Zinc and the Synthesis of Zinc Binding Proteins

描述（由申请人提供）：本项目的总体长期目标仍然是了解锌结合蛋白和与锌相互作用的蛋白质参与膳食锌的吸收、代谢和生物学功能。项目目标是相互关联的。目标1。金属硫蛋白（MT）在人类和小鼠细胞和组织中表达的功能意义。使用定量PCR（Q-PCR），将在人类受试者的受控锌耗竭和锌补充研究中使用SLC 30和SLC 39家族的hMT和特异性锌转运蛋白基因的测定，以评价单核细胞和T细胞中这些基因的锌反应性。作为亚硝化应激的一个因素，将评估由膳食锌调节的鼠MT的影响。这些实验将使用MT无效和诱导型一氧化氮（NO）合酶无效基因型，并集中于培养物中的分离的肝实质细胞和NO的靶点，即，肠细胞Aim II.锌应答基因的表征。这些研究将集中在THP 1细胞作为使用螯合剂诱导的锌耗竭和补充的人单核细胞的模型。将在锌响应基因和LPS激活之间进行比较。类似的研究将比较缺锌和补锌小鼠胸腺细胞LPS的锌反应基因。将通过微阵列进行分析，并通过蛋白质组学进行确认。将比较MTF 1和MTF 2转录因子对膳食锌的反应。Aim III.小鼠锌转运蛋白及其对膳食锌摄入的反应和生理介质。IL-6应答性转运蛋白Zip 14将进一步用转染的细胞表征，并在炎性低锌血症中发挥作用。将评价急性期反应期间网状内皮系统锌转运蛋白基因的表达。胰腺腺泡细胞和潘氏细胞用于内源性锌释放的锌加工步骤将受到密切关注。将广泛使用的转运蛋白定位使用免疫组织化学和免疫细胞化学和锌荧光团可视化荧光显微镜。

DESCRIPTION (provided by applicant): The overall long-term goal of this project continues to be directed at understanding the involvement of zinc binding proteins and those proteins that interact with zinc for the absorption, metabolism, and biological function of dietary zinc. The project aims are interrelated. Aim 1. Functional Significance of Metallothionein (MT) Expression in Human and Murine Cells and Tissues. Using quantitative PCR (Q-PCR), assays for hMT and specific zinc transporter genes of both the SLC30 and SLC39 families will be used in controlled zinc depletion and zinc supplementation studies with human subjects to evaluate the zinc responsiveness of these genes in monocytes and T cells. The influence of murine MT, as regulated by dietary zinc, will be evaluated as a factor in nitrosative stress. These experiments will use MT null and inducible nitric oxide (NO) synthase null genotypes and focus on isolated liver parenchymal cells in culture and targets of NO, viz., intestinal cells. Aim II. Characterization of Zinc Responsive Genes. These studies will focus on THP1 cells as a model for human mononuclear cells using chelator-induced zinc depletion and supplementation. Comparisons will be made between zinc-responsive genes ¿ LPS activation. Similar studies will compare zinc-responsive genes of thymocytes ¿ LPS from zinc-deficient and zinc-supplemented mice. Analysis will be by microarrays, with confirmation by proteomics. Responsiveness of the MTF1 and MTF2 transcription factors to dietary zinc will be compared. Aim III. Murine Zinc Transporters and Their Response to Dietary Zinc Intake and Physiologic Mediators. The IL-6 responsive transporter Zip14 will be further characterized with transfected cells and for a role in inflammatory hypozincemia. Expression of zinc transporter genes of the reticuloendothelial system during the acute phase response will be evaluated. The zinc processing steps the pancreatic acinar cells and Paneth cells use for endogenous zinc release will receive close attention. Extensive use will be made of transporter localization using immunohistochemistry and immunocytochemistry and zinc fluorophores as visualized by fluorescence microscopy.