金属反应转录因子-1（MTF-1）在铜和锌差异性影响黄颡鱼脂类代谢过程中的作用机制

Funded by the completed project from NSFC, occassionally we found that metal-responsive transcription factor-1 (MTF-1) was the potential key protein which combines metal elements (Cu and Zn) and lipid metabolism, but the further mechanism for MTF-1 remained unknown. Using the theory and methods of nutrition, toxicology, over-expression and siRNA, cellular transfection, double luciferase reporter system, EMSA and ChIP, quantitative RT-PCR and Western blot, the present project will explore the transcriptionally regulatory mechanism of MTF-1 and dig many key genes and proteins involved in absorption of metal elements and lipid metabolism, which have direct interactions with MTF-1. The project will also investigate the molecular mechanism of MTF-1 participating in the absorption and transport of Cu and Zn, and study the mechanism of MTF-1 mediating the Cu- and Zn-induced differential changes of lipid metabolism. Furthermore, the present project will elucidate the mechanism of MTF-1 during the Cu and Zn differentially influencing lipid metabolism of yellow catfish. The present project will build the direct bridge between metal elements and lipid metabolism, which provides powerful evidence for metal elements differentially influencing lipid metabolism, and meantime, provides the basis for investigating biological functions of MTF-1 and for exploring the roles of MTF-1 in lipid metabolism and its regulatory network in fish.

在前一个国家自然科学基金的资助下（已结题），申请者偶然发现MTF-1是连接铜和锌吸收转运与黄颡鱼脂类代谢的“桥梁”，但MTF-1的作用机制不清楚。本项目拟在前期研究基础上，采用营养学和毒理学的方法，辅以质粒过表达和siRNA干扰、细胞转染、双荧光素酶报告系统、EMSA和ChIP、荧光定量PCR和Western blot等技术，研究黄颡鱼MTF-1基因的转录调控机制；挖掘一批与MTF-1互作的、与铜和锌吸收转运及脂类代谢有关的关键基因和蛋白；探讨MTF-1调控黄颡鱼铜和锌吸收转运的分子机理；研究MTF-1介导铜和锌差异性影响黄颡鱼脂类代谢的机理及其异同，揭示MTF-1在铜和锌影响黄颡鱼脂类代谢过程中的作用机制。本项目将构建金属元素吸收转运和脂类代谢之间联系的直接“桥梁”，为金属元素影响脂类代谢的直接机制提供关键的实验证据，也为解析MTF-1的调控机制及在脂类代谢调控网络中的作用奠定基础。

MTF-1是目前已知的唯一调控锌吸收、转运和代谢的关键转录因子，然而，有关MTF-1转录调控机制、调控的靶基因及介导铜和锌影响脂类代谢的机制不清楚。本项目揭示黄颡鱼MTF-1基因的转录调控机制，发现它们的启动子上存在热应激启动子元件（HSE）和MTF-1反应元件（MRE）；铜和锌差异性影响MTF-1启动子的活性，及其mRNA和蛋白表达；鉴定得到10多个与MTF-1互作的、参与铜和锌代谢及脂类代谢的关键基因和蛋白，包括金属硫蛋白、ZnT家族和ZIP家族的一些成员，及调控脂类代谢的关键转录因子PPARr，也发现铜转运蛋白如Ctr1、Ctr2和Atox1启动子上存在MTF-1的结合元件；阐明铜通过氧化应激介导的自噬和Nrf2/PPARr通路增加黄颡鱼肝脏的脂肪沉积和代谢，纳米锌通过激活PPARr通路和生脂代谢增加黄颡鱼肠道的脂肪沉积。锌影响黄颡鱼脂肪沉积和代谢的机理，揭示MTF-1在铜和锌影响黄颡鱼脂类代谢过程中的作用机制。本项目为金属元素影响脂类代谢的直接机制提供关键的实验证据，也为解析MTF-1的调控机制及在脂类代谢调控网络中的作用奠定基础。通过本项目的研究，在J Nutr Biochem等期刊发表论文11篇（均标注本项目为第一资助），SCI论文10篇。