Defining the molecular basis of substrate selection by diverse Hsp104 homologues

通过不同的 Hsp104 同系物定义底物选择的分子基础

基本信息

批准号：
9755527
负责人：
Zachary March
金额：
$ 2.91万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-19 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9755527
关键词：
ATP phosphohydrolase Affinity Alabama Algorithms Amyloid Amyotrophic Lateral Sclerosis Binding Biochemical Biological Assay Caenorhabditis elegans Cellular biology Client Collaborations Disease Disease model Drosophila genus Engineering Eubacterium Eukaryota Genetic Goals Homologous Gene Human In Vitro Lead Longevity Measures Missense Mutation Modeling Modification Molecular Molecular Evolution Monitor Nerve Degeneration Neurodegenerative Disorders Parkinson Disease Pathogenesis Phenotype Plants Prions Protein Biochemistry Proteins Protozoa Saccharomyces cerevisiae Substrate Specificity System Testing Therapeutic Universities Variant Yeasts base combat design dopaminergic neuron fungus human disease innovation insight misfolded protein motor neuron function protein TDP-43 protein aggregate protein aggregation protein misfolding proteostasis proteotoxicity therapeutic candidate unfoldase yeast prion

项目摘要

Project Summary Hsp104 is a hexameric AAA+ protein disaggregase from yeast that can rapidly disassemble disordered aggregates, preamyloid oligomers, amyloids, and prions. These activities have allowed yeast to harness beneficial prions for adaptive purposes. However, humans and metazoan lack a direct Hsp104 homologue, and are vulnerable to protein misfolding, which underpins several fatal neurodegenerative diseases including Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). We previously engineered potentiated Hsp104 variants that antagonize misfolding of several proteins associated with neurodegenerative disease (TDP-43 implicated in ALS and αSyn implicated in PD). However, these variants lack substrate specificity and can be toxic in some circumstances. Thus, understanding how Hsp104 selects substrates remains an important objective. Hsp104 homologues are found in all nonmetazoan eukaryotes and eubacteria. However, the vast majority of these homologues remain unexplored. I have established that Hsp104 homologues from diverse lineages, including protozoa, fungi, and plants, are selective modifiers of TDP-43 and αSyn proteotoxic misfolding. Based on my preliminary findings, I hypothesize that differences in substrate recognition and binding among Hsp104 homologues underpins their substrate selectivity. To test this hypothesis, I will leverage molecular evolution algorithms to identify sequence motifs that modulate Hsp104 substrate-selectivity. I will use pure protein biochemistry to evaluate the ability of substrate-selective Hsp104 orthologues and engineered variants to disaggregate protein aggregates in vitro, and to gain direct mechanistic insight into and define parameters of Hsp104 substrate selection. Finally, I will evaluate the therapeutic potential of the selective Hsp014s I identify and engineer by (1) introducing the αSyn-selective Hsp104s into a C. elegans model of Parkinson's disease and monitoring protection of dopaminergic neurons in the worm and (2) introducing TDP-43-selective Hsp104s into a D. melanogaster model of TDP-43-opathy and monitoring the lifespan and motor neuron function of lies. These studies constitute an important step toward evolving enhanced disaggregases to combat protein misfolding in neurodegenerative disease.

项目概要 Hsp104 是一种来自酵母的六聚体 AAA+ 蛋白解聚酶，可以快速分解无序的聚集体、前淀粉样蛋白寡聚体、淀粉样蛋白和这些活动使酵母能够利用有益的朊病毒。然而，人类和后生动物缺乏直接的Hsp104。同源物，并且容易受到蛋白质错误折叠的影响，这是多种蛋白质的基础致命的神经退行性疾病，包括帕金森病 (PD) 和我们之前设计了增强型肌萎缩侧索硬化症（ALS）。 Hsp104 变体可拮抗与相关蛋白的错误折叠神经退行性疾病（TDP-43 与 ALS 相关，αSyn 与 ALS 相关）然而，这些变体缺乏底物特异性，并且在某些情况下可能有毒。因此，了解 Hsp104 如何选择底物仍然是一个问题。 Hsp104 同源物存在于所有非后生动物中。然而，绝大多数这些同源物。我已经确定 Hsp104 同源物来自不同的地方。谱系，包括原生动物、真菌和植物，是 TDP-43 的选择性修饰剂和 αSyn 蛋白毒性错误折叠。根据我的初步发现，我。 Hsp104 同源物之间底物识别和结合的差异为了验证这个假设，我将利用它们的底物选择性。用于识别调节 Hsp104 的序列基序的分子进化算法我将使用纯蛋白质生物化学来评估底物选择性的能力。底物选择性 Hsp104 直系同源物和工程变体以分解蛋白质在体外聚集，并获得直接的机制洞察和定义最后，我将评估 Hsp104 底物选择的参数。我通过 (1) 介绍选择性 Hsp014 的潜力来识别和设计 αSyn 选择性 Hsp104 进入帕金森病的秀丽隐杆线虫模型和监测线虫中多巴胺能神经元的保护，以及（2）引入 TDP-43 选择性 Hsp104 进入 TDP-43 病黑腹果蝇模型和这些研究监测谎言的寿命和运动神经元功能。发展的一个重要步骤是加强分类以打击神经退行性疾病中的蛋白质错误折叠。