The molecular determinants of zinc uptake mediated by hZIP4

描述(申请人提供)：锌和铁是生物系统中含量最丰富的两种过渡金属，但锌和非转铁蛋白结合铁进入细胞的分子机制尚未解决。根据与铁(酿酒酵母中的IRT)和锌(Zrt中的A.thaliana转运蛋白)的序列相似性，已经确定了14个人类ZIP家族成员。ZIP蛋白家族的这些亲代成员具有不同的阳离子选择性，目前还不清楚哪些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(H)锌离子导入蛋白ZIP4的分析，这一建议将确定跨膜、N末端和胞内结构域在离子渗透途径和ZIP家族阳离子选择性中的作用，并阐明为什么以前发现的锌转运蛋白突变会导致不同的疾病状态。该应用的长期目标是阐明真核ZIP蛋白的结构和功能 它直接与多种疾病状态有关，并解决了过渡金属转运体对具有相似离子半径、电荷和配位几何的第一排过渡金属的选择性。这一建议的目的是：1)检验跨膜结构域中的残基决定过渡金属渗透途径和ZIP转运蛋白选择性的假说，2)检验锌与胞液结构域配位引起构象变化从而调节生物金属转运速度的假说，3)检验AE导致N-末端结构域突变以及缺锌触发大的hZIP4 N-末端结构域裂解调节锌摄取的假说。为了达到目的一，我们将利用hZIP4突变体在非洲血吸虫卵母细胞中异源表达的摄取实验来阐明靶向跨膜区在hZIP4的离子渗透途径和阳离子选择性中的作用。为了达到目标二的目标，将鉴定胞浆结构域中与锌离子配位的残基，并将测量全长蛋白质胞质锌离子配位中断时锌离子摄取的变化。最后，目标三的目标将通过检测hZIP4和突变结构(包括截短的hZIP4)的锌吸收来实现，以直接测量hZIP4中N-末端结构域的变化对锌吸收的影响。由于锌是生命所必需的，这项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，并确定导致ZIP蛋白家族阳离子专一性不同的分子决定因素。利用多种方法，包括转运分析、结构生物学和生物物理方法，系统地阐明了一种直接与人类疾病有关的蛋白质的过渡金属渗透途径和选择性决定因素，这是新颖、及时和创新的。

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.