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：
9468119
负责人：
Zachary March
金额：
$ 4.4万
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 2020-09-18
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9468119
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.

项目摘要 HSP104是一种可以从酵母中的六聚体AAA+蛋白质分类酶迅速拆卸的无序聚集体，前蛋白低聚物，淀粉样蛋白和私人。这些活动使酵母可以利用自适应目的。但是，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，这是基础的几个致命的神经退行性疾病，包括帕金森氏病（PD）和肌萎缩性侧索硬化症（ALS）。我们以前设计了增强 HSP104变体拮抗与几种与之相关的几种蛋白质错误折叠的变体神经退行性疾病（在ALS中实施的TDP-43和αSyn实施 PD）。但是，这些变体缺乏底物特异性，在某些方面可能有毒情况。这，了解HSP104如何选择基材仍然是重要目标。 HSP104同源物都在所有非金属杂志中找到真核生物和精子。但是，绝大多数这些同源物保持出乎意料。我已经确定了潜水员的HSP104同源物谱系，包括原生动物，真菌和植物，是TDP-43的选择性修饰符和αSyn蛋白毒性错误折叠。根据我的初步发现，我假设 HSP104同源物之间的底物识别和结合差异支撑其底物选择性。为了检验这一假设，我将利用分子进化算法识别调节HSP104的序列基序底物选择性。我将使用纯蛋白质生物化学来评估底物选择性HSP104直系同源人员和工程变体，以分开体外蛋白质聚集体，并获得直接的机械洞察力并定义 HSP104基材选择的参数。最后，我将评估该疗法我通过（1）介绍选择性HSP014的潜力 αSyn选择性HSP104S进入帕金森氏病的秀丽隐杆线虫模型和监测蠕虫中多巴胺能神经元的保护和（2）引入 TDP-43选择性的HSP104S进入D. melanogaster tdp-43- opathy和监测谎言的寿命和运动神经元功能。这些研究构成了发展增强的分类酶的重要一步蛋白质在神经退行性疾病中的折叠率错误。