The overall objective is to clarify poorly understood aspects of 3- hydroxyisobutyrate formation and utilization. The work will focus on 3- hydroxyisobutyryl-CoA hydrolase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. It is proposed that the hydrolase responsible for 3-hydroxyisobutyrate formation has specificity for thiolytic cleavage of 3-hydroxyiso-butyryl-CoA at physiological concentrations at which CoA esters of metabolic pathways occur, that the hydrolase plays an important role in preventing the accumulation of potentially cytotoxic, mutagenic and clastogenic concentrations of 3- methacrylyl-CoA and acrylyl-CoA, that 3-hydroxyisobutyryl-CoA hydrolase also functions to prevent coenzyme A sequestration by drugs and in genetic/metabolic disorders, that the brain contains a broad-specificity, short chain acyl-CoA hydrolase for unique protection of this tissue against CoA sequestration, that colonic epithelial cells must have a very active catabolic pathway to protect against toxicity of isobutyrate produced by large bowel bacteria, and that both short-term (inhibition by acylation) and long-term (regulation of expression) mechanisms regulate the activity of methylmalonate semialdehyde dehydrogenase. The detailed specific aims are: (a) to purify and clone 3-hydroxyisobutyryl-CoA hydrolase; (b) to determine substrate specificity and tissue distribution of 3-hydroxyisobutyryl-CoA hydrolase and whether it (or enzymes of the same family) is responsible for protection against cytotoxic effects of methacrylyl-CoA as well as CoA sequestration by various drugs and blockage of metabolic pathways; (c) to purify and clone the broad-specificity short chain acyl-CoA hydrolase present in brain tissue; (d) to establish the metabolic fate of the isobutyrate produced by colonic bacteria; and (e) to establish the mechanisms responsible for short-term regulation of methylmalonate semialdehyde dehydrogenase activity and long-term regulation of methylmalonate semialdehyde dehydrogenase expression. The work on acyl-CoA hydrolase(s) is relevant to the teratogenic/clastogenic response to acrylate/methacrylate compounds and to elucidation of the cellular defense mechanisms against CoA sequestration caused by foreign compounds (ibuprofen, valproate, clofibrate) and genetic/metabolic disorders (methylmalonyl mutase deficiency, vitamin B12 deficiency, biotin deficiency). Overall the work will clarify poorly understood features of the valine, thymine, and beta-alanine catabolic pathways
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