Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells
| Autor: | MA Pedro, Lucia Rossetti Lopes, Alipio O. Carmo, Célio X.C. Santos, Ralf P. Brandes, Mariano Janiszewski, Francisco R.M. Laurindo |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
inorganic chemicals
Protein Folding Isomerase activity Recombinant Fusion Proteins Blotting Western Green Fluorescent Proteins Protein Disulfide-Isomerases Gene Expression Biology Transfection Biochemistry Muscle Smooth Vascular Cell Line chemistry.chemical_compound Bacitracin Superoxides Oxidoreductase Animals Homeostasis Humans Enzyme Inhibitors Protein disulfide-isomerase Molecular Biology Aorta Cell Line Transformed chemistry.chemical_classification Oxidase test Binding Sites Microscopy Confocal Superoxide Angiotensin II Cell Membrane NADPH Oxidases Cell Biology Oligonucleotides Antisense Molecular biology Enzyme Activation Protein Subunits chemistry NAD(P)H oxidase NOX1 Luminescent Measurements cardiovascular system Acridines Rabbits NAD+ kinase Oxidation-Reduction |
| Zdroj: | Journal of Biological Chemistry. 280:40813-40819 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m509255200 |
| Popis: | NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling. |
| Databáze: | OpenAIRE |
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