Regulation of Mammalian Copper Homeotasis

说明(申请人提供)：铜是一种必需的营养物质。然而，尽管铜对人类营养很重要，但人们对哺乳动物细胞中铜的稳态调节机制知之甚少。铜是几种重要的酶所必需的，然而，当铜的浓度过高时，它也是有毒的。因此，铜的动态平衡机制必须提供足够的铜来满足细胞的需求，同时防止这种营养的过度积累。哺乳动物细胞对铜的摄取是通过跨膜蛋白hCtr1实现的。目前，我们对hCtr1介导的铜摄取是否受铜供应变化的调控知之甚少。相比之下，我们对铜出口如何在哺乳动物细胞中进行调控有更广泛的了解。两个铜ATPase ATP7A和ATP7B通常位于跨高尔基体网络中，它们被升高的铜刺激重新定位到细胞质小泡或质膜上，以促进铜从细胞质中外流。在我们的初步研究中，我们发现hCtr1蛋白的位置受铜浓度的调节。高浓度的铜刺激细胞膜上hCtr1的快速内吞，这与转运蛋白的降解有关。我们推测，这一过程很可能是哺乳动物细胞高亲和力铜摄取的主要调节手段。然而，潜在的分子机制和涉及的信号还没有被定义。我们的长期目标是了解调控依赖hCtr1的铜摄取的分子基础。为了实现这一总体目标，我们提出了以下具体目标：1.确定铜刺激hCtr1内吞和降解的细胞内途径。2.鉴定hCtr1中与铜摄取、铜诱导的内吞和降解有关的氨基酸。3.评估hCtr1的定位、内吞和降解是否与细胞内铜水平有关。4.确定hCtr1蛋白在多种细胞类型中是否经历铜刺激的内吞和降解。我们的研究将极大地有助于理解细胞如何感知和响应铜可利用性的变化。铜在阿尔茨海默氏症、普里恩病和几种遗传性疾病中的作用表明，hCtr1的研究可能对改善人类健康具有深远的影响。

DESCRIPTION (provided by applicant): Copper is an essential nutrient. However, despite its importance to human nutrition, little is known about the mechanisms regulating copper homeostasis in mammalian cells. Copper is required by several important enzymes, however, it is also toxic when present in excess concentrations. Thus, copper homeostasis mechanisms must supply sufficient copper to meet cellular needs, while preventing the over-accumulation of this nutrient. Copper uptake in mammalian cells occurs via the membrane-spanning protein, hCtr1. Currently, we have limited knowledge of whether hCtr1-mediated copper uptake is regulated in response to varying copper availability. In contrast, we have a more extensive knowledge of how copper export is regulated in mammalian cells. Two copper ATPases, ATP7A and ATP7B, which are normally located in the trans-Golgi network, are stimulated to relocate to cytoplasmic vesicles or the plasma membrane by elevated copper to facilitate copper efflux from the cytoplasm. In our preliminary studies, we show that the location of the hCtr1 protein is regulated by copper concentrations. Elevated copper stimulates the rapid endocytosis of hCtr1 from the plasma membrane, and this is associated with degradation of the transporter. We hypothesize that this process is likely to be the principle means by which high affinity copper uptake is regulated in mammalian cells. However, the underlying molecular mechanisms and signals involved have not yet been defined. Our long-term goal is to understand the molecular basis for regulating hCtr1-dependent copper uptake. To achieve this overall goal, we propose the following specific aims: 1.To define the intracellular pathway for copper-stimulated endocytosis and degradation of hCtr1. 2. To identify amino acids within hCtr1 important for copper uptake, copper-induced endocytosis and degradation. 3. To assess whether the localization, endocytosis and degradation of hCtr1 is responsive to intracellular copper levels. 4. To determine whether the hCtr1 protein undergoes copper-stimulated endocytosis and degradation in a range of cell types. Our research will contribute greatly to understanding how cells sense and respond to changes in copper availability. The implication of copper in Alzheimer's disease, prion diseases, and several genetic disorders, suggests the study of hCtr1 may have far-reaching implications for the improvement of human health.