3 cells, a model of Sertoli cells. The responsive region to SOX9 and FOXL2 was identified within the Padi2 sequence by reporter assay. In fetal testes from Sox9 knockout (AMH-Cre:Sox9flox/flox) mice, Padi2 expression was greatly reduced, indicating SOX9 regulates Padi2 in vivo. In vitro analysis using siRNA suggested PADI2 modified transcriptional regulation by SOX9. However, Padi2-/- XY mice were fertile and showed no apparent reproductive anomalies. Although, PADI2 is known as an epigenetic transcriptional regulator through H3 citrullination, no significant difference in H3 citrullination between wildtype and Padi2-/- XY gonads was observed. These results suggest Padi2 is a novel gene involved in testis development that is specifically expressed in Sertoli cells through the regulation by SOX9 and FOXL2 and PADI2 supports regulation of target genes by SOX9. Analysis of the Padi2-/- XY phenotype suggested a redundant factor compensated for PADI2 function in testicular development.


Assuntos
Desiminases de Arginina em Proteínas/biossíntese , Fatores de Transcrição SOX9/metabolismo , Células de Sertoli/metabolismo , Testículo/embriologia , Animais , Linhagem Celular , Proteína Forkhead Box L2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Fatores de Transcrição SOX9/genética , Células de Sertoli/citologia , Testículo/metabolismo
18.
Free Radic Biol Med ; 129: 25-34, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30189264

RESUMO

The infiltration of activated leukocytes, including macrophages, at sites of inflammation and the formation and presence of hypochlorous acid (HOCl) are interlinked hallmarks of many debilitating disease processes, including atherosclerosis, arthritis, neurological and renal disease, diabetes and obesity. The production of extracellular traps by activated leukocytes in response to a range of inflammatory stimuli is increasingly recognised as an important process within a range of disease settings. We show that exposure of human monocyte-derived macrophages to pathophysiological levels of HOCl results in the dose-dependent extrusion of DNA and histones into the cellular supernatant, consistent with extracellular trap formation. Concurrent with, but independent of these findings, macrophage exposure to HOCl also resulted in an immediate and sustained cytosolic accumulation of Ca2+, culminating in the increased production of cytokines and chemokines. Polarisation of the macrophages prior to HOCl exposure revealed a greater propensity for inflammatory M1 macrophages to produce extracellular traps, whereas alternatively-activated M2 macrophages were less susceptible to HOCl insult. M1 macrophages also produced extracellular traps on exposure to phorbol myristate acetate (PMA), interleukin-8 (IL-8) and tumour necrosis factor α (TNFα). Taken together, these data indicate a potential role for macrophages in mediating extracellular trap formation, which may be relevant in pathological conditions characterised by chronic inflammation or excessive HOCl formation.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Ácido Hipocloroso/farmacologia , Interleucina-8/farmacologia , Macrófagos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Cálcio/metabolismo , Cátions Bivalentes , Diferenciação Celular , DNA/metabolismo , Espaço Extracelular/química , Armadilhas Extracelulares/metabolismo , Expressão Gênica , Histonas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Cultura Primária de Células , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
19.
J Transl Med ; 16(1): 214, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30064459

RESUMO

BACKGROUND: A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. METHODS: Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. RESULTS: Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. CONCLUSION: Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.


Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Citrulinação , Gengiva/enzimologia , Gengiva/microbiologia , Periodontite/enzimologia , Periodontite/microbiologia , Porphyromonas gingivalis/fisiologia , Desiminases de Arginina em Proteínas/metabolismo , Adulto , Artrite Reumatoide/microbiologia , Artrite Reumatoide/patologia , Exotoxinas/metabolismo , Gengiva/patologia , Humanos , Inflamação/patologia , Linfócitos/patologia , Pessoa de Meia-Idade , Periodontite/genética , Periodontite/patologia , Desiminases de Arginina em Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
J Cell Mol Med ; 22(10): 4732-4737, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30044533

RESUMO

The objective of our study was to evaluate the association between peptidylarginine deiminase 4 (PAD4) concentration and its polymorphisms with mortality in patients with septic shock. We prospectively evaluated 175 patients aged over 18 years with septic shock upon intensive care unit (ICU) admission. However, 48 patients were excluded. Thus, 127 patients were enrolled in the study. At the time of the patients' enrollment, demographic information was recorded. Blood samples were taken within the first 24 hours of the patient's admission to determine serum PAD4 concentrations and its polymorphism PADI4_89 [rs11203366], PADI4_94 [rs2240340] and PADI4_104 [rs1748033]. The mean age was 63.3 ± 15.2 years, 56.7% were male, PAD4 concentration was 4.62 (2.48-6.20) ng/mL and the ICU mortality rate was 67.7%. The patients who died in the ICU had higher APACHE II and Sequential Organ Failure Assessment (SOFA) scores. In addition, PAD4 concentration was higher in patients who died during ICU stay. However, there were no differences regarding PADI4 polymorphisms and ICU mortality. In the logistic regression models, PAD4 concentrations were associated with ICU mortality when adjusted for APACHE II score and lactate (OR: 1.477; CI 95%: 1.186-1.839; P < .001), and when adjusted for age, gender and APACHE II score (OR: 1.392; CI 95%: 1.145-1.692; P < .001). In conclusion, PAD4 concentration, but not PADI4_89, PADI4_94 and PADI4_104 polymorphisms, is associated with ICU mortality in septic shock patients.


Assuntos
Polimorfismo de Nucleotídeo Único , Desiminases de Arginina em Proteínas/genética , Choque Séptico/genética , Choque Séptico/mortalidade , APACHE , Idoso , Feminino , Expressão Gênica , Mortalidade Hospitalar/tendências , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Desiminases de Arginina em Proteínas/sangue , Choque Séptico/sangue , Choque Séptico/patologia , Análise de Sobrevida
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