Mechanisms of arsenical transport

说明(申请人提供)：砷从地质和人为两个来源进入人体。由于砷在环境中的普遍存在，每个生物体都发展了砷的外流和解毒的运输系统。长期接触砷与心血管和外周血管疾病、神经疾病、糖尿病和各种癌症有关。含砷药物被用作治疗白血病和寄生虫病的化疗药物。对砷化学和砷转运系统的分子细节的了解对于缓解砷毒性问题以及合理设计治疗耐药微生物和癌细胞的药物至关重要。我们已经确定水甘油卟啉是生物体从大肠杆菌到人类摄取三价砷[As(111)]的主要途径。我们还表明，在酵母和人类中，As(111)是通过己糖渗透膜运输的。这项建议的总体目标是阐明砷在微生物中运输的分子机制。到目前为止测序的每一种生物的基因组中都存在抗砷基因，这首先表明抗砷基因一定是古老的，其次表明砷肯定仍然存在于环境中，提供了维持它们在现代生物体中的选择压力。第一个具体目标是研究两种细菌(E.Coli和Sinorhizobium Meliloti)以及真核微生物酿酒酵母对三价砷的吸收机制。特别是，将确定允许砷运输的底物选择性的机制。第二个具体目标是详细分析ArsAB As(111)转位ATPase的结构和功能，该酶是最具特性的三价芳烃解毒系统。这个砷挤出泵由临床分离的耐药质粒R773的砷耐药操纵子编码。该项目需要对ARSA ATPase的核苷酸结合结构域、金属结合部位和信号转导结构域进行详细的分子分析，以及ARSA与泵的膜组件ARSB相互作用的方式。最后，我们将研究新型砷伴侣蛋白ARSD与ArsAB泵的相互作用。

DESCRIPTION (provided by applicant): Arsenic enters human body from both geological and anthropogenic sources. Because of the ubiquity of arsenic in the environment, every organism has developed transport systems for the efflux and detoxification of arsenic. Chronic exposure to arsenic has been linked to cardiovascular and peripheral vascular diseases, neurological disorders, diabetes and various cancers. Arsenic-containing drugs are used as chemotherapeutic agents for the treatment of leukemia and parasitic diseases. An understanding of both arsenic chemistry and the molecular details of arsenic transport systems is essential for alleviating the problems of arsenic toxicity, as well as for the rational design of drugs to treat drug-resistant microbes and cancer cells. We have identified aquaglyceroporins as a major pathway for trivalent arsenical [As(lll)] uptake in organisms from E. coli to humans. We have also shown that As(lll) is transported by hexose permeases in yeast and humans. The overall goal of this proposal is elucidation of the molecular mechanisms of arsenic transport in microorganisms. The presence of arsenic resistance (ars) genes in the genome of every living organism sequenced to date illustrates first that ars genes must be ancient and second that arsenic must still be ubiquitous in the environment, providing the selective pressure that maintains them in present-day organisms. The first specific aim proposes to study the mechanisms of uptake of trivalent arsenic by aquaglyceroporins and hexose permeases in two bacteria (E. coli and Sinorhizobium meliloti), and, for comparative purposes, in the eukaryotic microorganism Saccharomyces cerevisiae. In particular, the mechanism of substrate selectivity that allows arsenic transport will be determined. The second specific aim is a detailed structure-function analysis of the ArsAB As(lll)-translocating ATPase, the best-characterized detoxification system for trivalent arenic. This arsenic extrusion pump is encoded by the arsenical resistance operon of the clinically isolated resistance plasmid R773. The project entails detailed molecular analysis of the nucleotide binding domains, metal binding sites and signal transduction domains of the ArsA ATPase, as well as the way in which ArsA interacts with ArsB, the membrane component of the pump. Finally, the interaction of the new and novel arsenic chaperone protein, ArsD, with the ArsAB pump will be studied.