Regulation of Cellular Zinc Homeostasis

描述（由申请人提供）：锌是一种必需营养素，因为这种金属作为催化和结构辅助因子起着重要作用。一些锌依赖蛋白存在于细胞器中，如内质网（ER）、高尔基体和线粒体。因此，需要转运蛋白将锌分配到细胞内区室。由于过量的锌可能是有毒的，细胞也需要稳态机制来控制胞浆和细胞器内游离或不稳定锌的细胞内水平。细胞锌稳态是通过多种机制实现的，包括控制锌的摄取、外排、囊泡储存和金属硫蛋白的结合。在这个建议中，考虑了锌稳态的另一个重要方面，即控制细胞内细胞器内的锌水平。这些过程将以酵母酿酒酵母为模型真核细胞进行研究。初步结果提出了五个中心假设，为本提案的具体目标提供了基础：1)酵母Msc2和Zrg17蛋白形成异质复合物，将锌转运到内质网。在Aim 1中，该复合物的组成将被评估，其锌运输活性将被表征。2)锌指结构域将成为内质网和活细胞其他区室锌状态的有用探针。在目标2中，将基于荧光共振能量转移（FRET）开发新的体内锌传感器。这些和其他有待开发的检测将用于确定Msc2/Zrg17和其他转运体在维持内质网锌稳态中的作用。3)锌转运通过Msc2/Zrg17复合物进入内质网受到转录和翻译后控制机制的调控。在Aim 3中，这些调控系统的分子机制将被剖析，并确定它们在维持内质网锌状态中的作用。4)锌转运进入内质网除了由Msc2/Zrg17复合物介导外，还由转运体介导。在Aim 4中，将确定该隔室的其他锌转运蛋白。对哺乳动物细胞的几项研究表明，锌缺乏会导致氧化应激增加，而氧化应激的来源长期以来一直是个谜。在Aim 5中，将测试锌缺乏症的氧化应激是由内质网锌稳态丧失引起的假设。这些目标代表了真核细胞分泌途径中锌稳态的连贯和全面的分析。公共卫生相关性：细胞内锌转运和细胞器内的稳态过程对基本细胞功能、生理和人体健康至关重要。然而，尽管这很重要，我们对涉及的转运蛋白知之甚少，也不知道它们是如何响应锌状态和其他因素而受到调节的。通过我们的研究，我们将对所有真核生物分泌途径中的锌代谢有一个基本的了解，并产生新的细胞内锌探针，这将有助于我们的研究以及哺乳动物和其他生物的锌代谢分析。

DESCRIPTION (provided by applicant): Zinc is an essential nutrient because of the important roles this metal plays as a catalytic and structural cofactor. Several zinc-dependent proteins reside within organelles such as the endoplasmic reticulum (ER), Golgi, and mitochondria. Therefore, transporter proteins are needed to distribute zinc into intracellular compartments. Because excess zinc can be toxic, cells also require homeostatic mechanisms to control the intracellular levels of free or labile zinc in the cytosol and within organelles. Cellular zinc homeostasis is achieved by several mechanisms including the control of zinc uptake, efflux, vesicular storage, and binding by metallothionein. In this proposal, another important facet of zinc homeostasis is considered, i.e. the control of zinc levels within intracellular organelles. These processes will be studied using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Preliminary results have raised five central hypotheses that provide the foundation for the specific aims of this proposal: 1) The yeast Msc2 and Zrg17 proteins form a heteromeric complex that transports zinc into the ER. In Aim 1, the composition of this complex will be assessed and its zinc transport activity will be characterized. 2) It is proposed that zinc finger domains will make useful probes of zinc status in the ER and other compartments of living cells. In Aim 2, new in vivo zinc sensors will be developed based on fluorescence resonance energy transfer (FRET). These and other assays to be developed will be used to define the role of Msc2/Zrg17 and other transporters in maintaining ER zinc homeostasis. 3) Zinc transport into the ER via the Msc2/Zrg17 complex is regulated in response to zinc status by both transcriptional and post-translational control mechanisms. In Aim 3, the molecular mechanisms of these regulatory systems will be dissected and their roles in maintaining ER zinc status determined. 4) It is proposed that zinc transport into the ER is mediated by transporters in addition to the Msc2/Zrg17 complex. In Aim 4, other zinc transporters for this compartment will be identified. 5) Several studies of mammalian cells showed that zinc deficiency leads to increased oxidative stress, the source of which has long been a mystery. In Aim 5, the hypothesis that the oxidative stress of zinc deficiency arises from the loss of ER zinc homeostasis will be tested. These aims represent a cohesive and comprehensive analysis of zinc homeostasis in the secretory pathway of eukaryotic cells.7. PUBLIC HEALTH RELEVANCE: The processes of intracellular zinc transport and homeostasis within organelles are essential for basic cellular function, physiology, and human health. Despite this importance, however, we know little about the transporters involved and how they are regulated in response to zinc status and other factors. As a result of our proposed studies, we will obtain a fundamental understanding of zinc metabolism in the secretory pathway of all eukaryotes and generate new probes of intracellular zinc that will be useful in our studies as well as in the analysis of zinc metabolism in mammals and other organisms.