Structural insights into zinc homeostasis

项目概要/摘要 Zrt、Irt 样蛋白（ZIP 或溶质载体 39 (SLC39)）膜蛋白家族介导锌流入1。 锌稳态是多种生理过程的关键调节剂，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发挥关键作用2。因为两者都过剩 锌水平不足具有细胞毒性，哺乳动物细胞中的锌稳态通过丰富的锌离子得到严格控制。 作为锌输入蛋白（ZIP / SLC39 家族）或锌输出蛋白（ZnT / SLC30 家庭）。锌稳态蛋白是神经退行性疾病和神经退行性疾病的潜在药理学靶点 癌症，但由于缺乏结构和机制信息，进展受到阻碍。没有全长哺乳动物 ZIP 家族成员的结构已被解析，并且不存在体外功能测定。 ZIP9 (SLC39A9) 代表了前列腺癌的新治疗途径3。前列腺中锌的浓度最高 身体，促进细胞凋亡并有助于维持低水平的细胞增殖。在前列腺癌中， 锌水平下降，导致肿瘤增殖4。前列腺锌水平无法通过饮食来恢复 补充剂5,6。最近，ZIP9 被鉴定为膜雄激素受体 (mAR)，可介导 睾酮诱导的细胞凋亡7。 ZIP9 在前列腺癌中表达上调，但要实现其作为治疗药物的潜力 药理学目标需要完整的结构和功能表征。我的目标是阐明 ZIP9的结构和机制。目标 1 是开发一种基于蛋白脂质体的功能测定法 停流荧光光谱和定点突变来表征 ZIP9 的机制。 目标 2 是使用微晶电子衍射 (MicroED) 确定 ZIP9 锌流入的结构基础。 目标 3 是通过确定 ZIP9 的分子和结构基础来检查 ZIP9 的 mAR 特征。 利用生物化学和高分辨率单粒子低温与睾酮和 G 蛋白相互作用 电子显微镜（冷冻电镜）。这项研究将揭示 ZIP 介导的 Zn2 流入机制并阐明 ZIP9 作为膜雄激素受体的假定作用，拓宽了我们对锌稳态和 为使用 ZIP 作为前列腺癌及其他癌症的药理学靶点铺平了道路。

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.