Metals and Protein Structure in Protein-Folding Diseases

蛋白质折叠疾病中的金属和蛋白质结构

• 批准号: 6558040
• 负责人: LISA M MILLER
• 金额: $ 20.2万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-15 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6558040
• 关键词: Alzheimer's disease; X ray spectrometry; amyloid proteins; binding sites; brain; copper; fluorescence microscopy; heavy metals; image processing; infrared spectrometry; iron; metal complex; metalloproteins; molecular pathology; nuclear magnetic resonance spectroscopy; prions; protein binding; protein folding; protein structure; scrapie; zinc

DESCRIPTION (provided by applicant): A growing number of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, and Creutzfeldt-Jakob disease (CJD), involve the misfolding of normal proteins in the brain, which has recently been associated with the binding of metal ions such as iron, copper, and zinc. It is thought that metal dyshomeostasis is involved in protein misfolding and leads to oxidative damage and neuron degeneration. Yet, the functions of these metal ions and the misfolded proteins in the disease process are not well understood. To date, metal concentrations in brain tissue are generally measured via macroscopic (bulk) techniques, which cannot provide any spatial information on localized metal accumulation. Stains and/or fluorescently-tagged antibodies are used to identify misfolded proteins in tissue, but do not provide direct information on the protein's structure. Structural studies involving metal-binding and protein misfolding are primarily done in vitro. Thus, this proposal aims to bridge the gap between macroscopic methods of analyzing brain tissue and in vitro studies of metal-protein binding. It describes the development and utilization of a combination of spectroscopic imaging tools that directly investigate metal content and protein structure within intact tissues. The overall goal of this proposal is to obtain an in situ structural and mechanistic picture of how metal ions in the brain are involved in protein misfolding and aggregation in two protein-folding diseases: Alzheimer's disease and scrapie. We hypothesize that elevated concentrations of metal ions (notably Cu, Fe, and Zn) in the brain are involved in the disease pathogenesis. Using x-ray microspectroscopy, we will image the metal ion distribution, concentration, oxidation state, and local structure as a function of disease severity in intact brain tissue. By combining fluorescence microscopy and infrared microspectroscopy, we will image the location and secondary structure of the associated misfolded proteins. By combining these results, we will identify which metal ions accumulate before, concurrently, or after protein misfolding. We will test whether metal ion reduction is correlated with oxidation markers such as lipid peroxidation in the brain. Finally, we use the local structure and homogeneity of the in situ metal-protein complex to develop a possible mechanism for the complex formation and toxicity.

描述（由申请人提供）：越来越多的神经退行性疾病，包括阿尔茨海默病（AD）、帕金森病、亨廷顿病和克雅氏病（CJD），涉及脑中正常蛋白质的错误折叠，最近发现其与金属离子（如铁、铜和锌）的结合有关。 据认为，金属稳态失调涉及蛋白质错误折叠，并导致氧化损伤和神经元变性。 然而，这些金属离子和错误折叠的蛋白质在疾病过程中的功能还没有得到很好的理解。 迄今为止，脑组织中的金属浓度通常通过宏观（批量）技术测量，其不能提供关于局部金属积累的任何空间信息。 染色和/或荧光标记抗体用于识别组织中的错误折叠蛋白质，但不能提供有关蛋白质结构的直接信息。涉及金属结合和蛋白质错误折叠的结构研究主要在体外进行。因此，该建议旨在弥合脑组织宏观分析方法与金属-蛋白质结合体外研究之间的差距。 它描述了直接研究完整组织内金属含量和蛋白质结构的光谱成像工具组合的开发和利用。 这项提案的总体目标是获得一个原位的结构和机制的图片，在大脑中的金属离子是如何参与蛋白质的错误折叠和聚集在两个蛋白质折叠疾病：阿尔茨海默病和羊瘙痒症。 我们推测，脑中金属离子（特别是Cu，Fe和Zn）浓度升高参与了疾病的发病机制。 使用X射线显微光谱学，我们将成像金属离子分布，浓度，氧化状态和局部结构作为完整脑组织中疾病严重程度的函数。 通过结合荧光显微镜和红外显微光谱，我们将图像的相关错误折叠蛋白质的位置和二级结构。 通过结合这些结果，我们将确定哪些金属离子在蛋白质错误折叠之前，同时或之后积累。 我们将测试金属离子还原是否与氧化标记物相关，如大脑中的脂质过氧化。 最后，我们利用原位金属-蛋白质复合物的局部结构和均匀性来开发复合物形成和毒性的可能机制。