MAMMALIAN COPPER TRANSPORT, HOMEOSTASIS, AND ITS DEFECTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 铜是对正常生长和发育至关重要的酶的辅因子。虽然铜是一种基本的微量营养素，但铜的过度积累是有害的。铜代谢的遗传性疾病、与铜有关的退行性疾病和营养性铜缺乏提供了强有力的证据，表明稳态铜代谢是一个关键的生理过程。鉴于铜跨膜转运是铜代谢的中心步骤，明确铜转运蛋白的功能、作用方式和调控可能有助于更好地了解铜的动态平衡。我们的研究以及其他研究人员的实验表明，铜转运蛋白1(CTR1)家族是细胞摄取铜所必需的，它在从酵母到人类的真核生物中高度保守。然而，CTR1介导的铜运输的机制和调控对于铜的最佳获取可能是至关重要的，这一点仍然有待确定。为了表征与铜转运相结合的CTR1中的分子事件，我们使用了一种结合生理学、生物化学、细胞生物学、生物物理学和遗传学的多学科方法，使用酵母、哺乳动物细胞系和小鼠。为了将CTR1中的分子事件与其复杂的调控模式直接联系起来，我们建立了一种原位监测CTR1的构象变化和蛋白质-蛋白质相互作用的方法。我们的数据表明，CTR1的构象、活性、表达水平和亚细胞定位是以铜依赖的方式精细控制的。这支持了我们的中心假设，即CTR1的几层翻译后调节维持最佳的细胞铜获取。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Copper serves as a cofactor of enzymes that are vital for normal growth and development. While copper is an essential micronutrient, excess accumulation of copper is detrimental. Genetic disorders in copper metabolism, copper-implicated degenerative diseases, and nutritional copper deficiency provide striking evidence that homeostatic copper metabolism is a critical physiological process. Given that copper transport across the plasma membrane is a central step in copper metabolism, defining the function, mode of action, and regulation of copper transporter could lead to better insights into copper homeostasis. Our studies, along with experiments by other investigators, have shown that the copper transporter 1 (Ctr1) family of integral membrane proteins, which is highly conserved in eukaryotes ranging from yeast to humans, is necessary for cellular copper uptake. However, the mechanism and regulation of Ctr1-mediated copper transport that is likely critical for optimal copper acquisition remains to be determined. To characterize molecular events in Ctr1 that are coupled with copper transport, we have employed a multi-disciplinary approach combining physiology, biochemistry, cell biology, biophysics, and genetics using yeast, mammalian cell lines, and mice. To directly relate molecular events in Ctr1 with its complex modes of regulation, we have developed a method for monitoring conformational changes in Ctr1 and protein-protein interactions of Ctr1 in situ. Our data shows that the conformation, activity, expression levels, and subcellular localization of Ctr1 are delicately controlled in a copper-dependent manner. This supports our central hypothesis that several layers of post-translational regulation of Ctr1 maintain optimal cellular copper acquisition.