Regulation of Cellular Zinc Homeostasis

说明(申请人提供)：锌是一种必需的营养素，因为它作为催化剂和结构辅因子扮演着重要的角色。内质网(ER)、高尔基体和线粒体等细胞器中存在多种锌依赖蛋白。因此，需要转运蛋白将锌分布到细胞内的隔室中。因为过量的锌可能是有毒的，细胞还需要动态平衡机制来控制胞浆和细胞器中游离或不稳定锌的细胞内水平。细胞内锌稳态是通过多种机制实现的，包括锌的摄取、外排、囊泡储存和金属硫蛋白的结合。在这项建议中，锌稳态的另一个重要方面被考虑，即细胞内细胞器内锌水平的控制。这些过程将用酿酒酵母作为真核细胞的模型来研究。初步结果提出了五个中心假设，为该提议的具体目标提供了基础：1)酵母Msc2和Zrg17蛋白形成一个异构体复合体，将锌运输到内质网。在目标1中，将评估该络合物的组成，并表征其锌转运活性。2)锌指结构域可以作为活体细胞内质网和其他隔室中锌状态的有用探针。在目标2中，基于荧光共振能量转移(FRET)的新型体内锌传感器将被开发出来。这些和其他有待开发的检测方法将用于确定Msc2/Zrg17和其他转运蛋白在维持内质网锌稳态中的作用。3)锌通过Msc2/Zrg17复合体进入内质网，受转录和翻译后调控机制的调节。在目标3中，将剖析这些调控系统的分子机制，并确定它们在维持内质网锌状态中的作用。4)推测锌向内质网的转运除Msc2/Zrg17复合体外，还受转运体的介导。在目标4中，将确定该隔间的其他锌运输器。5)几项对哺乳动物细胞的研究表明，缺锌会导致氧化应激增加，其来源长期以来一直是个谜。在目标5中，将检验锌缺乏引起的氧化应激是由于内质网锌稳态的丧失而引起的假设。这些目标代表了对真核细胞分泌途径中锌的动态平衡的凝聚和全面的分析。与公共健康相关：细胞内锌的运输和细胞器内的动态平衡过程对基本的细胞功能、生理和人类健康至关重要。然而，尽管这很重要，我们对涉及的转运蛋白以及它们如何受到锌状态和其他因素的调节知之甚少。作为我们提出的研究的结果，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，这将对我们的研究以及对哺乳动物和其他生物中锌代谢的分析有用。

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.