Mechanistic insights into cellular metal detoxification

描述(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。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，镉是一种剧毒的环境污染物，与肾衰竭、癌症、生殖缺陷和内分泌紊乱等疾病有关。虽然接触镉是不可避免的和普遍的，但镉代谢的细胞机制，特别是镉的排泄系统，在很大程度上是未知的。该项目的长期目标是描述镉解毒的分子机制，并利用这一知识减少镉的摄入量。在寻找真核生物酿酒酵母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ATPase，该酶在镉敏感的酵母菌株中不起作用。几乎所有的生物都依赖于这个转运蛋白家族来维持各种离子的跨膜梯度，这对于营养吸收、神经传递、信号传递和/或防止离子的有毒积累至关重要。铜转运蛋白P型ATPase基因突变导致人类致死性遗传病，突显了P型ATPase在金属代谢中的重要作用。然而，金属转运P型ATPase的机制细节仍有待阐明。本应用重点研究了镉转运P型ATPase的功能、作用机制和调控。中心假设是这种P型ATPase是第一个镉特异性外排泵，在结构、底物特异性和调节模式上都是独一无二的。这一假设将使用多学科方法进行检验。首先，将阐明P-型ATPase的镉专一性，并确定该专一性的结构决定因素。这项研究将主要集中在金属结合结构域和残基的ATPase分析和结构功能分析上。其次，酵母遗传学、细胞生物学和生物物理方法将确定参与镉依赖的镉外排泵表达控制的独特模式中的反式作用调节因子和顺式作用元件。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ATPase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。与公众健康相关：镉是一种剧毒金属，与肾脏疾病、癌症、生殖缺陷和内分泌紊乱有关。鉴于镉对人体的暴露是不可避免的和普遍的，对细胞对镉的吸收、隔离和排出的机械性见解将有助于预防和治疗与镉相关的疾病，并帮助科学家开发减少人体镉摄入量的方法。这项应用通过鉴定和表征细胞镉外流机制来解决这个问题，该机制在防止镉的过度积累方面发挥着关键作用。

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. PUBLIC HEALTH RELEVANCE: Cadmium is a highly toxic metal that is implicated in kidney disease, cancer, reproductive defects, and endocrine disruption. Given that cadmium exposure to humans is unavoidable and widespread, mechanistic insights into cellular cadmium absorption, sequestration, and extrusion would facilitate prevention and treatment of cadmium-related disorders and help scientists develop methods for the reduction of cadmium intake in humans. This application addresses this problem through the identification and characterization of a cellular cadmium efflux mechanism that plays a critical role in preventing excess accumulation of cadmium.