Mechanistic insights into cellular metal detoxification

描述（由申请人提供）：非生理金属的去铁化和营养但有毒金属的稳态获取是基本的生物过程。许多与重金属毒性有关的健康问题强调了金属代谢的生理意义。例如，镉是一种高毒性的环境污染物，并与包括肾衰竭、癌症、生殖缺陷和内分泌干扰在内的疾病有关。虽然接触镉是不可避免的和广泛的，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。该项目的长期目标是表征镉解毒的分子机制，并利用这些知识减少镉的摄入量。在酵母酿酒酵母（一种模式真核生物）中寻找与金属抗性相关的基因期间，PI鉴定了一种新型镉挤压P型ATP酶，其在镉敏感酵母菌株中无功能。几乎所有的生物体都依赖于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性疾病，这突出了P型ATP酶在金属代谢中的重要作用。然而，金属转运P型ATP酶的机制细节仍有待阐明。这种应用程序的重点是表征的功能，作用机制，和镉转运P型ATP酶的调节。核心假设是，这种P型ATP酶是第一个镉特异性外排泵，其结构、底物特异性和调节模式都是独特的。这一假设将使用多学科方法进行检验。首先，镉特异性的P-型ATP酶将被阐明，并将确定特异性的结构决定因素。本研究将主要集中在ATP酶测定和金属结合结构域和残基的结构-功能分析。其次，酵母遗传学，细胞生物学和生物物理学的方法将确定反式作用的调节因子和顺式作用元件参与的镉依赖性表达控制的镉外排泵的独特模式。这些研究有望揭示真核生物中一种新的镉解毒系统，阐明P型ATP酶介导的金属转运机制，并最终提高对抗人类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.