Engineering and evolving substrate-specific Hsp104 variants

项目摘要 蛋白质错误折叠是包括肌萎缩侧索硬化症在内的多种致命神经退行性疾病的基础 (ALS)、帕金森氏病(PD)和阿尔茨海默氏症。目前还没有治疗蛋白质的有效方法。 错折的疾病。这些疾病中的每一种都与无序聚集体的积累有关，有毒的前 淀粉样寡聚体，以及淀粉样物或淀粉样物。虽然淀粉样蛋白通常与疾病有关，但 淀粉样蛋白折叠也被用于有益的目的，因此调节途径已经进化到 促进淀粉样蛋白分解。酵母已经进化到使用淀粉样蛋白来发挥特定的作用。HSP104，一种保守的 酵母中的六聚体AAA蛋白重塑因子，溶解无序的聚集体和淀粉样蛋白，但 仅对人类神经退行性疾病蛋白有有限的活性。而HSP104仅具有有限的能力 在人类细胞中聚集的救援蛋白，它可以被重新设计来溶解与疾病相关的 聚集体和淀粉样蛋白。已发现许多增强的Hsp104变体含有突变以 Hsp104中间结构域的保守和非保守残基。应用程序 由于Hsp104变体对神经元的毒性，动物模型中的Hsp104变体已经停滞不前。在试行中 研究中，我开发了新的筛选方法来分离两个Hsp104变种，它们拯救了 与肌萎缩侧索硬化症和帕金森病有关的蛋白质，而不会产生非靶点效应。此外，我还开发了 采用了新的选择策略来分离具有改进特性的Hsp104变体。我的下一步是 综合调查这些变种的收集，以确定增强和 我们屏幕上的选择趋势。我还将使用计算方法来更好地理解 Hsp104增强的基础。最后，我将建立一些技术来实现对大型数据的高通量分析 Hsp104变异体文库，以更好地了解Hsp104的机制和Hsp104底物的基础- 专一性。然后，我将评估α-syn fret生物传感器细胞系中顶级变体的治疗潜力。 HEK293T细胞和α-SYN聚集的原代神经元模型。具体地说，我将解决两个目标：1) 设计和进化底物特异性Hsp104变体和2)评估新开发的Hsp104的疗效 哺乳动物细胞中的变种。最终，我们预计微调的蛋白质解聚酶可能是 被开发用来逆转蛋白质的错误折叠，这些错误折叠是各种蛋白质错误折叠障碍的基础，并可能 同时对抗一种作用机制的损失或获得。不管它的治疗潜力如何 这些变种，它们将作为有用的探针来测试错误折叠被反转时会发生什么，从而帮助 勾勒出针对这些疾病的治疗目标。

Project Summary Protein misfolding underpins numerous fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer’s disease. Currently there are no effective treatments for protein misfolding diseases. Each of these disorders is linked to the accumulation of disordered aggregates, toxic pre- amyloid oligomers, and amyloid or amyloid-like conformers. While amyloid is typically implicated in disease, the amyloid fold has also been employed for beneficial purposes, and so regulatory pathways have evolved to promote amyloid disassembly. Yeast have evolved to employ amyloid for specific roles. Hsp104, a conserved hexameric AAA+ protein-remodeling factor from yeast, solubilizes disordered aggregates and amyloid but has only limited activity against human neurodegenerative disease proteins. While Hsp104 only has limited ability to rescue proteins that aggregate in human cells, it can be re-engineered to solubilize disease-associated aggregates and amyloid. Numerous potentiated Hsp104 variants have been discovered that harbor mutations to both conservative and non-conservative residues throughout the middle domain of Hsp104. Application of Hsp104 variants in animal models has been stalled due to the toxicity of Hsp104 variants in neurons. In pilot studies, I have developed new screening approaches to isolate two Hsp104 variants that rescue the toxicity of proteins implicated in ALS and PD without conferring off-target effects. In addition, I have developed and employed new selection strategies to isolate Hsp104 variants with improved properties. My next steps are to comprehensively survey the collection of these variants to determine the correlation between potentiation and the selection trends from our screens. I will also employ computational approaches to better understand the basis for Hsp104 potentiation. Finally, I will establish techniques that enable high-throughput analysis of large libraries of Hsp104 variants to better understand the mechanism of Hsp104 and the basis for Hsp104 substrate- specificity. I will then assess the therapeutic potential of top variants in an α-syn FRET biosensor cell line of HEK293T cells and a primary neuron model of α-syn aggregation. Specifically, I will address two aims: 1) Engineer and evolve substrate-specific Hsp104 variants and 2) Assess the efficacy of newly developed Hsp104 variants in mammalian cells. Ultimately, we anticipate that finely-tuned protein disaggregases could be developed to reverse the misfolding of proteins that underpin diverse protein-misfolding disorders and could simultaneously counter both a loss or gain of function mechanism. Regardless of the therapeutic potential of these variants, they will serve as useful probes for testing what happens when misfolding is reversed, helping to delineate the therapeutic goals for targeting these disorders.