Molecular mechanism of ATP-dependent copper transporters

描述(申请人提供)：这些研究的长期目标是了解调节人类细胞中铜浓度的基本机制。铜在人体生理中起着重要的作用，是胚胎发育、呼吸、神经递质生物合成等重要生理过程所必需的。人类铜转运酶ATP7A和ATP7B在细胞内铜的调节中起着核心作用。这些转运蛋白的突变分别导致严重的代谢紊乱、门克斯病和威尔逊病。最近的研究还表明，ATP7A(Menkes病蛋白，MNKP)和ATP7B(威尔逊病蛋白，WNDP)参与了细胞对铂类化疗药物的耐药，因此可能成为癌症治疗的重要药理靶点。尽管MNKP和WNDP在人体生理中起着重要的作用，但我们对这些由ATP驱动的转运蛋白如何调节细胞内铜的了解极其有限。在拟议的一系列实验中，利用PI实验室建立的一套独特的生化工具来剖析控制WNDP和MNKP在细胞中的功能和调节的特定分子机制。提出了四个具体目标。第一个目标是通过表征WNDP和MNKP蛋白N-末端结构域中多个金属结合位点的功能作用，来全面了解这些蛋白对铜的依赖调节。第二个目标是获得必要的结构信息，以了解WNDP和MNKP与核苷酸的结合以及开发这些转运蛋白的特定抑制剂。第三，最近发现的一种蛋白质Murr1在WNDP的功能和调节中的作用将使用生物化学和细胞生物学方法相结合来确定。最终目的是剖析MNKP/WNDP相关顺铂耐药的分子机制。这些研究将产生有关铜在人体细胞中的调节的重要机制信息，并将有助于更好地理解和治疗与铜失衡相关的疾病。

DESCRIPTION (provided by applicant): The long-term goal of these studies is to understand the fundamental mechanisms that regulate the concentration of copper in human cells. Copper plays an essential role in human physiology and is required for embryonic development, respiration, neurotransmitter biosynthesis, and other important physiological processes. Human copper-transporting ATPases ATP7A and ATP7B play a central role in the regulation of intracellular copper. Mutations in these transporters result in the severe metabolic disorders, Menkes disease and Wilson's disease, respectively. Recent studies also suggest that ATP7A (Menkes disease protein, MNKP) and ATP7B (Wilson's disease protein, WNDP) are involved in cellular resistance to platinumbased chemotherapeutic drugs, and therefore could be important pharmacological targets in cancer treatment. Despite the essential role of MNKP and WNDP in human physiology, our knowledge of how these ATP-driven transporters regulate intracellular copper is extremely limited. In the proposed series of experiments, a unique set of biochemical tools established by the PI's laboratory is utilized to dissect specific molecular mechanisms that control the function and regulation of WNDP and MNKP in cells. Four specific aims are proposed. The first objective is to generate a comprehensive picture of copper-dependent regulation of WNDP and MNKP by characterizing the functional roles of the multiple metal-binding sites in the N-terminal domain of these proteins. The second goal is to obtain structural information necessary for an understanding of nucleotide binding by WNDP and MNKP and for the development of specific inhibitors for these transporters. Thirdly, the role of a recently discovered protein Murr1 in the function and regulation of WNDP will be determined using a combination of biochemical and cell biological approaches. The final objective is to dissect the molecular mechanism of the MNKP/WNDP-related cisplatin resistance. These studies will yield important mechanistic information about the regulation of copper in human cells and will contribute to a better understanding and treatment of disorders associated with copper misbalance.