Structural insights into zinc homeostasis

锌稳态的结构见解

• 批准号: 10315746
• 负责人: SARA JEAN WEAVER
• 金额: $ 6.6万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10315746
• 关键词: Affinity; Agonist; Androgen Receptor; Apoptosis; Binding; Biochemistry; Biological Assay; Biopsy; Brain; Breast; Castration; Catalysis; Cell Communication; Cell Culture Techniques; Cell Proliferation; Cellular biology; Characteristics; Charge; Crystallization; Cytosol; Detergents; Diabetes Mellitus; Dietary Zinc; Drug Design; Electron Microscopy; Elements; Environment; Family; Family member; Fluorescence Spectroscopy; Foundations; Future; GTP-Binding Proteins; Genome; Goals; Hand; Homeostasis; Human; Immunity; In Vitro; Individual; Insulin; Ions; Length; Lipids; Literature; Maintenance; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Maps; Measures; Mediating; Membrane; Membrane Proteins; Messenger RNA; Metals; Molecular; Molecular Structure; Mutagenesis; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Pathway interactions; Pharmacology; Phase; Physiological Processes; Play; Process; Production; Prostate; Proteins; Protocols documentation; Research; Resistance; Resolution; Role; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structural Protein; Structure; Testing; Testosterone; Therapeutic; Therapeutic Intervention; Tissue Sample; Work; Zinc; base; cancer cell; cryogenics; cytotoxic; dietary; egg; electron diffraction; experimental study; immune function; insight; insulin secretion; member; nanomolar; neurotransmission; novel; novel therapeutic intervention; particle; prostate cancer cell; protein structure; proteoliposomes; proteostasis; solute; stopped-flow fluorescence; tool; tumor; uptake

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.

项目总结/摘要 Zrt-，Irt-样蛋白（ZIP，或溶质载体39（SLC 39））家族的膜蛋白介导锌流入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、胰岛素抵抗等。 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。因为这两种过度 缺乏锌水平是细胞毒性的，锌稳态在哺乳动物细胞中通过丰富的 作为锌输入者（ZIP /SLC 39家族）或锌输出者（ZnT /SLC 30）的膜蛋白网络 家庭）。锌稳态蛋白是神经退行性疾病的潜在药理学靶点， 癌症，但进展受到缺乏结构和机制信息的阻碍。无全长哺乳动物 ZIP家族成员的结构已经被解决，并且不存在体外功能测定。ZIP9（SLC39A9） 代表了前列腺癌的一种新的治疗途径3。前列腺中锌的浓度最高， 它能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。前列腺锌水平不能用饮食来挽救 5，6.最近，ZIP 9被鉴定为膜雄激素受体（mAR），其介导 睾酮诱导的睾丸激素缺乏症7. ZIP 9在前列腺癌中上调，但要实现其作为治疗前列腺癌的潜力， 药理学靶点需要完整的结构和功能表征。我的目的是阐明 ZIP 9的结构和机制。目的1是开发一种基于蛋白脂质体的功能测定方法， 停流荧光光谱法和定点诱变来表征ZIP 9的机制。 目的2是利用微晶电子衍射（MicroED）确定ZIP 9内流锌的结构基础。 目的3是通过确定ZIP 9的分子和结构基础来检查ZIP 9的mAR特征。 使用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（cryoEM）。本研究将阐明ZIP介导的Zn 2+内流机制， ZIP 9作为膜雄激素受体的假定作用，拓宽了我们对锌稳态的理解， 为ZIPs作为前列腺癌及其他癌症的药理学靶点铺平了道路。