STRUCTURE/FUNCTION OF THE HUMAN PON1 POLYMORPHISM

人血清对氧磷酶（PON1）是一种高密度脂蛋白（HDL）相关酶。除了脂质代谢外，它还在许多有机磷（OP）杀虫剂的解毒中起主要作用。PON1在对硫磷、毒死蜱和二嗪农以及神经毒剂索曼和沙林的毒性代谢物水解中表现出底物依赖性多态性。现有的证据支持这样的假设，即个体的PON1状态在通过细胞色素P450/PON1途径处理的有机磷酯（OPs）解毒过程中很重要。多态性的基础是PON1在192位的一个氨基酸取代。Arg192指定了一种具有高抗对氧磷活性的异构体，而Gln192指定了一种具有低抗对氧磷活性的异构体。对重氮氮农、索曼和沙林的水解，多态性的影响是相反的，从而预测对对硫磷耐药的个体对重氮氮农、索曼和沙林敏感，而对硫磷敏感的个体对重氮氮农、索曼和沙林的抗性更强。除了活性多态性外，个体之间酶表达水平的差异很大，且随时间稳定。基因型和表型的结合被称为PON1状态。本研究的目的是：1)确定活性多态性的分子结构基础；2)完善小鼠模型，研究体内PON1多态性的功能基因组方面；3)确定导致个体间PON1表达水平差异很大的基因表达调控机制，这在发育中也很重要。为了研究活性多态性的分子基础，我们将测定Arg192和Gln192 PON1亚型的晶体结构。纯化的和重组的PON1都将用于这些研究。为了改进小鼠模型，我们将使用敲除PON1的小鼠，其中一些小鼠将被给予纯化的人PON1 （Gln192或Arg192）。此外，我们将利用表达人Arg192或Gln192同种异构体的转基因小鼠作为半合子、纯合子和杂合子。几种OP化合物对脑、隔膜和血液乙酰胆碱酯酶的影响将作为模拟人类PON1状态小鼠的急性毒性指标进行评估。为了研究在人群中观察到的酶水平的巨大个体差异的分子基础，上游和下游的候选调节序列将用于控制表达对氧磷酶信息的肝细胞系（HepG2）中报告基因荧光素酶的表达。来自表达非常高酶水平的个体的序列磁带将与来自表达非常低酶水平的个体的序列磁带进行比较，以确定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.