Defining the molecular basis of substrate selection by diverse Hsp104 homologues

项目摘要 Hsp 104是来自酵母的六聚体AAA+蛋白解聚酶， 快速分解无序聚集体、类淀粉低聚物、淀粉样蛋白， 朊病毒这些活动使酵母能够利用有益的朊病毒， 适应性目的。然而，人类和后生动物缺乏直接的Hsp 104， 同源物，并且容易发生蛋白质错误折叠，这是几个 致命的神经退行性疾病，包括帕金森病（PD）和 肌萎缩侧索硬化症（ALS）。我们以前设计了增强 Hsp 104变异体拮抗几种蛋白质的错误折叠， 神经退行性疾病（TDP-43与ALS有关，αSyn与ALS有关， PD）。然而，这些变体缺乏底物特异性，并且在某些情况下可能是有毒的。 情节因此，理解Hsp 104如何选择底物仍然是一个问题。 重要目标。HSP 104同源物存在于所有非后生动物中， 真核生物和真细菌。然而，这些同系物中的绝大多数 仍然未被探索。我已经确定了来自不同来源的Hsp 104同源物， 包括原生动物、真菌和植物在内的谱系是TDP-43的选择性修饰剂 和αSyn蛋白毒性错误折叠。基于我的初步发现，我假设 热休克蛋白104同源物之间底物识别和结合的差异 支持它们的底物选择性。为了验证这个假设，我将利用 分子进化算法识别调控Hsp 104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Hsp 104直向同源物和工程化变体 蛋白质聚集体在体外，并获得直接的机制洞察和定义 Hsp 104底物选择的参数。最后，我将评估治疗 选择性Hsp 014的潜力，我通过以下方式确定和设计：（1）引入 α-Syn选择性热休克蛋白104转化为C. elegans帕金森病模型， 监测蠕虫中多巴胺能神经元的保护，以及（2）引入 TDP-43选择性热休克蛋白104转化D. TDP-43病的黑胃模型， 监测谎言的寿命和运动神经元功能。这些研究 这是朝着发展增强型分解气体以对抗 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.