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J Immunol ; 197(7): 2854-63, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27534558


The loss of tolerance and the presence of circulating autoantibodies directed against nuclear Ags is the hallmark of systemic lupus erythematosus (SLE). Many of these Ags are complexed with short, noncoding RNAs, such as U1 and Y1. The amount of U1 and Y1 RNA complexed with SLE patient Abs and immune complexes was measured in a cross-section of 228 SLE patients to evaluate the role of these RNA molecules within the known biochemical framework of SLE. The study revealed that SLE patients had significantly elevated levels of circulating U1 and/or Y1 RNA compared with healthy volunteers. In addition, the blood-borne RNA molecules were correlated with SLE disease activity and increased expression of IFN-inducible genes. To our knowledge, this study provides the first systematic examination of the role of circulating RNA in a large group of SLE patients and provides an important link with IFN dysregulation.

Regulação da Expressão Gênica , Interferons/imunologia , Lúpus Eritematoso Sistêmico/genética , RNA/sangue , Adulto , Reações Antígeno-Anticorpo , Autoanticorpos/imunologia , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Pessoa de Meia-Idade , RNA/imunologia , RNA Citoplasmático Pequeno/sangue , RNA Nuclear Pequeno/sangue
J Biol Chem ; 286(48): 41530-8, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21987572


Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.

Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Artrite Reumatoide/metabolismo , Células HeLa , Humanos , Fosforilação/fisiologia , Proteômica/métodos
Biochem Soc Symp ; (70): 39-52, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14587281


Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

Alanina/metabolismo , Metaloendopeptidases/metabolismo , Valina/metabolismo , Proteínas ADAM , Proteína ADAM17 , Indução Enzimática , Metaloendopeptidases/biossíntese , Metaloendopeptidases/química , Especificidade por Substrato
Biochem Biophys Res Commun ; 308(2): 331-8, 2003 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-12901873


Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease disintegrin that cleaves a variety of membrane proteins, releasing ("shedding") their extracellular domains from cells. Most TACE-mediated shedding events occur at low basal rates that are enhanced by treatment of cells with a variety of stimuli. To study the mechanism of induced shedding, we developed a peptide-cleavage assay that measures the cellular TACE activity. In unstimulated cells, cleavage of a TNFalpha processing-site peptide was mediated mainly by enzymes other than TACE. However, stimulation of cells with phorbol-12-myristate-13-acetate (PMA) increased peptide cleavage in a TACE-dependent manner. PMA treatment did not increase the amount of TACE on the cell surface. Moreover, the cytoplasmic domain of TACE was not required for the induced activity. Based on these observations, induction of TACE-mediated shedding events occurs at least in part via an increase in the enzymatic activity of cellular TACE, independent of its cytoplasmic domain.

Metaloendopeptidases/metabolismo , Proteínas ADAM , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Metaloendopeptidases/química , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Transdução Genética