Carboxylesterases represent a large class of hydrolytic enzymes that play important roles in the metabolism of endogenous lipids, pharmacological determination of therapeutic agents and detoxication of organophosphorus insecticides. The focus of the current grant period has been on molecular cloning, enzyme kinetics and xenobiotic regulation. Enzymatic characterization and molecular modeling have revealed that carboxylesterases contain several functional subsites (structures) that likely play determinant roles in substrate recognition and inhibitor reactivity. Carboxylesterases, with the same functional subsites exhibit the same substrate preference and a similar sensitivity to serine enzyme inhibitors. Studies on xenobiotic regulation have demonstrated that dexamethasone suppresses the expression of rat hydrolase A, B/C and S, whereas the expression of human HCE-1 and HCE-2 is induced by this drug. Suppression requires only nanomolar whereas induction requires micromolar levels. The proposed studies are designed to test the hypotheses that non-conserved residues assumed to form functional subsites among carboxylesterases contribute significantly to the observed differences on the substrate preference and that the dexamethasone-directed suppression of rat carboxylesterases is mediated by the glucocorticoid receptor whereas the induction of human carboxylesterases is mediated by the pregnane X receptor.
The specific aims of the proposed studies are: (1) to characterize functional subsites determining substrate and inhibitor selectivity; and (2) to elucidate molecular basis for species-dependent regulation by dexamethasone. Site-direct mutagenesis will be performed to replace a single residue or an entire putative subsite, and the mutants will be tested for the altered hydrolytic activity toward structurally dissimilar substrates. Studies on cell proliferation and toxicity will be performed to determine whether changes in catalytic properties translate into actual perturbations in biological functions. In order to specify the receptor involved in the species-dependent regulation by dexamethasone, antiglucocorticoids and dominant regulators will be used to selectively block or alter either pathway and the role of each receptor will be established in regulating carboxylesterase expression. Establishment of the importance of the subsites for substrate recognition will provide information to elucidate the catalytic action of carboxylesterases and to ascertain isoform-based pharmacological and toxicological relevance. Specification of a receptor involved in dexamethasone-mediated regulation will provide molecular mechanisms to predict potential drug-drug interactions as a result of altered expression of carboxylesterases. ? ? ?
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