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

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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.