Engineering mitochondrial protein disaggregases for neurodegenerative disease

工程线粒体蛋白解聚酶治疗神经退行性疾病

• 批准号: 9982740
• 负责人: RYAN R CUPO
• 金额: $ 3.27万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9982740
• 关键词: ATP phosphohydrolase; Address; Affect; Animal Model; Biochemical; Biochemistry; Biological Models; Caenorhabditis elegans; Cell Survival; Cellular biology; Cytoplasm; Data; Disease; Engineering; Enzymes; Exhibits; Frontotemporal Dementia; Functional disorder; Genetic; Genetic Engineering; Homologous Gene; Human; Hyperactive behavior; In Vitro; Lewy Body Dementia; Luciferases; Measures; Mitochondria; Mitochondrial Proteins; Modeling; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Parkinson Disease; Pathogenesis; Play; Production; Protein Engineering; Proteins; RNA-Binding Protein FUS; Reactive Oxygen Species; Role; Saccharomyces cerevisiae; Structure; Substrate Specificity; TDP-43 aggregation; Techniques; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Variant; Yeast Model System; Yeasts; alpha synuclein; combat; innovation; insight; interest; mitochondrial dysfunction; new therapeutic target; novel; prevent; protein TDP-43; protein aggregation; protein misfolding; protein structure; proteostasis; response; synucleinopathy; tool; yeast genetics; yeast protein

PROJECT SUMMARY Protein aggregates accumulate in several neurodegenerative diseases. For example, the proteins FUS and TDP-43 aggregate in frontotemporal dementia, while α-synuclein (αSyn) aggregates in synucleinopathies such as dementia with Lewy bodies and Parkinson’s disease. Protein disaggregases, such as the yeast AAA+ protein Hsp104, directly disassemble aggregated protein structures and soluble oligomers and therefore hold great therapeutic potential for protein misfolding diseases. We have previously potentiated Hsp104 to disaggregate αSyn, FUS, and TDP-43 in vitro and in yeast models. Our potentiated Hsp104 variants exhibit some toxicity when expressed in higher metazoan model systems, perhaps due to a lack of substrate specificity. Thus, I became interested in studying Hsp78 (the yeast mitochondrial homologue of Hsp104) because its divergent sequence might provide clues to mitigating Hsp104 toxicity and it has a putative human mitochondrial orthologue, Skd3. Little is known about the cell biology or biochemistry of Skd3. Three key pieces of data underlie my aims: (1) I targeted mitochondrial (mt) Hsp78 (mtHsp78) to the cytoplasm (cHsp78) and potentiated its activity via homologous mutations that nonselectively potentiate Hsp104. I found that three cHsp78 variants selectively rescue α-synuclein, FUS, or TDP-43 toxicity in yeast. (2) Surprisingly, I discovered that three different mtHsp78 variants rescue αSyn toxicity in yeast without affecting cytoplasmic αSyn aggregation. (3) I have purified Skd3 and shown that Skd3 has robust protein disaggregase activity, in vitro. Three unaddressed problems impede harnessing these protein disaggregases therapeutically. On the basis of my preliminary data, I hypothesize that: (1) the differences in rescue by the cHsp78 variants for αSyn, FUS, and TDP-43 is due to differences in substrate selectivity for disaggregation; (2) the mtHsp78 variants rescue αSyn toxicity by supplementing mitochondrial proteostasis thereby preventing mitochondrial dysfunction and promoting cell survival; (3) the human mitochondrial protein disaggregase Skd3 can be potentiated to increase activity and that potentiated Skd3 variants will rescue toxicity in metazoan model systems of synucleinopathies. Therefore, I will address three aims: (1) I will test the selective disaggregase activity of the cHsp78 variants in vitro using biochemical techniques innovated in our lab; (2) I will determine the mechanism of mtHsp78 rescue of αSyn toxicity using yeast genetics and cell biology; (3) I will characterize and potentiate Skd3 against αSyn toxicity in metazoan model systems using biochemical, cell biology, and genetic engineering techniques. These studies are an important step towards understanding the mitochondrial protein disaggregase machinery and their role as a potential therapeutic for synucleinopathies such as dementia with Lewy bodies and Parkinson’s disease.

项目摘要 蛋白质聚集体在几种神经退行性疾病中积累。例如，蛋白质FUS 和TDP-43在额颞叶痴呆中聚集，而α-突触核蛋白（αSyn）在突触核蛋白病中聚集 如路易体痴呆症和帕金森病。蛋白质分解剂，如酵母AAA+ 蛋白Hsp 104，直接分解聚集的蛋白质结构和可溶性寡聚体， 蛋白质错误折叠疾病的巨大治疗潜力。我们之前已经增强了HSP 104， 在体外和酵母模型中解聚αSyn、FUS和TDP-43。我们的增强的Hsp 104变体表现出 当在高等后生动物模型系统中表达时，可能由于缺乏底物， 的特异性因此，我对研究Hsp 78（Hsp 104的酵母线粒体同源物）产生了兴趣。 因为它的不同序列可能提供减轻Hsp 104毒性的线索， 线粒体直系同源物，Skd 3。人们对Skd 3的细胞生物学或生物化学知之甚少。三个关键 目的：（1）将线粒体Hsp 78（mtHsp 78）靶向于细胞质（cHsp 78）， 并通过非选择性增强HSP 104的同源突变增强其活性。我发现三个 cHsp 78变体选择性地拯救酵母中的α-突触核蛋白、FUS或TDP-43毒性。(2)令人惊讶的是，我发现 三种不同mtHsp 78变体在酵母中拯救αSyn毒性而不影响胞质αSyn 聚合来(3)我已经纯化了Skd 3，并表明Skd 3在体外具有强大的蛋白解聚酶活性。 三个未解决的问题阻碍了利用这些蛋白质分解的治疗。的基础上 根据我的初步数据，我假设：（1）cHsp 78变体对αSyn，FUS， 和TDP-43是由于底物选择性的差异解聚;（2）mtHsp 78变异体拯救 αSyn毒性通过补充线粒体蛋白质稳态，从而防止线粒体功能障碍， 促进细胞存活;（3）人线粒体蛋白解聚酶Skd 3可被增强以增加 活性，并且增强的Skd 3变体将拯救突触核蛋白病的后生动物模型系统中的毒性。 因此，我将提出三个目标：（1）我将测试cHsp 78变体在大肠杆菌中的选择性解聚酶活性。 （2）探讨mtHsp 78的拯救机制 利用酵母遗传学和细胞生物学研究αSyn的毒性;（3）我将表征并增强Skd 3对αSyn的作用 毒性后生动物模型系统使用生物化学，细胞生物学和遗传工程技术。 这些研究是理解线粒体蛋白解聚酶机制的重要一步 以及它们作为突触核蛋白病如路易体痴呆的潜在治疗剂的作用， 帕金森氏症。