The role of ZIP12 in zinc homeostasis and associated neurodegenerative pathologies

锌是一种必需的微量营养素，参与催化和结构功能，几乎涉及到 细胞中的每一个新陈代谢过程。而神经元锌离子分布的改变与 有多种疾病状态，包括阿尔茨海默病(AD)、肌萎缩侧索硬化症、精神分裂症和抑郁症， 神经元内锌离子稳态的分子机制在很大程度上尚不清楚。锌和铁-REG- 调节转运蛋白(ZIP)介导第一排过渡金属进入细胞质。 HZIP12是人类14个拉链之一，是在大脑中表达的一种锌离子摄取转运蛋白。HZIP12 CSNPs(编码单核苷酸多态)与阿尔茨海默病和AD相关。 尾状核。重要的是，我们之前对同源hZIP4的结构功能研究使 对观察到的cSNP可能引起的功能改变的假设。这项建议是基于 假设对hZIP12和与神经退行性疾病相关的变体的研究将作为 这是建立神经元锌离子稳态的可测试机制模型的切入点。Aim One测试 假设与神经元疾病相关的hZIP12突变影响转运活性和/或超 面部表情。HZIP12介导的锌离子转运将在神经元SH-SY5Y细胞中被检测到 野生型和突变蛋白。膜靶向和二聚化研究将表征预测的 观察到的cSNPs的结构效应。给出了三种变异体的结构和功能后果。 将检查蚂蚁：1)一个与AD有关的cSNP，2)两个与形态改变相关的cSNP 和3)两个cSNP假设改变金属的选择性。目标二测试hy- 假设hZIP12功能障碍改变了细胞内的锌离子池和金属分布。胞内锌离子水平， 以及跨膜转运蛋白的表达，将在早期稳定状态和非 有毒的锌离子水平。为此，将采用有针对性和不偏不倚的全球方法：1)规模 排除层析-电感耦合等离子体质谱(SEC-ICPMS)分析 蛋白质组将与质谱仪(LC-MS/MS)相结合，以鉴定锌离子结合蛋白。2) 参与反应的补偿基因的表达将通过无偏见的方式进行评估 已知与锌离子有关的基因的RNA-Seq和直接Q-PCR方法 动态平衡/分配这些研究的结果将开创分子和 脑内锌离子动态平衡的亚细胞机制，提供了hZIP12的详细描述 在神经元锌离子亚细胞分布中的作用，并阐明hZIP12在 神经退行性疾病。

Zinc is an essential micro-nutrient that participates in catalytic and structural functions touching nearly every metabolic process in the cell. While alterations in neuronal Zn2+ distribution have been associated with multiple disease states including Alzheimer’s Disease (AD), ALS, schizophrenia, and depression, the molecular mechanism of Zn2+ homeostasis in neurons is largely unexplored. The Zinc and Iron-reg- ulated transport Proteins (ZIP) mediate the entrance of first row transition metals into the cytoplasm. hZIP12, one of the fourteen human ZIPs, is a Zn2+ uptake transporter expressed in the brain. hZIP12 cSNPs (coding single nucleotide polymorphisms) have been associated with AD and abnormalities in the caudate nucleus. Importantly, our previous structural functional studies of the homologous hZIP4 enables postulation of the likely functional alteration introduced by observed cSNP. This proposal is based on the hypothesis that studies of hZIP12 and variants associated with neurodegenerative diseases will serve as an entrance point to build testable mechanistic models of neuronal Zn2+ homeostasis. Aim one tests the hypothesis that hZIP12 mutations associated with neuronal diseases impact transport activity and/or sur- face expression. hZIP12-mediated Zn2+ transport will be measured in neuronal SH-SY5Y cells expressing wild type and mutated proteins. Membrane targeting and dimerization studies will characterize predicted structural effects of observed cSNPs. The structural and functional consequence of three types of vari- ants will be examined: 1) one cSNP linked to AD, 2) two cSNPs associated with morphological alterations of the caudate nucleus; and 3) two cSNPs hypothesized to alter metal selectivity. Aim two tests the hy- pothesis that hZIP12 dysfunction modifies cellular Zn2+ pools and metal distribution. Cytosolic Zn2+ levels, as well as transmembrane transporter expression, will be assessed under early steady state and non- toxic Zn2+ levels. To this end, targeted as well as unbiased global approaches will be employed: 1) size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) profiling of the proteome will be combined with mass spectrometry (LC-MS/MS) to identify Zn2+ binding proteins. 2) The expression of compensatory genes participating in the response will be evaluated via unbiased approaches (RNA-Seq) as well as directed Q-PCR of genes known to be involved in Zn2+ homeostasis/distribution. Results from these studies will initiate the definition of molecular and subcellular mechanisms of Zn2+ homeostasis in the brain, provide a detailed description of hZIP12 function in neuronal Zn2+ subcellular distribution, and elucidate the role of hZIP12 in relevant neurodegenerative diseases.