Probing early protein aggregation mechanisms and their relationship to disease ef

描述（由申请人提供） 摘要：蛋白质折叠和聚集的问题是许多最具破坏性的疾病的根本原因，挑战着全世界的公共卫生。因此，更令人沮丧的是，目前还没有治疗这些神经退行性疾病的方法，在许多情况下，甚至没有可靠的延迟症状。可以肯定地说，这些蛋白质开始聚集、形成可溶性寡聚体并伸长成纤维的许多分子机制尚不清楚，神经毒性的原因是否是聚集蛋白质的原始功能丧失，可溶性寡聚体可能渗透并破坏细胞膜，重要的生物空间被纤维聚集体窒息，或这些或其他因素的组合。解决这些问题的一个关键挑战是缺乏物理工具，可以瞬时表征早期蛋白质聚集事件，包括蛋白质构象变化和可溶性蛋白质寡聚体的形成，并且可以在关键生物成分存在下瞬时地并最终在体内以位点特异性这样做。我建议开发一套创新的仪器和新的方法，用于提高磁共振检测的灵敏度和选择性，与现有的核磁共振（NMR）和电子顺磁共振（EPR）实验相比，提高了两个数量级以上。我们的目标是蛋白质聚集过程中的分子界面的时间分辨可视化，在脂质膜的存在下，作为突变和化学信号的函数，例如分子伴侣和渗透剂。我们将伴随我们的体外研究，探测活细胞中的蛋白质聚集事件和跟踪活细胞的存活率。这些研究是有可能与界面蛋白质位点的选择性自旋标记和瞬态分子相互作用的灵敏检测，通过测量界面水合水动力学，这是敏感地调制后的分子方法在远程10 <$s的自旋标记的网站，以及通过增强测量电子自旋-自旋距离和动态。我将通过新颖的Overhauser动态核极化技术实现前一项创新，并通过前所未有的脉冲整形能力实现后一项创新，以显着增强脉冲EPR性能。在最初的几年里，我将专注于研究tau蛋白，它的纤维缠结在阿尔茨海默氏症和其他神经退行性疾病中被发现。我们将与两个全长人类同种型，tau-3R和tau-4 R，以及半胱天冬酶切割的tau蛋白，因为最近已经发现在体内缠结形成之前。我们的工具和策略的优势在于，它们通常适用于与神经退行性疾病有关的所有聚集蛋白。我们提出的研究重点是揭示蛋白质寡聚体和聚集体在疾病相关效应中的作用，并寻求解决一个重要的生物医学问题：开发合理的治疗方法和早期诊断神经退行性疾病。 公共卫生相关性：我建议开发一套创新的仪器和方法，用于将传统磁共振光谱工具的灵敏度提高两个以上的数量级，这将允许对现有技术基本上不可见的早期蛋白质聚集机制进行前所未有的研究。这将导致解开错误折叠的蛋白质，寡聚体或纤维沉积物与疾病相关影响的关系，并解决了一个重要的生物医学问题，即为阿尔茨海默病等神经退行性疾病制定合理的治疗方法和早期诊断策略。

DESCRIPTION (Provided by the applicant) Abstract: Problems in protein folding and aggregation are the root cause of many of the most devastating diseases, challenging public health worldwide. Therefore, it is even more devastating to understand that there is currently no treatment for these neurodegenerative diseases-in many cases not even for reliably delaying symptoms. It is safe to say that much of the molecular mechanisms by which these proteins begin to aggregate, form soluble oligomers and elongate to fiber are not understood, nor is the debate settled whether the cause of neurotoxicity is the loss of the original function of the protein that is being recruited for aggregation, the soluble oligomer that may permeate through and destroy cell membranes, the suffocation of important biological space by fibrous aggregates, or the combination of these or other factors. A key challenge for tackling these questions is the lack of physical tools that can transiently characterize early protein aggregation events, including protein conformational changes and the formation of soluble protein oligomers, and can do so with site-specificity, transiently, in the presence of key biological constituents, and ultimately in vivo. I propose to develop a set of innovative instrumentations and novel approaches for enhancing the sensitivity and selectivity of magnetic resonance detection by more than two orders of magnitude compared to existing nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) experiments. Our target is the time-resolved visualization of molecular interfaces during protein aggregation, in the presence of lipid membranes and as a function of mutation and chemical signals, e.g. of molecular chaperones and osmolytes. We will accompany our in vitro studies with probing protein aggregation events in live cells and tracking live cell survivability. These studies are made possible with selective spin labeling of interfacial protein sites and the sensitive detection of transient molecular interactions, through the measurements of interfacial hydration water dynamics that is sensitively modulated upon molecular approach within distant 10 ¿ of spin labeled sites, as well as through the enhanced measurement of electron spin-spin distances and dynamics. I will achieve the former innovation with the novel Overhauser dynamic nuclear polarization technique, and the latter with unprecedented pulse shaping capability to significantly enhance pulsed EPR performance. I will focus in the first years on the study of tau whose fibrous tangles are found in Alzheimer's and other neurodegenerative diseases. We will work with the two full length human isoforms, tau-3R and tau-4R, as well as the caspase-cleaved tau proteins, as has been recently found to precede tangle formation in vivo. The strength of our tools and strategies is that they are generally applicable to all aggregating proteins implicated in neurodegenerative diseases. Our proposed studies focus on unraveling the roles of protein oligomers and aggregates in disease-related effects, and pursue to address a significant biomedical problem: the development of rational treatments and earlier diagnosis for neurodegenerative diseases. Public Health Relevance: I propose to develop a set of innovative instrumentations and methods for enhancing the sensitivity of conventional magnetic resonance spectroscopic tools by more than two orders of magnitude that will allow for unprecedented studies of early protein aggregation mechanisms that are largely invisible to existing techniques. This will lead to unraveling the relationships of misfolded proteins, oligomers or fibrous deposits to disease-related effects, and addresses a significant biomedical problem of developing rational treatments and earlier diagnosis strategies for neurodegenerative diseases such as Alzheimer's disease.