Engineering and evolving substrate-specific Hsp104 variants

工程和发展底物特异性 Hsp104 变体

• 批准号: 10531129
• 负责人: Jeremy Ryan
• 金额: $ 4.77万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531129
• 关键词: Aging; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Amyotrophic Lateral Sclerosis; Animal Model; Bacteria; Biosensor; Cell Line; Cells; Chemosensitization; Collection; DNA Sequence Alteration; Data; Development; Disease; Engineering; Ensure; Failure; Fluorescence Resonance Energy Transfer; Future; Generations; Goals; Homologous Gene; Human; Learning; Lewy Bodies; Libraries; Life; Link; Mammalian Cell; Modeling; Mutation; Neurodegenerative Disorders; Neuroglia; Neurons; Parkinson Disease; Pathology; Patients; Pilot Projects; Plants; Prions; Property; Protein Dynamics; Protein Engineering; Proteins; Proteome; Regulatory Pathway; Role; Structure; Substrate Specificity; Surveys; System; Techniques; Technology; Testing; Therapeutic; Toxic effect; Variant; Yeasts; alpha synuclein; amyloid fibril formation; conformer; deep sequencing; design; disease phenotype; dopaminergic neuron; effective therapy; efficacy evaluation; efficacy validation; experimental study; fungus; gain of function; high throughput analysis; human disease; improved; innovation; insight; motor neuron degeneration; novel therapeutics; protein TDP-43; protein aggregation; protein folding; protein misfolding; proteostasis; screening; trend; yeast prion; yeast protein

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.

项目摘要 蛋白质错误折叠是许多致命神经退行性疾病的基础，包括肌萎缩侧索硬化症 (ALS)帕金森病（PD）和阿尔茨海默病。目前还没有有效的蛋白质治疗方法 错误折叠疾病这些疾病中的每一种都与无序聚集体的积累有关， 淀粉样蛋白低聚物和淀粉样蛋白或淀粉样蛋白样构象异构体。虽然淀粉样蛋白通常与疾病有关， 淀粉样蛋白折叠也被用于有益的目的，因此调节途径已经发展到 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是一种保守的 来自酵母的六聚AAA+蛋白质重塑因子，溶解无序聚集体和淀粉样蛋白，但具有 对人神经变性疾病蛋白质仅有有限的活性。虽然Hsp104仅具有有限的能力， 拯救在人类细胞中聚集的蛋白质，它可以被重新改造以溶解疾病相关的蛋白质。 聚集体和淀粉样蛋白。已经发现了许多增强的Hsp104变体，其具有突变， 在整个HSP 104的中间结构域的保守和非保守残基。的应用 由于Hsp104变体在神经元中的毒性，Hsp104变体在动物模型中的研究已经停滞。在试点 研究中，我已经开发了新的筛选方法，以分离两种Hsp104变体， 与ALS和PD有关的蛋白质，而不赋予脱靶效应。此外，我还开发并 采用新的选择策略，分离具有改进性质的Hsp104变体。我的下一步是 全面调查这些变体的集合，以确定增强和 从我们的屏幕上选择趋势。我还将采用计算方法来更好地理解 Hsp104增强的基础。最后，我将建立技术，使高通量分析的大 Hsp104变体的文库，以更好地理解Hsp104的机制和Hsp104底物的基础。 的特异性然后，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。具体而言，我将提出两个目标：1） 工程化和进化底物特异性Hsp104变体和2）评估新开发的Hsp104的功效 哺乳动物细胞中的变异体。最终，我们预计，微调蛋白质解聚酶可能是 开发用于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疾病的基础， 同时对抗功能机制的丧失或获得。不管治疗潜力如何， 这些变体，它们将作为有用的探针，用于测试当错误折叠逆转时会发生什么，有助于 描述针对这些疾病的治疗目标。