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• W2748441281 abstract "Introduction: The ubiquitin-proteasome pathway is responsible for the majority of cell cycle protein degradation and is centrally involved in regulating cell proliferation. Ubiquitin activating-, conjugating-, and ligase enzymes attach a poly-ubiquitin chain to proteins which can be identified and degraded by the 26S proteasome. Alternatively, the poly-ubiquitin chain can be removed and de-ubiquinated by one of several enzymes, such as isopeptidase T. This enzyme hydrolyzes isopeptide bonds in unanchored poly-ubiquitin chains, which results in recycling of ubiquitin monomers. A buildup of poly-ubiquitin chains results in inhibition of the 26S proteasome. We previously showed that nitric oxide (NO) directly inhibits the catalytic activity of the 26S proteasome. Our hypothesis was that NO also affects the activity of isopeptidase T. The purpose of this study was two-fold: 1) to evaluate the effect of NO on the activity of isopeptidase T; and 2) to determine the effect of NO on protein expression and localization of isopeptidase T in vascular smooth muscle cells (VSMC). Methods: An activity assay was established to determine the functional activity of isopeptidase T under various conditions. Reaction buffer containing ethylene diamine tetra-acetic acid (EDTA) was added to recombinant isopeptidase T, +/- increasing concentrations of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP). To this reaction, the fluorogenic peptide-substrate ubiquitin-AMC was added. Isopeptidase T activity was measured using a fluorescent microplate reader. Additionally, protein expression and intracellular localization of isopeptidase T in mouse aortic VSMC was determined using Western blot analysis and immunofluorescence staining, respectively. Results: The activity of isopeptidase T (2nM) was inhibited by SNAP (125-1000uM) in a time- and concentration-dependent manner. At baseline, isopeptidase T activity was 1001 +/- 16, however with the addition of SNAP activity was inhibited by 83% (172 +/- 10, P<0.05). With the addition of DTT (5mM), a reducing agent, there was significant reversal of the SNAP-mediated inhibition of activity (838 +/- 8, P<0.05). Pre-treatment with glutathione, a sulfhydryl-specific reducing agent, also prevented much of the SNAP-mediated inhibition of isopeptidase T activity (822 +/- 30, P<0.05). Western blot analysis of VSMC treated with SNAP (125-1000uM) for 24 hours showed no difference in protein expression of isopeptidase T versus control. Immunofluorescence staining for isopeptidase T revealed both nuclear and cytoplasmic localization. NO exposure did not alter the intracellular localization of the enzyme. Conclusions: Nitric oxide inhibits the activity of isopeptidase T, and this is likely secondary to modification of a critical cysteine residue. This inhibition of isopeptidase T by NO is yet another mechanism through which NO inhibits the 26S proteasome. These data provide further insight into the effect of NO on the ubiquitin-proteasome pathway and may provide one mechanism by which NO affects cellular proliferation of VSMC." @default.
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• W2748441281 date "2007-02-01" @default.
• W2748441281 modified "2023-10-14" @default.
• W2748441281 title "58" @default.
• W2748441281 doi "https://doi.org/10.1016/j.jss.2006.12.069" @default.
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