CDF-1 Regulation of Zinc Homeostasis

描述(申请人提供)：这项建议的长期目标是描述多细胞动物锌代谢和锌离子稳态调节的机制。锌离子代谢对人类健康具有重要的意义，因为饮食不足或先天代谢缺陷导致的锌缺乏会导致许多病理变化。锌离子还调节细胞增殖等重要过程，锌离子代谢可能会影响癌症等疾病。第一个特定目的是研究线虫CDF-1基因在完整动物体内锌离子代谢中的作用。CDF-1编码一种阳离子扩散促进蛋白，促进锌离子跨质膜外流。研究设计和方法包括开发线虫体内锌含量、分布、吸收和排泄的分析方法。这些分析将被用来确定饮食中锌的变化如何影响锌的动态平衡。CDF-1在锌代谢中的作用将通过分析在特定组织中缺乏CDF-1活性、过度表达CDF-1或表达CDF-1的突变体来确定。CDF-1活性的调节将通过分析完整动物的CDF-1mRNA和蛋白质，并确定这些产物如何受到膳食锌的调节来表征。第一个特定目标的最后部分是研究CDF-1促进锌离子跨质膜转运的生化机制。研究设计和方法包括利用纯化的组分或细胞系统建立锌离子转运的分析方法，以及表征CDF-1在锌离子转运中的作用。与CDF-1结合的蛋白质将使用酵母双杂交系统进行鉴定，并将分析CDF-L相互作用蛋白在锌离子转运中的作用。CDF-1相互作用蛋白在完整动物中的作用将使用遗传学方法进行研究。第二个具体目标是确定一个调节锌离子内稳态的基因网络。研究设计和方法包括对影响线虫锌代谢的突变进行基因筛查。遗传学方法将被用来确定新发现的基因在锌代谢中的具体作用。这些基因在细胞命运决定中的作用将被确定，以表征锌代谢和RAS介导的信号之间的关系。分子方法将被用来克隆受影响的基因，并揭示这些蛋白质调节锌代谢的机制。这些研究可能通过建立CDF-1在完整动物中的作用，阐明CDF-1的生化机制，以及鉴定和表征调控锌离子的新蛋白，为研究锌的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+.