Regulation of Mammalian Copper Homeotasis

说明（申请人提供）：铜是人体必需的营养素。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稳态的调节机制知之甚少。几种重要的酶都需要铜，然而，当铜浓度过高时，它也是有毒的。因此，铜稳态机制必须提供足够的铜来满足细胞的需要，同时防止这种营养物质的过度积累。哺乳动物细胞中的铜摄取通过膜跨越蛋白hCtr1发生。目前，我们对hctr1介导的铜摄取是否受不同铜可用性的调节了解有限。相比之下，我们对哺乳动物细胞中铜的输出是如何调节的有更广泛的了解。两种铜atp酶，ATP7A和ATP7B，通常位于反式高尔基网络中，被升高的铜刺激重新定位到细胞质囊泡或质膜上，促进铜从细胞质外排。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。升高的铜刺激质膜上hCtr1的快速内吞，这与转运体的降解有关。我们假设这一过程可能是哺乳动物细胞中高亲和力铜摄取调节的主要手段。然而，潜在的分子机制和信号尚未被定义。我们的长期目标是了解调控hctr1依赖性铜摄取的分子基础。为实现这一总体目标，我们提出以下具体目标：定义铜刺激下hCtr1的胞内吞噬和降解途径。2. 鉴定hCtr1中对铜摄取、铜诱导的内吞和降解有重要作用的氨基酸。3. 评估hCtr1的定位、内吞和降解是否对细胞内铜水平有反应。4. 确定hCtr1蛋白是否在一系列细胞类型中经历铜刺激的内吞作用和降解。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用性的变化。铜在阿尔茨海默病、朊病毒疾病和几种遗传疾病中的意义表明，对hCtr1的研究可能对改善人类健康具有深远的意义。

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.