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）对抗的。在神经元中，蛋白质错误折叠和聚集可导致神经变性疾病，包括肌萎缩性侧索硬化（ALS）、阿尔茨海默病、额颞叶痴呆、帕金森病、亨廷顿病和脊髓小脑共济失调。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。由于蛋白质质量控制是所有生物体所必需的，因此简单的模型系统（如酵母）是以快速且具有成本效益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，以对抗帕金森病和ALS的毒性蛋白质聚集体。首先，我将测试的假设，人类热休克蛋白110，热休克蛋白70，和热休克蛋白40分子伴侣的独特组合可以赋予解聚酶底物特异性的细胞。我将在帕金森和肌萎缩侧索硬化症的酵母模型中建立和测试已知hsp 110/70/40基因所有可能的三联体组合的质粒文库。其次，我将使用eMAGE，我在博士期间发明的一种技术，来设计先前表征的由hsp 110（Apg-2），hsp 70（Hsc 70）和hsp 40（Hdj 1）组成的人类解聚酶机制。最后，我将在帕金森病和ALS的人类神经元细胞模型中验证酵母的发现。该提案中概述的实验管道利用酵母遗传学的规模和力量来鉴定表现出毒性拯救的Hsp 110/70/40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酶机制的理解，并可能为帕金森病和ALS的候选疗法确定新的机制。

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.