Molecular Toxicology of Carboxylesterases

描述（由申请人提供）：羧酸酯酶是一大类水解酶，在内源性脂质代谢、治疗药物的药理学测定和有机磷杀虫剂的解毒中发挥重要作用。目前资助期的重点是分子克隆、酶动力学和异种生物调控。酶学表征和分子模型表明，羧酸酯酶含有几个功能亚位点（结构），可能在底物识别和抑制剂反应性中起决定性作用。具有相同功能亚位的羧酸酯酶表现出相同的底物偏好和对丝氨酸酶抑制剂的相似敏感性。外源调控研究表明，地塞米松抑制大鼠水解酶A、B/C和S的表达，而诱导人HCE-1和HCE-2的表达。抑制只需要纳摩尔水平，而诱导需要微摩尔水平。本研究旨在验证以下假设：在羧酸酯酶之间形成功能亚位的非保守残基对观察到的底物偏好差异有重要影响；地塞米松对大鼠羧酸酯酶的抑制是由糖皮质激素受体介导的，而对人类羧酸酯酶的诱导是由妊娠X受体介导的。拟议研究的具体目的是：(1)表征确定底物和抑制剂选择性的功能亚位；(2)阐明地塞米松对物种依赖性调控的分子基础。位点直接诱变将用于替换单个残基或整个假定的亚位点，并且将测试突变体对结构不同底物的水解活性的改变。将对细胞增殖和毒性进行研究，以确定催化性质的变化是否会转化为生物功能的实际扰动。为了明确参与地塞米松物种依赖性调节的受体，将使用抗糖皮质激素和优势调节因子选择性地阻断或改变其中一种途径，并确定每种受体在调节羧酸酯酶表达中的作用。建立亚位对底物识别的重要性将为阐明羧酸酯酶的催化作用和确定基于异构体的药理学和毒理学相关性提供信息。指定参与地塞米松介导的调节的受体将提供分子机制来预测由于羧酸酯酶表达改变而导致的潜在药物-药物相互作用。

DESCRIPTION (provided by applicant): 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.