Defining the molecular basis of substrate selection by diverse Hsp104 homologues

项目摘要 Hsp104是酵母中的一种六聚体AAA+蛋白解聚酶，可以 快速分解无序的聚集体、前叶低聚物、淀粉样蛋白和 普里恩。这些活性使酵母能够利用有益的普鲁恩 适应目的。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，这是几个 致命的神经退行性疾病，包括帕金森氏病(PD)和 肌萎缩侧索硬化症(ALS)。我们之前设计了增强的 HSP104变异体对抗几种与以下相关的蛋白质的错误折叠 神经退行性疾病(肌萎缩侧索硬化症与tdp-43有关，α-syn与 Pd)。然而，这些变种缺乏底物特异性，在某些情况下可能是有毒的。 情况。因此，了解Hsp104如何选择底物仍然是一个 重要的目标。在所有非后生动物中都发现了Hsp104同系物 真核生物和真细菌。然而，这些同源中的绝大多数 仍未被开发。我已经确定Hsp104的同源物来自不同的 包括原生动物、真菌和植物在内的谱系是TDP-43的选择性修饰物 和αSyn蛋白毒素错误折叠。根据我的初步发现，我假设 Hsp104同系物之间底物识别和结合的差异 支撑了它们的底物选择性。为了检验这一假设，我将利用 识别Hsp104调控序列基序的分子进化算法 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同源基因和工程变体可解聚 蛋白质在体外聚集，并获得直接的机械洞察和定义 Hsp104底物选择的参数。最后，我会评估治疗 选择性Hsp014的潜力我通过(1)引入 αSYN选择性HSP104导入帕金森病线虫模型 监测虫体中多巴胺能神经元的保护和(2)引入 TDP-43-选择Hsp104进入TDP-43-病的黑腹果蝇模型 监测谎言的寿命和运动神经元功能。这些研究 构成了朝着进化增强解聚体的重要一步 神经退行性疾病中的蛋白质错误折叠。

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.