smFRET analysis of TDP-43 conformation under the effect of Hero proteins

Hero蛋白作用下TDP-43构象的smFRET分析

• 批准号: 22KJ0814
• 负责人: Lam Andy Yat Wung
• 金额: $ 1.09万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2023
• 资助国家: 日本
• 起止时间: 2023-03-08 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22KJ0814/
• 关键词: TDP-43; Hero protein; HSP40; JDP; aggregation; chaperone; IDP; smFRET

In FY2022 I validated that some HSP40 family chaperones are effective suppressors of overexpressed TDP-43 aggregation in HEK293T cells. This is used as a positive control for testing the effectiveness of Hero protein aggregation suppression in cells and confirms contemporary reports of DNAJA2 effectiveness for TDP-43 aggregation. Interestingly, while overexpression of these passive HSP40 proteins suppressed aggregation, the HSP70 and HSP90 chaperones did not, or even increased aggregation, suggesting the importance of passive chaperoning mechanisms. Overall, these cell-based results set a foundation for further studies.I also observed that a familial-associated ALS mutation in the TDP-43 LCD affects the conformational distribution at the single-molecule level. This is a direct observation that such a mutation has important effects at the intramolecular level, before oligomerization and later aggregation. This result furthers our understanding of the effects of mutations in TDP-43 Amyotrophic lateral sclerosis and frontotemporal lobar degeneration, both of which are fatal neurodegenerative diseases, with no known cures.Nonetheless, Hero and HSP40 affected conformation of this mutant, similar to their effects on the WT TDP-43 LCD. This is in line with their ability to efficiently suppress the aggregation of this mutant in cells. This suggests that single-molecule conformational effects can explain the effects of these two proteins on the bulk aggregation of TDP-43 at the cellular level. Overall, this work advances understanding of TDP-43 aggregation and chaperoning.

在FY 2022中，我验证了一些HSP 40家族分子伴侣是HEK 293 T细胞中过表达TDP-43聚集的有效抑制剂。这被用作阳性对照，用于测试Hero蛋白质聚集抑制在细胞中的有效性，并证实了DNAJA 2对TDP-43聚集的有效性的当代报道。有趣的是，虽然这些被动HSP 40蛋白的过表达抑制聚集，但HSP 70和HSP 90分子伴侣没有，甚至增加聚集，这表明被动分子伴侣机制的重要性。总的来说，这些基于细胞的结果为进一步的研究奠定了基础。我还观察到TDP-43 LCD中的家族相关ALS突变影响单分子水平的构象分布。这是一个直接的观察，这样的突变在分子内水平，寡聚化之前，后来的聚集有重要的影响。这一结果进一步加深了我们对TDP-43肌萎缩侧索硬化症和额颞叶变性的突变效应的理解，这两种疾病都是致命的神经退行性疾病，没有已知的治愈方法。尽管如此，Hero和HSP 40影响了这种突变体的构象，类似于它们对WT TDP-43 LCD的影响。这与它们有效抑制该突变体在细胞中聚集的能力一致。这表明单分子构象效应可以解释这两种蛋白质在细胞水平上对TDP-43的大量聚集的影响。总的来说，这项工作促进了对TDP-43聚集和陪伴的理解。

项目成果

Single-molecule observation of the effects of Hero proteins on TDP-43 conformation

Hero蛋白对TDP-43构象影响的单分子观察

• 发表时间: 2022
• 作者: Lam;A. Y. W.;Tsuboyama;K.;Tadakuma;H.;Tomari;Y.
• 通讯作者: Y.