Functional Testing of Conserved Regions in Eukaryotic Divalent Metal Transporters

描述（由申请人提供）：铁是人体必需的微量元素，缺铁会导致缺铁性贫血，而铁过多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调节铁通过跨膜转运蛋白（Nramp2，铁转运蛋白）的运动。我的初步工作已经确定了Nramp2和运铁蛋白中的特定位点，这些位点可能对它们的功能至关重要。这个项目会让我有机会获得新的技能，使我能够直接测试我在计算研究中产生的假设。这些结果可能为更好地理解铁转运和调控提供关键信息（即hepcidin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，检验Nramp 1和Nramp p2 （DMT1）环E3中保守氨基酸残基的功能作用。2. 测定环E3特性赋予的金属特异性决定因素。3. 检查环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. 通过生物信息学方法继续研究NRAMP1、NRAMP2和铁转运蛋白在进化中的作用。

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.