Regulation of Cellular Zinc Homeostasis

描述（由申请人提供）：锌是一种必需营养素，但如果过度积累可能有毒。因此，生物体具有稳态机制来维持足够的细胞内锌供应，并防止随着细胞外水平的变化而过度积累。本研究的长期目标是了解真核细胞中锌稳态的分子机制。这一过程涉及质膜中的锌吸收和流出转运蛋白、将锌移入和移出细胞内隔室的细胞器转运蛋白、细胞内锌结合蛋白和螯合剂以及控制其活性的调节系统。尽管锌的营养重要性，我们仍然对锌稳态知之甚少。该建议描述了一个综合的遗传，分子，生物化学和生物物理的方法来研究锌稳态的酵母酿酒酵母。这种生物体是理解许多生物过程的有用模型，并且被证明对于锌的研究同样是非常宝贵的。酵母基因组中>40个基因的表达在锌缺乏细胞中由锌应答转录激活因子Zap 1诱导。Zap 1是负责这种调节的细胞内锌传感器，其活性由锌直接结合到蛋白质来调节。Zap 1有几个确定的功能域。在其C-末端是DNA结合结构域（DBD）。该结构域特异性结合Zapl靶基因启动子中的位点。Zapl还含有两个激活结构域，AD 1和AD 2，两者都激活转录。我们的研究结果表明，Zap 1是由锌通过三个，甚至可能是四个，不同的机制。首先，Zap 1通过正性转录自调节来控制其自身的表达。这种自动调节控制细胞中Zapl蛋白的水平和ZRE结合。此外，Zn独立地控制AD 1和AD 2的活性。因此，锌对Zap 1活性的调节需要两个独立的锌传感器来控制这些活动。我们的初步研究使我们对这些传感器和这些调控事件的分子机制提出了假设。这些假设将在本研究中进行验证。我们将评估的第四种机制是DBD功能本身是否受锌调节。这些研究将导致对金属传感和锌稳态的基本见解。

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.