Mechanism of Aggregation and Disaggregation of Huntingtin

项目摘要/摘要 亨廷顿病(HD)是一种致命的神经退行性疾病，影响身体、精神和情感 大约每10,000人中就有1人的状态。这种疾病没有治愈或有效的治疗方法，这是一个因素 这直接源于对这种疾病背后的机制缺乏了解。HD是由 由Huntingtin外显子1(Htt_EX1)的多谷氨酰胺(PolyQ)结构域基因突变所致。这种突变 导致多发性肌束病理性扩张(&gt；36Q)，原因不明，这 膨胀会改变蛋白质的功能，使其变得有毒，容易发生错误折叠和 聚合。为了理解PolyQ扩展是如何导致疾病的，我将结合使用 用光谱技术鉴定和表征受亨廷顿蛋白(Htt)影响的分子特征 单体和纤维。单体和纤维是聚集途径中的重要构象， 是主要的毒性来源。具体地说，我的目标是研究这些基因的C-末端结构域是如何 构象受多聚Q扩展的影响，以及这个结构域(C-末端)是否促进 HTT和伴侣DNAJB1。越来越多的证据，包括我们实验室最近的发现，都暗示了 这一区域在有毒Htt原纤维物种的整体组织中的重要性。这一区域的特征 因此，可能掌握着理解Htt聚集机制的关键，并揭示了潜在的目标 纤维的解聚。在目标1中，我将描述突变体(&gt；36q)和野生型(&lt；35q)的结构。 固体核磁共振单体及其创新的样品制备 允许捕获处于单体状态的Htt的方法。在此过程中，我计划发现构象 参与HD发病机制的变化。在目标2中，我将利用单核磁共振和电子顺磁 共振(EPR)，以确定促进伴侣DNAJB1识别Htt原纤维的位点。 这类站点的识别将是理解聚集识别和 解体。最终，我的发现将使我们能够更深入地了解推动 亨廷顿病中蛋白质错误折叠、解聚和毒性的机制，从而提供了关键 有效疗法发展的目标。

PROJECT SUMMARY/ABSTRACT Huntington's disease (HD) is a fatal neurodegenerative disorder affecting the physical, mental, and emotional state of approximately 1 in 10,000 individuals. There is no cure or effective treatment for this disease, a factor that stems directly from the lack of knowledge regarding the mechanism underlying this disorder. HD is caused by a genetic mutation in the polyglutamine (polyQ) domain of Huntingtin exon 1 (Htt_ex1). This mutation causes the polyQ tract to become pathologically expanded (>36Q), and for reasons that remain unknown, this expansion alters the function of the protein and causes it to become toxic and prone to misfolding and aggregation. In order to understand how a polyQ expansion leads to disease, I will utilize a combination of spectroscopic techniques to identify and characterize the molecular features that are affected in huntingtin (Htt) monomers and fibrils. Monomers and fibrils represent important conformers in the aggregation pathway and are prominent sources of toxicity. Specifically, I aim to investigate how the C-terminal domain of these conformers is affected by a polyQ expansion and whether this domain (C-terminus) facilitates the interaction of Htt and the chaperone DnaJB1. Mounting evidence, including recent findings from our lab, has alluded to the importance of this region in the overall organization of toxic Htt fibril species. Characterization of this region may therefore hold the key to understanding the mechanism of Htt aggregation and reveal potential targets for the disaggregation of fibrils. In aim 1, I will characterize the structure of mutant (>36Q) and wild type (<35Q) monomers using solid-state Nuclear Magnetic Resonance (ssNMR) and an innovative sample preparation method that allows for the trapping of Htt in its monomeric state. In doing so, I plan to uncover conformational changes that contribute to the pathogenesis of HD. In aim 2, I will utilize ssNMR and Electron Paramagnetic Resonance (EPR) to identify the sites that facilitate Htt fibril recognition by the chaperone DnaJB1. Identification of such sites will be key in understanding the mechanism underlying aggregate identification and disaggregation. Ultimately, my findings will allow for greater insight into the molecular features driving the mechanism of protein misfolding, disaggregation, and toxicity in Huntington's disease, and thereby provide key targets for the development of efficacious therapeutics.