CDF-1 Regulation of Zinc Homeostasis

描述（由申请人提供）：本提案的长期目标是表征多细胞动物中锌代谢和Zn2+稳态调节的机制。锌离子代谢对人类健康具有重要意义，因为由于饮食不足或先天代谢错误引起的锌缺乏会导致许多疾病。Zn2+还调节细胞增殖等重要过程，Zn2+代谢可能影响癌症等疾病。第一个具体目的是表征秀丽隐杆线虫cdf-1基因在完整动物体内Zn2+代谢中的作用。cdf-1编码一种阳离子扩散促进蛋白，促进Zn2+在质膜上的外排。研究设计和方法包括建立线虫体内锌含量、分布、吸收和排泄的测定方法。这些试验将用于确定饮食中锌的变化如何影响Zn2+体内平衡。cdf-1在Zn2+代谢中的作用将通过分析缺乏cdf-1活性、过表达cdf-1或在特定组织中表达cdf-1的突变体来确定。通过分析完整动物的CDF-1 mRNA和蛋白，并确定膳食锌如何调节这些产物，将对CDF-1活性的调节进行表征。第一个具体目标的最后一部分是表征CDF-1在促进Zn2+跨质膜运输中的生化作用机制。研究设计和方法包括使用纯化组分或细胞系统开发Zn2+运输的分析，以及表征CDF-1在Zn2+运输中的作用。利用酵母双杂交系统鉴定与CDF-1结合的蛋白，并分析CDF-1相互作用蛋白在Zn2+转运中的作用。cdf -1相互作用蛋白在完整动物中的作用将通过遗传学方法进行研究。第二个具体目标是确定调控Zn2+稳态的基因网络。研究设计和方法包括对影响秀丽隐杆线虫Zn2+代谢的突变进行基因筛选。遗传学方法将用于确定新发现的基因在Zn2+代谢中的具体作用。这些基因在细胞命运规范中的作用将被确定，以表征Zn2+代谢和ras介导的信号传导之间的关系。分子方法将用于克隆受影响的基因，并揭示这些蛋白调节Zn2+代谢的机制。这些研究可能通过在完整动物中建立CDF-1的作用，阐明CDF-1的生化机制，以及鉴定和表征调节Zn2+的新蛋白，为Zn2+代谢提供重要的新见解。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to characterize the mechanisms of zinc metabolism and the regulation of Zn2+-homeostasis in multicellular animals. Zn2+ metabolism has important implications for human health because zinc deficiencies caused by inadequate diet or inborn errors of metabolism result in many pathologies. Zn2+ also regulates important processes such as cell proliferation, and Zn2+ metabolism may affect diseases such as cancer. The first specific aim is to characterize the role of the C. elegans cdf-1 gene in Zn2+ metabolism in an intact animal. cdf-1 encodes a cation diffusion facilitator protein that promotes Zn2+ efflux across the plasma membrane. The research design and methods include developing assays of zinc content, distribution, uptake and excretion in nematode worms. These assays will be used to determine how changes in dietary zinc affect Zn2+-homeostasis. The role of cdf-1 in Zn2+ metabolism will be determined by analyzing mutants that lack CDF-1 activity, overexpress CDF-1, or express CDF-1 in specific tissues. The regulation of CDF-1 activity will be characterized by analyzing cdf-1 mRNA and protein in intact animals and determining how these products are regulated by dietary zinC. The final part of the first specific aim is to characterize the biochemical mechanism of action of CDF-1 in promoting Zn2+ transport across the plasma membrane. The research design and methods include developing assays of Zn2+ transport using purified components or cellular systems and characterizing the role of CDF-1 in Zn 2+ transport. Proteins that bind to CDF-1 will be identified using the yeast two-hybrid system and the role of CDF-l-interacting proteins in Zn2+ transport will be analyzed. The role of CDF-1-interacting proteins in intact animals will be investigated using genetic approaches. The second specific aim is to identify a network of genes that regulate Zn2+- homeostasis. The research design and methods include conducting genetic screens for mutations that affect C. elegans Zn2+ metabolism. Genetic methods will be used to determine the specific role of newly identified genes in Zn2+ metabolism. The role of these genes in cell fate specification will be determined to characterize the relationship between Zn2+ metabolism and Ras-mediated signaling. Molecular approaches will be used to clone the affected genes and reveal the mechanisms used by these proteins to regulate Zn2+ metabolism. These studies are likely to provide significant new insights into Zn2+ metabolism by establishing the role of CDF-1 in an intact animal, elucidating the biochemical mechanism of CDF-1, and identifying and characterizing new proteins that regulate Zn2+.