Functional Testing of Conserved Regions in Eukaryotic Divalent Metal Transporters

说明(申请人提供)：铁是一种必需的微量元素：太少会导致缺铁性贫血，而太多则会导致器官损伤和感染。细胞控制铁水平的一种方式是通过跨膜转运蛋白(Nramp2，铁蛋白)调节铁的移动。我的初步工作已经确定了Nramp2和铁蛋白中可能对其功能至关重要的特定位点。这个项目将让我有机会获得新的技能，使我能够直接测试我的计算研究产生的假设。这一结果可能会为更好地理解铁的运输和调控提供关键信息(即海普西丁相互作用和Nramp2的小分子抑制)。具体目标是：1.通过外源表达研究和比较动力学分析，验证Nramp1和Nramp2(DMT1)环E3中保守氨基酸残基的功能作用。2.确定环E3特性赋予的金属特异性决定因素。3.研究环E3与铁转运调节剂和小分子转运抑制剂相互作用的潜在作用。4.利用生物信息学方法继续研究NRAMP1、NRAMP2和铁蛋白在进化中的作用。 与公共健康相关：缺铁在美国和世界各地都是一个公共健康问题。NRAMP2是肠上皮细胞中非血红素铁的主要进口者，因此需要对其功能和调控有更好的了解。

DESCRIPTION (provided by applicant): Iron is an essential trace element: too little contributes to iron deficiency anemia, while too much iron can lead to organ damage and infection. One way cells control iron levels is by regulating the movement of iron through transmembrane transporters (Nramp2, ferroportin). My preliminary work has identified specific sites in Nramp2 and ferroportin that could be essential to their functions. This project will give me the opportunity to acquire new skills, enabling me to directly test the hypotheses generated by my computational studies. The results may provide key pieces of information (i.e., hepcidin interactions and small molecule inhibition of Nramp2) toward a better understanding of iron transport and regulation. The specific goals are: 1. Test the functional role of conserved amino acid residues in loop E3 of Nramp 1 and Nramp2 (DMT1) through exogenous expression studies and comparative kinetic analysis. 2. Determine metal specificity determinants imparted by loop E3 characteristics. 3. Examine the potential role of loop E3 interactions with iron transport regulators and small molecule transport inhibitors. 4. To continue work on the roles of NRAMP1, NRAMP2, and ferroportin in evolution through bioinformatics methods. PUBLIC HEALTH RELEVANCE: Iron deficiency is a public health problem in the United States and around the world. NRAMP2 is the primary importer of non-heme iron in intestinal epithelium, so a better understanding is needed of its function and regulation.