RESUMO
The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.
Assuntos
Autoantígenos/metabolismo , Hidrolases/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Ribonucleoproteínas/metabolismo , Glândulas Salivares/fisiologia , Síndrome de Sjogren/metabolismo , Trifosfato de Adenosina/imunologia , Animais , Apoptose , Células Cultivadas , Citrulinação , Citocinas/metabolismo , Ativação Enzimática , Fibrinogênio/metabolismo , Regulação da Expressão Gênica , Humanos , Hidrolases/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/genética , Antígeno SS-BRESUMO
To explore the CENP-B centromere protein in beans, carrots, onions and potatoes, total RNA was isolated and reverse transcribed by PCR, and the cDNA encoding the CENP-B amino terminus domain amplified using CENP-B oligonucleotides. Blots containing PCR products were hybridized with a nick-translated pG/CNPB probe containing a complete human CENP-B gene. In all the plant species, anti-CENP-B antibodies recognized an 80-kDa protein. A 360-bp sequence encoding for the amino terminus region of the CENP-B protein was amplified by PCR in all the species and the nick translated pG/CNPB probe hybridized with the PCR products. Apparently the CENP-B centromere protein or an equivalent protein is widely distributed in the vegetal kingdom.
Assuntos
Humanos , Proteínas Cromossômicas não Histona/genética , Verduras/genética , Autoanticorpos/imunologia , Western Blotting , Proteínas Cromossômicas não Histona/imunologia , Daucus carota , DNA Complementar/genética , Fabaceae , Hibridização In Situ , Cebolas , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA , Solanum tuberosum , Verduras/químicaRESUMO
To explore the CENP-B centromere protein in beans, carrots, onions and potatoes, total RNA was isolated and reverse transcribed by PCR, and the cDNA encoding the CENP-B amino terminus domain amplified using CENP-B oligonucleotides. Blots containing PCR products were hybridized with a nick-translated pG/CNPB probe containing a complete human CENP-B gene. In all the plant species, anti-CENP-B antibodies recognized an 80-kDa protein. A 360-bp sequence encoding for the amino terminus region of the CENP-B protein was amplified by PCR in all the species and the nick translated pG/CNPB probe hybridized with the PCR products. Apparently the CENP-B centromere protein or an equivalent protein is widely distributed in the vegetal kingdom.