Human Zinc Transporter Zip 13 and Cellular Zinc Homeostasis

说明(申请人提供)：锌是许多细胞过程所必需的营养物质，然而，过量的锌会导致细胞死亡。调控细胞锌稳态的一种机制涉及锌转运蛋白家族中的ZIP，它的功能是将锌输入细胞质。这项研究计划阐述了人类转运蛋白Zip13(HZip13)在维持人类细胞锌稳态中的作用。为了研究hZip13在人类细胞中的作用，将使用免疫荧光显微镜在基础条件下将hZip13定位于其在成纤维细胞和极化上皮细胞中的作用部位。此外，将监测高锌和低锌条件下hZip13的定位，以确定锌是否调节hZip13的定位。根据hZip13的基本定位信息，将使用荧光化学传感器来分析hZip13的功能，以监测锌向细胞质中的动员。报告分析也将被用来监测胞浆、内质网和分泌途径锌状态的变化。为了研究低锌条件下hZip13基因表达上调的机制，我们将对编码hZip13蛋白的基因SLC39A13启动子区域的序列元件或转录产物进行分析。核连续分析将被用来确定在低细胞锌时转录起始速率是否改变，或者hZip13 mRNA的半衰期是否因低细胞锌而改变。如果转录起始控制hZip13 mRNA的丰度，转录起始位置将通过快速扩增cDNAEnds确定，并通过S1核酸酶保护试验确认。一旦转录起始点已知，hZip13启动子区域将被用荧光素酶报告基因分析来解剖，其中系统缺失的hZipl 3启动子与荧光素酶基因融合将在低锌的人类细胞中表达。相反，如果mRNA的稳定性控制转录水平，将进行类似的实验，以确定在低锌条件下调控转录水平的重要序列元件。对控制低锌条件下hZip13基因表达的序列元件的鉴定最终将有助于确定在低锌条件下调控锌稳态的分子机制。 这项拟议的研究将确定人类锌转运蛋白Zip13在人类细胞中的功能以及它在维持锌稳态中所起的作用。这项提议将开始识别控制人类Zip13的遗传机制，以响应低细胞锌，为人类细胞如何调控锌代谢提供线索。

DESCRIPTION (provided by applicant): Zinc is an essential nutrient needed for many cellular processes, however, excess zinc leads to cell death. One mechanism to regulate cellular zinc homeostasis involves the Zrt-, Irt-like, Protein (ZIP) family of zinc transporters which function to import zinc into the cytoplasm. This research proposal addresses the role of the human transporter Zip13 (hZip13) in maintaining zinc homeostasis in human cells. To address the role of hZip13 in human cells, immunofluorescence microscopy will be used to localize hZip13 to its subcellular site of action in basal conditions in both fibroblasts and polarized epithelial cells. Furthermore, hZip13 localization will be monitored in high and low zinc conditions to determine if zinc regulates hZip13 localization. With basic localization information about hZip13, the function of hZip13 will be analyzed using fluorescent chemosensors to monitor zinc mobilization into the cytoplasm. Reporter assays will also be used to monitor changes in cytosolic, endoplasmic reticulum, and secretory pathway zinc status. To characterize the mechanism upregulating hZip13 mRNA expression levels in low zinc, sequence elements in the promoter region of SLC39A13, the gene which encodes for the hZipl 3 protein, or the mRNA transcript will be analyzed. Nuclear run-on assays will be used to determine if the rate of transcript initiation changes in low cellular zinc or whether the half-life of hZip13 mRNA half-life changes in response to low cellular zinc. Should transcription initiation control hZip13 mRNA abundance, the transcription start site will be determined using rapid amplification of cDNA ends and confirmed using S1 nuclease protection assays. Once the transcription initiation site is known, the hZip13 promoter region will be dissected using a luciferase reporter assay where systematic deletions of the hZipl 3 promoter fused to the luciferase gene will be expressed in human cells in low zinc. In contrast, should mRNA stability control transcript levels, analogous experiments will be done to identify the sequence element important for regulating transcript level in low zinc. The identification of the sequence elements controlling hZip13 mRNA expression in low zinc will eventually lead to determining the molecular mechanisms involved in regulating zinc homeostasis in low zinc conditions. The proposed study will determine the function of the human zinc transporter Zip13 in human cells and the role it plays in maintaining zinc homeostasis. This proposal will begin to identify the genetic mechanisms controlling human Zip13 in response to low cellular zinc providing clues to how human cells regulate zinc metabolism.