Molecular Aspects of Copper Binding to the Prion Protein

描述（由申请人提供）：本研究计划的目的是在分子水平上阐明铜在朊病毒蛋白中结合的机制和功能，以及这种相互作用与朊病毒介导的神经系统疾病传播的关系。朊病毒蛋白（PrP）的错误折叠形式导致一类称为传染性海绵状脑病（TSE）的致命神经退行性疾病，其中包括人类的疯牛病和克雅氏病。PrP是在所有哺乳动物和鸟类物种中发现的膜结合糖蛋白。尽管对这种非凡的蛋白质进行了近25年的研究，但其正常的生理功能仍然未知。然而，最近的发现表明，PrP结合铜与高亲和力在其保守的N-末端octrepeat结构域，这种相互作用赋予一个未知来源的神经保护功能。在上一个资助期间，Pi的实验室确定了所有相关的Cu（II）位点，在该区域发现了一个与错误折叠相关的新位点，并提供了铜占据全长PrP的详细分子表征。此外，它表明，铜（II）的协调环境的变化显着取决于铜蛋白质的精确比例。拟议的研究将建立在这些发现的基础上，使用蛋白质设计，生物物理技术，如电子顺磁共振和神经细胞培养。目的#1侧重于确定野生型PrP和与疾病相关的八重复扩增突变体中的铜结合亲和力和协同性。目标#2将使用蛋白质设计来选择特定的铜结合模式。反过来，这些突变体将在细胞培养中进行测试，以确定与神经保护功能相关的PrP形式。目标#3将研究铜如何影响合成朊病毒的组装动力学和结构。这些研究将确定铜是否在疾病中发挥作用。目的#4将开发PrP融合蛋白，旨在优化X射线结构测定的结晶。TSE与流行的、与年龄相关的神经退行性疾病（如阿尔茨海默病和帕金森病）有共同的病理学特征。此外，朊病毒在食物中或通过医疗程序传播的可能性使TSE成为持续的健康威胁。因此，这项研究计划将有助于改善公共健康和安全，并将为与衰老相关的神经退行性疾病提供重要的新见解。

DESCRIPTION (provided by applicant): The objective of this research program is to elucidate, at the molecular level, the mechanism and function of copper binding in the prion protein and the relationship of this interaction to the propagation of prion mediated neurological disease. A misfolded form of the prion protein (PrP) is responsible for a class of fatal neurodegenerative diseases termed the Transmissible Spongiform Encephalopathies (TSEs), which include mad cow disease and Creutzfeldt-Jakob disease in humans. PrP is a membrane bound glycoprotein found in all mammals and avian species. Despite nearly twenty-five years of research on this remarkable protein, its normal physiological function remains unknown. Recent discoveries, however, demonstrate that PrP binds copper with high affinity in its conserved N-terminal octarepeat domain, and that this interaction confers a neuroprotective function of unknown origin. Over the last funding period, the Pi's laboratory identified all relevant Cu(ll) sites, discovered a new site in the region associated with misfolding and provided a detailed molecular characterization of copper occupied full-length PrP. Moreover, it was shown that the Cu(ll) coordination environment changes significantly depending on the precise ratio of copper to protein. The proposed research will build on these discoveries using protein design, biophysical techniques such as electron paramagnetic resonance, and neuronal cell culture. Aim #1 focuses on determining the copper binding affinity and cooperativity in wild type PrP and in mutants with octarepeat expansions associated with disease. Aim #2 will use protein design to select for specific copper binding modes. In turn, these mutants will be tested in cell culture to identify the form of PrP associated with neuroprotective function. Aim #3 will examine how copper influences the assembly kinetics and structure of synthetic prions. These studies will determine whether copper plays a role in disease. Aim #4 will develop of PrP fusion proteins designed to optimize crystallization for X-ray structure determination. The TSEs share pathologies with prevalent, age- related neurodegenerative illnesses such as Alzheimer's and Parkinson's disease. In addition, the potential of prions in food or transmission through medical procedures make the TSEs a continuing health threat. Consequently, this research program will help improve public health and safety, and will provide critical new insights into neurodegenerative diseases associated with aging.