The molecular determinants of zinc uptake mediated by hZIP4

描述（由申请人提供）：锌和铁是生物系统中最丰富的两种过渡金属，然而锌和非转铁蛋白结合的铁转运到细胞中的分子机制尚未解决。基于与铁的序列相似性，已经鉴定了14个人ZIP家族成员（S.酿酒酵母）和锌（A. thaliana运输公司。ZIP蛋白家族的这些亲本成员具有不同的阳离子选择性，目前还不清楚是什么分子决定因素定义了ZIP蛋白家族的阳离子选择性。通过对人类（h）Zn 2+输入者ZIP 4的分析，该提议将定义跨膜、N-末端和胞质结构域在ZIP蛋白家族的离子渗透途径和阳离子选择性中的贡献，以及阐明为什么先前发现的锌转运蛋白突变导致不同的疾病状态。本申请的长期目标是阐明真核ZIP蛋白的结构和功能 其直接涉及多种疾病状态，并解决了过渡金属转运蛋白如何对具有相似离子半径、电荷和配位几何形状的第一行过渡金属具有选择性。这项建议的目的是：1）检验跨膜结构域内的残基限定过渡金属渗透途径和ZIP转运蛋白的选择性的假设，2）检验锌与胞质结构域的配位诱导调节生物金属易位速度的构象变化的假设，和3）检验引起N-末端结构域内突变的AE以及锌缺乏触发大的hZIP 4 N-末端结构域的裂解，调节锌摄取的假设。为了解决目标一，在X.将使用非洲乳鼠卵母细胞来阐明靶向跨膜结构域在hZIP 4的离子渗透途径和阳离子选择性中的作用。为了获得目标二的目标，将鉴定在胞质结构域中配位Zn 2+的残基，并测量全长蛋白质的胞质Zn 2+配位破坏后Zn 2+摄取的变化。最后，通过检查hZIP 4和突变体构建体（包括截短的hZIP 4）的锌摄取，以直接测量hZIP 4中N-末端结构域的改变对Zn 2+摄取的影响，来实现目标三的目标。由于锌是生命所必需的，因此本研究的结果将具有重要意义，因为它将详细描述细胞外、跨膜和细胞内结构域对ZIP蛋白阳离子渗透途径的贡献，并定义有助于ZIP蛋白家族中不同阳离子特异性的分子决定簇。使用的方法，包括运输测定，结构生物学和生物物理方法的组合，系统地阐明过渡金属渗透途径和蛋白质的选择性决定因素，这是直接涉及几种人类疾病，是新颖的，及时的和创新的。

DESCRIPTION (provided by applicant): Zinc and iron are the two most abundant transition metals in biological systems, however the molecular mechanism of zinc and non-transferrin bound iron transport into cells is not resolved. Fourteen human ZIP family members have been identified based on sequence similarity to iron (Irt in S. cerevisiae) and zinc (Zrt in A. thaliana transporters. These parent members of the ZIP protein family have differing cation selectivity and it is currently unclear what molecular determinants define cation selectivity for the ZIP family of proteins. Through analysis of the human (h) Zn2+ importer, ZIP4, this proposal will define the contribution of the transmembrane, N-terminal and cytosolic domains in the ion permeation pathway and cation selectivity of the ZIP family of proteins as well as elucidate why previously discovered mutations in zinc transporters lead to distinct disease states. The long-term goal of the application is to elucidate the structure and function of a eukaryotic ZIP protein which is directly implicated in multiple disease states and resolve how transition metal transporters are selective for first row transition metals which have similar ionic radii, charge ad coordination geometry. The aims of this proposal are: 1) Test the hypothesis that residues within the transmembrane domains define both the transition metal permeation pathway and selectivity of ZIP transporters, 2) Test the hypothesis that zinc coordination to the cytosolic domain induces a conformational change which regulates the velocity of biometal translocation, and 3) Test the hypothesis that AE causing mutations within the N- terminal domain as well as zinc deficiency triggered cleavage of the large hZIP4 N-terminal domain, regulates zinc uptake. To address aim one, an uptake assay following heterologous expression of hZIP4 mutants in X. laevis oocytes will be employed to elucidate the contribution of targeted transmembrane domains in the ion permeation pathway and cation selectivity of hZIP4. To obtain the goals of aim two, residues that coordinate Zn2+ in the cytosolic domain will be identified and changes in Zn2+ uptake upon disruption of cytosolic Zn2+ co- ordination for the full-length protein will be measured. Finally, the goals of aim three will be accomplished by examining zinc uptake for hZIP4 and mutant constructs, including truncated hZIP4, to directly measure the effects of alterations of the N-terminal domain in hZIP4 on Zn2+ uptake. As zinc is required for life, the results from this study will be significant as it will provide a detailed description of the contribution of the extracellular, transmembrane and intracellular domains to the cation permeation pathway of ZIP proteins as well as define the molecular determinants which contribute to differing cation specificity among the ZIP family of proteins. The use of a combination of approaches, including transport assays, structural biology and biophysical methods, to systemically elucidate the transition metal permeation pathway and the selectivity determinants of a protein which is directly implicated in several human diseases, is novel, timely and innovative.