STRUCTURE/FUNCTION OF THE HUMAN PON1 POLYMORPHISM

人血清对氧磷酶（PON 1）是一种高密度脂蛋白（HDL）相关酶。 它除了在脂质代谢中起作用外，还在许多有机磷（OP）杀虫剂的解毒中起重要作用。 PON 1在水解敌百虫、毒死蜱、二嗪农以及神经毒剂梭曼和沙林等有毒代谢物方面表现出底物依赖的多态性。 现有的证据支持这一假设，即一个人的PON 1状态是重要的解毒有机磷酯（OP）处理通过细胞色素P450/PON 1途径。 多态性的基础是PON 1第192位的氨基酸取代。 Arg 192指定对氧磷具有高活性的同种型，而Gln 192指定对氧磷具有低活性的同种型。 多态性的影响是逆转二嗪农，梭曼和沙林的水解，并导致预测的个人耐二嗪农，梭曼和沙林敏感，而个人敏感的二嗪农，梭曼和沙林将更耐二嗪农。 除了活性多态性之外，个体之间的酶表达水平存在很大的变异性，并且随时间推移而稳定。 基因型和表型的组合被称为PON 1状态。 拟议研究的目标是：1）确定活性多态性的分子结构基础; 2）完善用于研究体内PON 1多态性的功能基因组方面的小鼠模型; 3）确定导致个体之间PON 1表达水平差异很大的基因表达调节机制，这在发育中也很重要。 为了检查活性多态性的分子基础，将测定Arg 192和Gln 192 PON 1同种型的晶体结构。 纯化的和重组的PON 1都将用于这些研究。 为了完善小鼠模型，我们将利用PON 1敲除小鼠，其中一些将给予纯化的人PON 1（Gln 192或Arg 192）。 此外，我们将利用表达人Arg 192或Gln 192同种型的转基因小鼠作为半合子、纯合子和杂合子。 将评估几种OP化合物对脑、膈肌和血液乙酰胆碱酯酶的影响，作为模拟人PON 1状态小鼠的急性毒性指标。 为了检查在人群中观察到的酶水平的较大个体变异性的分子基础，上游和下游候选调控序列将用于控制表达对氧磷酶信息的肝细胞系（HepG 2）中报告基因荧光素酶的表达。 将来自表达非常高酶水平的个体的序列盒与来自表达非常低酶水平的个体的序列盒比较它们调节报告基因表达的能力。

Human serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme. It plays a major role in the detoxication of a number of organophosphate (OP) insecticides in addition to its role in lipid metabolism. PON1 exhibits a substrate dependent polymorphism for the hydrolysis of the toxic metabolites of parathion, chlorpyrifos and diazinon as well as the nerve agents soman and sarin. The available evidence supports the hypothesis that an individual's PON1 status is important in the detoxication of organophosphorus esters (OPs) processed through the cytochrome P450/PON1 pathway. The basis of the polymorphism is an amino acid substitution in PON1 at position 192. Arg192 specifies an isoform with high activity against paraoxon while Gln192 specifies an isoform with low activity against paraoxon. The effect of the polymorphism is reversed for the hydrolysis of diazinon, soman and sarin and leads to the prediction that individuals resistant to parathion will be sensitive to diazinon, soman and sarin, whereas individuals sensitive to parathion will be more resistant to diazinon, soman and sarin. In addition to the activity polymorphism, there is a large variability in the level of enzyme expression between individuals that is stable over time. The combination of genotype and phenotype is referred to as PON1 status. The goals of the proposed research are to: 1) determine the molecular structural basis of the activity polymorphism; 2) refine the mouse model for investigating the functional genomic aspects of the PON1 polymorphism in vivo; and 3) determine the mechanism of regulation of gene expression that results in widely different levels of expression of PON1 between individuals, and is also important in development. To examine the molecular basis of the activity polymorphism, the crystal structure of the Arg192 and Gln192 PON1 isoforms will be determined. Both purified and recombinant PON1 will be used in these studies. For the refinement of the mouse model, we will utilize the PON1 knockout mice, some of which will be given purified human PON1 (Gln192 or Arg192). In addition, we will utilize transgenic mice that express the human Arg192 Or Gln192 isoform as hemizygotes, homozygotes and heterozygotes. The effects of several OP compounds on brain, diaphragm and blood acetylcholinesterases will be assessed as an index of acute toxicity with mice of simulated human PON1 status. To examine the molecular basis of the large individual variability of enzyme levels observed in human populations, the upstream and downstream candidate regulatory sequences will be used to control the expression of the reporter gene luciferase in a liver cell line (HepG2) that expresses paraoxonase message. Sequence cassettes from individuals expressing very high enzyme levels will be compared with those from individuals expressing very low enzyme levels for their ability to regulate the expression of the reporter gene.