Mechanistic insights into cellular metal detoxification

描述（由申请人提供）：非生理性金属的解毒和营养但有毒金属的稳态获取是基本的生物过程。与重金属中毒有关的一些健康问题强调了金属代谢的生理意义。例如，镉是一种剧毒的环境污染物，与肾功能衰竭、癌症、生殖缺陷和内分泌紊乱等疾病有关。虽然镉暴露是不可避免的和普遍的，但镉代谢的细胞机制，特别是镉排泄系统，在很大程度上是未知的。本项目的长期目标是研究镉解毒的分子机制，并利用这些知识来减少镉的摄入。在寻找酵母（一种真核生物模型）中与金属抗性相关的基因时，PI发现了一种新的镉挤出p型atp酶，该酶在镉敏感酵母菌株中不起作用。几乎所有的生物体都依赖这个转运蛋白家族来维持各种离子的跨膜梯度，这对于营养摄取、神经传递、信号传导和/或防止离子的毒性积累至关重要。铜转运p型atp酶的突变导致人类致命的遗传疾病，这突出了p型atp酶在金属代谢中的重要作用。然而，金属运输p型atp酶的机制细节仍有待阐明。本应用着重于镉转运p型atp酶的功能、作用机制和调控的表征。核心假设是，这种p型atp酶是第一个具有独特结构、底物特异性和调节模式的镉特异性外排泵。这一假设将采用多学科方法进行检验。首先，将阐明p型atp酶的镉特异性，并确定特异性的结构决定因素。本研究将主要集中在atp酶分析和金属结合结构域和残基的结构功能分析上。其次，酵母遗传学、细胞生物学和生物物理学方法将确定参与镉依赖表达控制的镉外排泵独特模式的反式调节因子和顺式作用元件。这些研究有望揭示真核生物中新的镉解毒系统，揭示p型atp酶介导的金属转运机制，并最终提高人类对抗金属相关疾病的能力。

DESCRIPTION (provided by applicant): Detoxification of non-physiological metals and homeostatic acquisition of nutritional yet toxic metals are fundamental biological processes. A number of health issues linked to heavy metal toxicity underscore the physiological significance of metal metabolism. For example, cadmium is a highly toxic environmental contaminant and implicated in disorders, including kidney failure, cancer, reproductive defects, and endocrine disruption. While exposure to cadmium is unavoidable and widespread, the cellular mechanisms of cadmium metabolism, especially cadmium excretion systems, are largely unknown. The long-term goals of this project are the characterization of molecular mechanisms of cadmium detoxification and employing this knowledge to reduce cadmium intake. During the search for genes involved in metal resistance in yeast Saccharomyces cerevisiae, a model eukaryote, the PI identified a novel cadmium extruding P-type ATPase that is non- functional in cadmium sensitive yeast strains. Virtually all organisms rely on this family of transporters for maintaining a transmembrane gradient of various ions, which is vital for nutrient uptake, neurotransmission, signaling, and/or prevention of toxic accumulation of ions. Mutations in copper transporting P-type ATPases lead to lethal genetic diseases in humans, which highlights the essential role for P-type ATPases in metal metabolism. However, mechanistic details of metal-transporting P-type ATPases remain to be elucidated. This application focuses on the characterization of the function, mechanisms of action, and regulation of a cadmium transporting P-type ATPase. The central hypothesis is that this P-type ATPase is the first cadmium-specific efflux pump that is unique in structure, substrate specificity, and mode of regulation. This hypothesis will be tested using a multi-disciplinary approach. First, cadmium specificity of the P-type ATPase will be elucidated, and structural determinants of the specificity will be identified. This study will largely focus on ATPase assays and structure-function analysis of metal-binding domains and residues. Second, yeast genetics, cell biology, and biophysical approaches will identify trans-acting regulatory factors and cis-acting elements involved in the unique mode of cadmium-dependent expression control of this cadmium efflux pump. The proposed studies are expected to reveal a novel cadmium detoxification system in a eukaryote, shed light on the mechanism of P-type ATPase-mediated metal transport, and ultimately advance the ability to combat metal-related disorders in humans.