REGULATION OF CELLULAR ZINC HOMEOSTASIS

描述：(摘自申请者的摘要)锌是一种必需的营养素 对所有生物体都适用，但如果累积过多可能是有毒的。因此， 随着细胞外锌浓度的变化，细胞必须保持足够的 细胞内锌的供应，以满足代谢需求，同时防止其 过度积累。这项研究的长期目标是了解 真核细胞锌稳态的分子机制。这 这一过程涉及锌结合蛋白和锌离子之间复杂的相互作用。 螯合剂，以及控制这些活动的调节机制。尽管 锌对人类健康的重要性，我们对锌的动态平衡知之甚少。 这项建议描述了一种结合了遗传、分子、生化和 锌和锌转运蛋白研究的生物物理方法。他们的 锌反应蛋白Zap1在缺锌时的诱导表达 转录激活蛋白。ZAP1被认为是细胞内的 锌传感器及其活性受锌(II)的控制 直接与蛋白质结合以控制其DNA结合活性。一秒钟 建议研究的中心假设是Zap1靶基因在 此外，ZRT1和ZRT2在锌的动态平衡中起着关键作用。为了测试 这些假设，提出了以下具体目的：a)确定在 锌对Zap1 DNA结合的体内影响，b)识别潜在的附件 调节ZAP1活性所需的因素，c)用于体内遗传分析 为了确定Zap1中锌反应调节所需的残基， D)建立体外系统研究锌(II)、e)MAP和 在体外鉴定Zap1锌反应结构域中的金属结合部位， F)表征Zap1锌反应中锌离子诱导的分子开关 结构域，g)定义ZRc1、Cot1和Zrt3转运蛋白在液泡中的作用 锌的储存和解毒，以及h)表征新的转运蛋白 负责细胞内锌的转运和外流。这些研究应该 更深入地了解锌的动态平衡过程 真核生物。

DESCRIPTION: (adapted from applicant's abstract) Zinc is an essential nutrient for all organisms but can be toxic if accumulated in excess amounts. Therefore, as extracellular zinc concentrations change, cells must maintain an adequate intracellular supply of zinc to meet metabolic demands while preventing its over accumulation. The long-range goal of this research is to understand the molecular mechanisms of cellular zinc homeostasis in eukaryotic cells. This process involves a complex interaction between zinc binding proteins and chelators, and the regulatory mechanisms that control these activities. Despite the importance of zinc to human health, we know little about zinc homeostasis. This proposal describes a combined genetic, molecular, biochemical, and biophysical approach to the study of zinc and zinc transporters. Their expression is induced in zinc deficiency by Zap1, a zinc-responsive transcriptional activator protein. Zap1 is proposed to be the intracellular zinc sensor for this regulation and its activity is controlled by Zn(II) binding directly to the protein to control its DNA binding activity. A second central hypothesis to the proposed research is that Zap1 target genes in addition to ZRT1 and ZRT2 play critical roles in zinc homeostasis. To test these hypotheses, the following specific aims are proposed: a) determine the in vivo effects of zinc on Zap1 DNA binding, b) identify potential accessory factors required for regulating Zap1 activity, c) use in vivo genetic analysis to identify residues in Zap1 that are required for zinc-responsive regulation, d) establish in vitro systems to study Zap1 regulation by Zn(II), e) map and characterize metal-binding sites in the Zap1 zinc-responsive domain in vitro, f) characterize the Zn(II)-induced molecular switch in the Zap1 zinc-responsive domain, g) define the role of the Zrc1, Cot1, and Zrt3 transporters in vacuolar zinc storage and detoxification, and h) characterize new transporters responsible for intracellular zinc transport and efflux. These studies should provide greater insight into the processes of zinc homeostasis in all eukaryotes.