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

• 批准号: 10238101
• 负责人: Edward Matthew Barbieri
• 金额: $ 6.75万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/10238101
• 关键词: Alzheimer' 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

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.

细胞压力导致蛋白质错误折叠和聚集，这是由蛋白质伴侣酶（分解气体）对抗的。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，包括肌萎缩性侧索硬化症（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。由于所有生物体都需要蛋白质质量控制，因此简单的模型系统（如酵母）是以快速和经济有效的方式研究类似人类过程的有力工具。该项目将利用酵母的高通量基因工程来研究和设计人类分解酶系统，以对抗导致帕金森病和渐冻症的有毒蛋白质聚集。首先，我将验证人类hsp110、hsp70和hsp40伴侣蛋白的独特组合可以在细胞中赋予解聚酶底物特异性的假设。我将在帕金森氏症和渐冻症酵母模型中创建并测试已知hsp110/70/40基因的所有可能的三重组合的质粒文库。其次，我将使用eMAGE，这是我在博士期间发明的一种技术，来设计先前表征的由hsp110 (Apg-2), hsp70 （Hsc70）和hsp40 （Hdj1）组成的人类分解酶机制。最后，我将在帕金森氏病和ALS的人类神经元细胞模型中验证酵母的发现。本提案中概述的实验管道利用酵母遗传学的规模和能力来鉴定表现出毒性拯救的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。该项目将通过全面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渐冻症的候选治疗方法确定新的机制。