Structure and Mechanism of Zinc Efflux Transporters

 描述(由申请人提供)：锌转运蛋白通过细胞内细胞器之间明显的跨膜锌梯度来调节亚细胞锌的分布。锌的时空动态提供了关键的细胞信号转导机会，但也挑战了具有广泛疾病含义的细胞内锌稳态。我们的长期目标是了解锌转运蛋白的结构和机制及其功能调节。这项提议是一个持续研究项目的竞争性更新，该项目专注于阳离子扩散促进剂(CDF)家族的锌外流转运体。在过去的十年里，我们已经阐明了细菌CDF同系物的分子结构、配位化学和结构动力学。虽然我们在基础研究方面取得了进展，但人类CDF hZnT8已被确定为1型糖尿病的主要自身抗原，以及与2型糖尿病相关的主要危险因素。HZnT8治疗性翻译的关键障碍 缺乏对糖尿病关联的分子机制和易感性hZnT8变异的功能后果的了解。为了填补这一知识空白，我们在下一个预算周期的研究重点将从细菌CDF模型转移到hZnT8，具体目标有四个：(1)了解变构对锌运输的调控机制。(2) (3)研究hZnT8及其遗传变异体在胰岛β细胞中的功能作用；(4)制备hZnT8特异性单抗并研究其对胰岛素储存和分泌的影响。计划中的研究将围绕CDF的功能调控展开，以了解锌运输的变构调节如何实时发生(AIM-1)，hZnT8如何受到糖尿病相关基因变异的影响(AIM-2)，hZnT8如何调节囊泡锌动态和胰岛素分泌以响应葡萄糖刺激(AIM-3)，以及针对细胞外表位的构象特异性单抗(AIM-4)是否能阻断hZnT8。所获得的知识将用于阐明基于hZnT8的糖尿病治疗的分子基础。

 DESCRIPTION (provided by applicant): Zinc transporters regulate subcellular zinc distributions with sharp transmembrane zinc gradients among intracellular organelles. The spatiotemporal zinc dynamics provides crucial cellular signaling opportunities, but also challenges intracellular zinc homeostasis with broad disease implications. Our long-term goal is to understand structures and mechanisms of zinc transporters and their functional regulations. This proposal is a competitive renewal of a continuing research project focusing on zinc-efflux transporters from the Cation Diffusion Facilitator (CDF) family. In the past decade, we have elucidated the molecular architecture, coordination chemistry and structural dynamics of a bacterial CDF homolog. While we are making progress on the basic research front, a human CDF, hZnT8 has been identified as a major autoantigen in type-1 diabetes as well as a major risk factor associated with type-2 diabetes. A critical barrier to therapeutic translation of hZnT8 is the lack of knowledge about the molecular mechanism responsible for diabetes association and the functional consequences of susceptibility hZnT8 variations. To fill in this knowledge gap, our research focus in the next budget cycle will shift from a bacterial CDF model to hZnT8 with four specific aims: (1) to understand the mechanism of allosteric regulation of zinc transport. (2) to characterize transport kinetics of purified hZnT8 and its genetic variants, (3) to define the functional roles of hZnT8 and its genetic variants in pancreatic beta cells, (4) to develop hZnT8-specifc mAbs and determine their effects on insulin storage and secretion. The planned research will revolve around functional regulation of CDFs to understand how allosteric regulation of zinc transport occurs in real time (aim-1), how hZnT8 is affected by diabetes-associated genetic variations (aim-2), how hZnT8 modulates vesicular zinc dynamics and insulin secretion in response to glucose stimulations (aim-3), and whether hZnT8 can be blocked by conformation-specific monoclonal antibodies targeting extracellular epitopes (aim-4). The knowledge gained will be used to elucidate the molecular underpinning of hZnT8-based diabetes therapy.