Structure and Function of the Human PON1 Polymorphism

描述（由申请人提供）：拟议研究的主要目的是了解人类PON1基因遗传变异的生理后果，并确定导致个体血浆PON1水平巨大差异的因素。大肠杆菌PON1表达和纯化方案的建立为表征序列变异对PON1催化效率的影响提供了快速手段，同时为毒理学实验和结构/功能研究提供了充足的重组PON1蛋白。证明重组人PON1治疗急性OP暴露病例的治疗潜力，为拟议研究的具体目标1和2提供了动力。本申请中提出的研究有5个具体目标。Specific Aim 1的目标是改进在大肠杆菌表达系统中产生天然和变异人类pon1的表达和纯化方案。特异性目标2的目标是产生具有足够催化效率的PON1变体，以防止暴露于三甲酰基磷酸和对氧磷。特异性目标3的目标是鉴定和验证有机磷（OP）化合物暴露的生物标志物。目前迫切需要暴露的生物标志物，以延长暴露分析的时间框架，而不是测量尿液或血液代谢物提供的几天窗口。特异性目标4解决了母体PON1状态在保护胎儿免受妊娠期OP（重氮唑松）暴露中的重要性。迄今为止产生的数据表明，PON1状态在确定对重氮嗪/重氮唑和毒死蜱/毒死蜱的敏感性方面很重要。特异性目的5研究启动子区域表观遗传甲基化对PON1血浆水平的影响。迄今为止，我们的研究已经发现了显著影响血浆PON1水平的启动子多态性。对大约200个新多态性的鉴定表明，只有30%的血浆PON1水平变异性似乎依赖于启动子区多态性。对PON1启动子区域CpGs甲基化的初步检查揭示了可变性，这可能解释了调控的另一个重要来源。来自转基因PON1小鼠系谱的数据与这种额外的调控水平一致。公共卫生相关性：本项目产生的数据已协助环保署就减少二嗪农和毒死蜱接触进行谈判。PON1状态分析已经确定低PON1状态是颈动脉疾病的危险因素，并且还确定了男性帕金森患者和对照组之间的差异。工程重组人PON1将为治疗神经毒剂和杀虫剂暴露提供治疗方法。

DESCRIPTION (provided by applicant): A major objective of the proposed research is to understand the physiological consequences of genetic variability in the human PON1 gene and to identify factors that contribute to the large differences in plasma PON1 levels among individuals. Development of E. coli PON1 expression and purification protocols provides a rapid means for characterizing the effects of sequence variants on the catalytic efficiency of PON1 and at the same time provides sufficient recombinant PON1 protein for toxicology experiments and structure/function studies. Demonstration of the therapeutic potential of recombinant human PON1 for treating cases of acute OP exposure has provided the impetus for Specific Aims 1 and 2 of the proposed research. The research proposed in this application has 5 specific aims. The goal of Specific Aim 1 is to improve the expression and purification protocols for generating native and variant human PON1s in the E. coli expression system. The goal of Specific Aim 2 is to generate PON1 variants that have sufficient catalytic efficiency to protect against exposures to tricresyl phosphate and paraoxon. The goal of Specific Aim 3 is to identify and validate biomarkers for exposure to organophosphorus (OP) compounds. There is a pressing need for biomarkers of exposure that extend the time-frame of exposure analyses beyond the few day window that measuring urinary or blood metabolites provides. Specific Aim 4 addresses the importance of maternal PON1 status in protecting the fetus from OP (diazoxon) exposure during gestation. The data generated to date indicate that PON1 status is important in determining sensitivity to diazinon/diazoxon and chlorpyrifos/chlorpyrifos oxon. Specific Aim 5 examines the influence of promoter region epigenetic methylation on PON1 plasma levels. Our research to date has characterized promoter polymorphisms that significantly influence plasma PON1 levels. Identification of approximately 200 new polymorphisms has shown that only 30% of the variability of plasma PON1 levels appears to be dependent on promoter-region polymorphisms. Preliminary examination of methylation of CpGs in the PON1 promoter region revealed variability that may explain another significant source of regulation. Data from transgenic PON1 mouse pedigrees are consistent with this additional level of regulation. PUBLIC HEALTH RELEVANCE: Data generated from this project has assisted the EPA in negotiating a reduction in diazinon and chlorpyrifos exposure. The PON1 status analyses have identified low PON1 status as a risk factor for carotid artery disease and have also identified differences between male Parkinson's patients and controls. Engineering recombinant human PON1 will provide therapeutics for treating nerve agent and insecticide exposures.