Structure and Function of the Human PON1 Polymorphism

描述(由申请人提供)：拟议研究的一个主要目标是了解人类PON1基因遗传变异的生理后果，并确定导致个体间血浆PON1水平差异较大的因素。E.ColiPON1表达和纯化方案的建立为快速表征序列变异对PON1催化效率的影响提供了一种手段，同时也为毒理学实验和结构/功能研究提供了充足的重组PON1蛋白。证明了重组人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。具体目标1的目的是改进在大肠杆菌表达系统中产生天然和变异型人PON1的表达和纯化方案。具体目标2的目标是产生具有足够催化效率的PON1变体，以防止接触三甲酚和对氧磷。具体目标3的目标是识别和验证接触有机磷(OP)化合物的生物标志物。迫切需要接触生物标志物，将接触分析的时间范围延长到测量尿液或血液代谢物提供的几天窗口之外。具体目标4阐述了母亲PON1状态在怀孕期间保护胎儿免受OP(重氮酮)暴露的重要性。迄今为止产生的数据表明，PON1的状态在确定对二氮磷/重氮磷和毒死蜱/毒死蜱的敏感性方面很重要。特异性目的5检测启动子区表观遗传甲基化对PON1血浆水平的影响。到目前为止，我们的研究已经确定了显著影响血浆PON1水平的启动子多态性。对大约200个新的多态的鉴定表明，血浆PON1水平的变异性中只有30%似乎依赖于启动子区域的多态。对PON1启动子区域CPGS甲基化的初步检查发现了可变性，这可能解释了另一个重要的调控来源。来自转基因PON1小鼠家系的数据与这一额外水平的调控是一致的。公共卫生相关性：该项目产生的数据帮助环境保护局就减少二氮磷和毒死磷暴露进行谈判。PON1状态分析已经确定低PON1状态是颈动脉疾病的风险因素，并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药物。

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.