Programming Human Chaperone Systems Against Neurodegenerative Disease

对人类伴侣系统进行编程以对抗神经退行性疾病

基本信息

批准号：
10026294
负责人：
Edward Matthew Barbieri
金额：
$ 6.42万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-30 至 2022-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10026294
关键词：
Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Bar Codes Biochemical Biochemistry Biological Assay Biological Models Cell model Cell physiology Cells Cellular Stress Chaperone Gene Clustered Regularly Interspaced Short Palindromic Repeats DNA Sequence Alteration Disease Doctor of Philosophy Engineering Enzymes Exhibits Frontotemporal Dementia Gene Combinations Genes Genetic Genetic Engineering Human Human Engineering Huntington Disease In Vitro Lead Libraries Modeling Molecular Chaperones Neurodegenerative Disorders Neurons Organism Parkinson Disease Plasmids Population Process Proteins Quality Control Quantitative Microscopy Spinocerebellar Ataxias Substrate Specificity System Techniques Testing Therapeutic Toxic effect Transgenes Triplet Multiple Birth Validation Work Yeast Model System Yeasts alpha synuclein combat combinatorial cost efficient deep sequencing effective therapy exhaustion invention mutant new therapeutic target novel novel strategies professor protein TDP-43 protein aggregation protein misfolding screening therapeutic candidate tool vector yeast genetics

项目摘要

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.

细胞应激会导致蛋白质错误折叠和聚集，而蛋白质伴侣酶（解聚集酶）可对抗蛋白质错误折叠和聚集。在神经元中，蛋白质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、额颞叶痴呆、帕金森病、亨廷顿舞蹈症。和脊髓小脑性共济失调缺乏可行的治疗方案反映出我们对细胞过程缺乏了解。由于所有生物体都需要蛋白质质量控制，因此酵母等简单模型系统是快速且经济高效地研究类似人类过程的强大工具。该项目将利用高通量遗传技术。首先，我将测试人类 hsp110、hsp70 和 hsp40 分子伴侣的独特组合这一假设。可以在细胞中赋予解聚酶底物特异性。我将在帕金森病和 ALS 酵母模型中创建并测试已知 hsp110/70/40 基因的所有可能三联体组合的质粒库。在攻读博士学位期间，设计了先前表征的由 hsp110 (Apg-2)、hsp70 (Hsc70) 和 hsp40 组成的人类解聚酶机器(Hdj1). 最后，我将验证酵母在帕金森病和 ALS 的人类神经细胞模型中的发现，该提案中概述的实验流程利用酵母遗传学的规模和能力来识别 Hsp110/70/40 突变体和基因组合。表现出毒性的拯救，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组合空间，极大地增强目前对人类分解酶机制的理解。三基因伴侣相互作用，它可能会确定帕金森病和 ALS 候选疗法的新机制。