Programming Human Chaperone Systems Against Neurodegenerative Disease

细胞应激导致蛋白质错误折叠和聚集，这是由蛋白质伴侣酶(解聚酶)来对抗的。在神经元中，蛋白质的错误折叠和聚集可导致神经退行性疾病，包括肌萎缩侧索硬化症(ALS)、阿尔茨海默病、额颞痴呆、帕金森氏病、亨廷顿病和脊髓小脑性共济失调。缺乏可行的治疗方案反映了我们对这些疾病中出错的细胞过程缺乏了解。由于所有活着的生物体都需要蛋白质质量控制，所以酵母等简单的模型系统是以快速和经济高效的方式研究类似人类过程的强大工具。该项目将利用酵母中的高通量基因工程来研究和设计人类解聚酶系统，以对抗帕金森氏症和ALS背后的有毒蛋白质聚集体。首先，我将测试这一假设，即人类hsp110、hsp70和hsp40伴侣蛋白的独特组合可以在细胞中传递解聚酶底物专一性。我将为帕金森氏症和肌萎缩侧索硬化症酵母模型中已知的hsp110/70/40基因的所有可能的三联体组合创建并测试质粒库。其次，我将使用Emage，这是我在博士期间发明的一项技术，来设计以前描述的人类解聚酶机器，包括hsp110(apg-2)、hsp70(Hsc70)和hsp40(Hdj1)。最后，我将在帕金森氏病和肌萎缩侧索硬化症的人类神经细胞模型中验证酵母的发现。这项提案中概述的实验管道利用酵母遗传学的规模和能力来识别表现出挽救毒性的Hsp110/70/40突变体和基因组合，然后在真正的人类神经元中进行实验验证。该项目将通过彻底筛选三基因伴侣相互作用的组合空间，极大地提高目前对人类解聚机制的理解，并可能为帕金森氏病和ALS的候选治疗找到新的机制。

Cellular stress causes protein misfolding and aggregation, which is combatted by protein chaperone enzymes (disaggregases). In neurons, protein misfolding and aggregation can lead to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, frontotemporal dementia, Parkinson's disease, Huntington's disease, and spinocerebellar ataxias. The lack of viable therapeutic options reflects the dearth of our understanding regarding the cellular processes that go awry in these diseases. Since protein quality control is required for all living organisms, simple model systems such as yeast are powerful tools to study the analogous human process in a rapid and cost-efficient way. This project will leverage high-throughput genetic engineering in yeast to study and engineer human disaggregase systems to combat toxic protein aggregates that underlie Parkinson’s disease and ALS. First, I will test the hypothesis that unique combinations of human hsp110, hsp70, and hsp40 chaperones can impart disaggregase substrate specificity in a cell. I will create and test plasmid libraries for all possible triplet combinations of the known hsp110/70/40 genes in yeast models of Parkinson’s and ALS. Second, I will use eMAGE, a technique that I invented during my PhD, to engineer the previously characterized human disaggregase machinery comprised of hsp110 (Apg-2), hsp70 (Hsc70) and hsp40 (Hdj1). Lastly, I will validate the findings from yeast in human neuronal cell models of Parkinson’s disease and ALS. The experimental pipeline outlined in this proposal leverages the scale and power of yeast genetics to identify Hsp110/70/40 mutants and gene combinations that exhibit rescue of toxicity, which are then experimentally validated in a bona fide human neuron. This project will greatly enhance the current understanding of human disaggregase mechanisms by exhaustively screening the combinatorial space of three-gene chaperone interactions and it will likely identify new mechanisms for candidate therapeutics of Parkinson’s disease and ALS.