Regulation of Cellular Zinc Homeostasis

描述(由申请人提供)：锌是一种必需的营养物质，但如果过量积累可能会中毒。因此，生物体具有维持足够的细胞内锌供应和防止随着细胞外水平的变化而过度积累的动态平衡机制。本研究的长期目标是了解真核细胞锌稳态的分子机制。这一过程涉及细胞质膜中锌的摄取和外排转运体、将锌运入和运出细胞内的细胞器转运体、细胞内锌结合蛋白和螯合剂，以及控制其活性的调控系统。尽管锌具有重要的营养价值，但我们对锌的动态平衡知之甚少。这项建议描述了一种遗传学、分子、生化和生物物理相结合的方法来研究酿酒酵母中锌的动态平衡。这种生物已经成为了解许多生物过程的有用模型，并被证明对锌的研究同样具有无价的价值。酵母基因组中&gt；40基因的表达是由Zap1诱导的，Zap1是一种锌反应转录激活因子。ZAP1是负责这一调节的细胞内锌感受器，其活性受锌直接与蛋白质结合的调节。ZAP1有几个已确定的功能结构域。在它的C末端是一个DNA结合域(DBD)。该结构域与Zapl靶基因启动子中的位点特异结合。Zap1还含有两个激活结构域，即ad1和ad2，这两个结构域都能激活转录。我们的结果表明，锌通过三种，甚至四种不同的机制来调节Zap1。首先，Zap1通过正转录自我调节来控制自身的表达。这种自动调节控制细胞内ZAPL蛋白的水平和ZRE的结合。此外，锌独立控制AD1和AD2的活性。因此，锌对Zap1活性的调节需要两个独立的锌传感器来控制这些活性。我们的初步研究使我们得出了关于这些传感器和这些调控事件背后的分子机制的假设。这些假设将在这项拟议的研究中得到检验。我们将评估的第四个机制是DBD功能本身是否受锌的调节。这些研究将导致对金属传感和锌动态平衡的基本见解。

DESCRIPTION (provided by applicant): Zn is an essential nutrient but can be toxic if overaccumulated. Therefore, organisms have homeostatic mechanisms to maintain adequate intracellular supplies of Zn and prevent overaccumulation as extracellular levels change. The long-range goal of this research is to understand the molecular mechanisms of Zn homeostasis in eukaryotic cells. This process involves Zn uptake and efflux transporters in the plasma membrane, organellar transporters that move Zn into and out of intracellular compartments, intracellular Zn binding proteins and chelators, and the regulatory systems that control their activities. Despite the nutritional importance of Zn, we still know little about Zn homeostasis. This proposal describes a combined genetic, molecular, biochemical, and biophysical approach to the study of Zn homeostasis in the yeast Saccharomyces cerevisiae. This organism has been a useful model for understanding many biological processes and is proving to be equally invaluable for the study of Zn. The expression of >40 genes in the yeast genome is induced in Zn-deficient cells by Zap1, a Zn-responsive transcriptional activator. Zap1 is the intracellular Zn sensor responsible for this regulation and its activity is modulated by Zn binding directly to the protein. Zap1 has several identified functional domains. At its C-terminus is a DNA binding domain (DBD). This domain binds specifically to sites found in the promoters of Zapl's target genes. Zapl also contains two activation domains, AD1 and AD2, both of which activate transcription. Our results indicate that Zap1 is regulated by Zn via three, possibly even four, different mechanisms. First, Zap1 controls its own expression through positive transcriptional autoregulation. This autoregulation controls the level of Zapl protein in the cell and ZRE binding. In addition, Zn independently controls the activities of AD1 and AD2. Thus, regulation of Zap1 activity by Zn requires two independent Zn sensors to control these activities. Our preliminary studies have led us to hypotheses regarding these sensors and the molecular mechanisms underlying these regulatory events. These hypotheses will be tested in this proposed research. The fourth mechanism we will assess is if DBD function per se is Zn regulated. These studies will lead to fundamental insights into metallosensing and Zn homeostasis.