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2022财年，我证实了一些Hsp40家族伴侣蛋白是HEK293T细胞中过表达的TDP-43聚集的有效抑制因子。这被用作一种阳性对照，用于测试细胞中Hero蛋白质聚集抑制的有效性，并证实了DNAJA2对TDP-43聚集的有效性。有趣的是，虽然这些被动HSP40蛋白的过度表达抑制了聚集，但HSP70和HSP90伴侣蛋白没有，甚至增加了聚集，这表明被动伴侣机制的重要性。总体而言，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中的家族性ALS突变在单分子水平上影响构象分布。这是一个直接的观察，这种突变在分子内水平上有重要的影响，在寡聚和后来的聚集之前。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突变的影响的理解，这两种疾病都是致命的神经退行性疾病，目前还没有已知的治疗方法。这与他们有效抑制该突变体在细胞中聚集的能力是一致的。这表明单分子构象效应可以在细胞水平上解释这两种蛋白质对TDP-43聚集的影响。总体而言，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.