Molecular Toxicology of Carboxylesterases

性状（由申请方提供）：羧酸酯酶是一大类水解酶，在内源性脂质代谢、治疗剂药理学测定和有机磷杀虫剂解毒中发挥重要作用。目前资助期的重点是分子克隆、酶动力学和外源性调节。酶的特性和分子模拟表明，羧酸酯酶含有几个功能亚位点（结构），可能发挥决定性作用的底物识别和抑制剂的反应。具有相同功能亚位点的羧酸酯酶表现出相同的底物偏好性和对丝氨酸酶抑制剂的相似敏感性。外源性调节的研究表明，地塞米松抑制大鼠水解酶A、B/C和S的表达，而人HCE-1和HCE-2的表达由该药物诱导。抑制只需要纳摩尔，而诱导需要微摩尔水平。拟议的研究旨在测试假设，假定形成羧酸酯酶之间的功能亚位点的非保守残基显着贡献的底物偏好上观察到的差异，地塞米松定向抑制大鼠羧酸酯酶是由糖皮质激素受体介导的，而人类羧酸酯酶的诱导介导的甾烷X受体。拟议研究的具体目的是：（1）表征决定底物和抑制剂选择性的功能亚位点;（2）阐明地塞米松种属依赖性调节的分子基础。将进行定点诱变以替换单个残基或整个推定亚位点，并将检测突变体对结构不同底物的水解活性改变。将对细胞增殖和毒性进行研究，以确定催化特性的变化是否转化为生物功能的实际扰动。为了指定参与地塞米松的物种依赖性调节的受体，将使用抗糖皮质激素和显性调节剂选择性阻断或改变任一途径，并确定每种受体在调节羧酸酯酶表达中的作用。建立的重要性，底物识别的亚位点将提供信息，以阐明羧酸酯酶的催化作用，并确定异构体为基础的药理学和毒理学的相关性。参与地塞米松介导的调节的受体的规范将提供分子机制，以预测潜在的药物相互作用，作为改变羧酸酯酶的表达的结果。

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.