Structural insights into zinc homeostasis

项目摘要/摘要 膜蛋白家族中的类IRT蛋白(ZIP，又称溶质载体39，SLC39)介导锌离子内流。 锌稳态是多种生理过程的关键调节器，包括免疫功能、胰岛素 分泌和细胞通讯，并在蛋白质结构和催化中起关键作用。因为这两种过剩 而缺乏锌水平是细胞毒性的，在哺乳动物细胞中锌的稳态是通过丰富的 作为锌进口体(ZIP/SLC39家族)或锌出口体(ZNT/SLC30)的膜蛋白网络 家庭)。锌稳态蛋白是治疗神经退行性疾病的潜在药理靶点 癌症，但由于缺乏结构性和机械性的信息，进展受到阻碍。没有全长的哺乳动物 ZIP家族成员的结构已经被解决，并且没有体外功能分析存在。ZIP9(SLC39A9) 代表了前列腺癌的一种新的治疗途径。前列腺中锌含量最高的是 促进细胞凋亡，帮助维持低水平的细胞增殖。在前列腺癌方面， 锌水平降低，导致肿瘤增殖。饮食不能挽救前列腺癌的锌水平 补充5，6.最近，ZIP9被鉴定为膜雄激素受体(MAR)，它介导 睾丸激素诱导的细胞凋亡7。ZIP9在前列腺癌中上调，但要实现其作为一种 药理靶标需要完整的结构和功能表征。我的目的是要澄清 ZIP9的结构和作用机制。目标1是开发一种基于蛋白质脂质体的功能分析方法，使用 停流荧光光谱分析和定点突变研究ZIP9的作用机制。 目的二是用微晶电子衍射(MicroED)确定ZIP9分子内锌离子的结构基础。 目的3是通过确定ZIP9的S MAR的分子和结构基础来研究其MAR特性 生物化学和高分辨率单粒子低温与睾酮和G蛋白的相互作用 电子显微镜(低温电子显微镜)。这项研究将揭开ZIP介导的锌离子内流的机制并澄清 ZIP9的S作为膜雄激素受体的作用，拓宽了我们对锌的动态平衡和 为将拉链用作前列腺癌及其他疾病的药理靶点铺平了道路。

Project Summary / Abstract The Zrt-, Irt-like Protein (ZIP, or Solute Carrier 39 (SLC39)) family of membrane proteins mediate zinc influx1. Zinc homeostasis is a critical regulator of a variety of physiological processes, including immune function, insulin secretion, and cell communication, and plays key roles in protein structure and catalysis2. Because both excess and deficient zinc levels are cytotoxic, zinc homeostasis is tightly controlled in mammalian cells via an abundant network of membrane proteins that act as zinc importers (ZIP / SLC39 family) or zinc exporters (ZnT / SLC30 family). Zinc homeostasis proteins are potential pharmacological targets in neurodegenerative disorders and cancer, but progress is hampered by a lack of structural and mechanistic information. No full length mammalian structures of ZIP family members have been solved and no in vitro functional assays exist. ZIP9 (SLC39A9) represents a novel treatment avenue for prostate cancer3. The prostate has the highest concentration of zinc in the body, which promotes apoptosis and helps maintain low levels of cellular proliferation. In prostate cancer, zinc levels decrease, leading to tumor proliferation4. Prostate zinc levels cannot be rescued with dietary supplements5,6. Recently, ZIP9 was identified as a membrane Androgen Receptor (mAR) which mediates testosterone-induced apoptosis7. ZIP9 is upregulated in prostate cancer, but to realize its potential as a pharmacological target a full structural and functional characterization is required. My objective is to elucidate the structure and mechanism of ZIP9. Aim 1 is to develop a proteoliposome-based functional assay using stopped-flow fluorescence spectroscopy and site-directed mutagenesis to characterize the mechanism of ZIP9. Aim 2 is to determine the structural basis of zinc influx by ZIP9 using Microcrystal Electron Diffraction (MicroED). Aim 3 is to examine ZIP9's mAR characteristics by determining the molecular and structural basis of its interactions with testosterone and G proteins using biochemistry and high resolution single particle cryogenic electron microscopy (cryoEM). This research will unravel the mechanism of ZIP-mediated Zn2+ influx and clarify ZIP9's putative role as a membrane Androgen Receptor, broadening our understanding of zinc homeostasis and paving the way for the use of ZIPs as a pharmacological target in prostate cancer and beyond.