REGULATION OF CELLULAR ZINC HOMEOSTASIS

描述：锌是必不可少的，但过多的锌可能是有毒的。 因此，我们认为， 存在控制细胞内 这种金属的浓度。 这项研究的长期目标是 确定真核细胞锌稳态的分子基础。 这 该提案描述了一种结合遗传、分子、生物化学和 生物物理方法来研究这些过程中的酵母 酿酒酵母 酵母已被证明是一种理想的模型系统， 最近的铁和铜稳态的研究，毫无疑问，将被证明是 对锌代谢的研究同样有价值。 锌在酵母中的吸收是 由两个独立的系统组成。 一个系统对锌有很高的亲和力 ZRT1基因编码该系统的转运蛋白。 的 第二个系统对底物具有较低的亲和力，ZRT2基因编码 它的运输机。 酵母中锌稳态的一个重要方面是 通过这两个基因的转录控制来调节锌的摄取。 ZRT1和ZRT2在锌限制细胞中均以高水平表达，但在锌缺乏细胞中表达水平较高。 在锌含量丰富的细胞中含量很低。 初步的研究已经确定了一个新的基因，ZAP 1（锌响应 激活蛋白），在这种调节中起着关键作用。 为 顾名思义，目前的假设是ZAP 1编码一种转录因子， 结合ZRT 1和ZRT 2启动子中序列（ZRE）的激活剂。 DNA与ZRE的结合由Zap1p中的锌指结构域赋予， 这些基因的转录速率通过以下作用增加： 蛋白质中的酸性激活结构域。 还建议Zap1p 作为锌“传感器”，其转录激活结构域是 通过锌离子直接与蛋白质结合来抑制。 测试 这一假设的各个方面，以下具体目标是 建议：1）精确鉴定ZRE并确定Zap1p是否结合 这些序列。 2)对Zap1p蛋白进行功能解剖。 3)确定锌对Zap1p功能的体内影响。 4)识别 Zap1p活性的潜在分子间阻遏物。 5)表征 Zap1p体外锌结合。 这些实验将导致一个更大的 了解人体细胞中的锌稳态。

DESCRIPTION: Zinc is essential yet too much zinc can be toxic. Therefore, regulatory mechanisms are present that control the intracellular concentration of this metal. The long-range goal of this research is to determine the molecular basis of zinc homeostasis in eukaryotic cells. This proposal describes a combined genetic, molecular, biochemical, and biophysical approaches to the study of these processes in the yeast Saccharomyces cerevisiae. Yeast has proven to be an ideal model system for recent studies of iron and copper homeostasis and will no doubt prove to be equally valuable for the study of zinc metabolism. Zinc uptake in yeast is mediated by two separate systems. One system has a high affinity for zinc and the ZRT1 gene encodes the transporter protein of this system. The second system has a lower affinity for substrate and the ZRT2 gene encodes its transporter. An important facet of zinc homeostasis in yeast is the regulation of zinc uptake by transcriptional control of these two genes. ZRT1 and ZRT2 are both expressed at high levels in zinc-limited cells but at low levels in zinc-replete cells. Preliminary studies have identified a new gene, ZAP1 (for Zinc-responsive Activator Protein), that plays a critical role in this regulation. As the name implies, the current hypothesis is that ZAP1 encodes a transcriptional activator that binds to sequences (ZREs) in the promoters of ZRT1 and ZRT2. DNA binding to the ZREs is conferred by zinc finger domains in Zap1p and the rates transcription of these genes are increased through the action of acidic activation domains in the protein. It is also proposed that Zap1p acts as the zinc "sensor" and its transcriptional activation domains are repressed by binding of zinc ions directly to the protein. To test the various aspects of this hypothesis, the following specific aims are proposed: 1) Precisely identify the ZREs and determine if Zap1p binds to these sequences. 2) Perform a functional dissection of the Zap1p protein. 3) Determine the in vivo effects of zinc on Zap1p function. 4) Identify potential intermolecular repressors of Zap1p activity. 5) Characterize zinc-binding by Zap1p in vitro. These experiments will lead to a greater understanding of zinc homeostasis in human cells.