Engineering mitochondrial protein disaggregases for neurodegenerative disease

项目总结 蛋白质聚集体在几种神经退行性疾病中积聚。例如，蛋白质FUS α-突触核蛋白(α-Syn)在突触核病中聚集 例如路易体痴呆症和帕金森氏症。蛋白质解聚酶，如酵母AAA 蛋白质Hsp104，直接分解聚集的蛋白质结构和可溶性低聚物，因此保持 对蛋白质错误折叠疾病的巨大治疗潜力。我们之前已将Hsp104增强为 在体外和酵母模型中解聚αsyn、fus和tdp-43。我们的增强型Hsp104变种展示 在较高的后生动物模型系统中表达时的一些毒性，可能是由于缺乏底物 专一性。因此，我对研究Hsp78(Hsp104的酵母线粒体同源物)产生了兴趣。 因为它的不同序列可能为减轻Hsp104的毒性提供线索，而且它有一个假定的人类 线粒体同源基因，Skd3。对Skd3的细胞生物学或生物化学知之甚少。三把钥匙 我的目标是：(1)我将线粒体(Mt)Hsp78(MtHsp78)靶向细胞质(CHsp78)。 并通过非选择性地增强Hsp104的同源突变来增强其活性。我发现有三个 CHsp78变异体选择性地挽救酵母中的α-突触核蛋白、FUS或TDP-43毒性。令人惊讶的是，我发现 三种不同的mtHsp78突变体在不影响细胞质α同步的情况下挽救酵母中的α同步毒力 聚合。(3)对Skd3进行了纯化，结果表明Skd3在体外具有较强的蛋白解聚酶活性。 三个未解决的问题阻碍了这些蛋白质解聚的治疗。在…的基础上 我的初步数据，我假设：(1)cHsp78变体对αSYN，FUS， 而TDP-43则是由于底物对解聚的选择性不同所致；(2)mtHsp78突变体的挽救 补充线粒体蛋白平衡从而预防线粒体功能障碍和α的SYN毒性 促进细胞存活；(3)人线粒体蛋白解聚酶Skd3可被增强 活性和增强的Skd3变异体将挽救后生动物模型系统中的毒性。 因此，我将解决三个目标：(1)我将测试cHsp78变体的选择性解聚酶活性 利用本实验室创新的生化技术进行体外实验；(2)确定mtHsp78的解救机制 利用酵母菌遗传学和细胞生物学研究α突触蛋白的毒性；(3)我将对α突触蛋白3进行鉴定和增强。 使用生化、细胞生物学和基因工程技术的后生动物模型系统的毒性。 这些研究是了解线粒体蛋白解聚酶机制的重要一步。 以及它们作为一种潜在的治疗联合核病的作用，如路易体痴呆和 帕金森氏症。

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.