Small heat shock proteins HspB1 and HspB5 differentially alter the condensation and aggregation of the TDP-43...

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein that regulates processes of mRNA metabolism, during which it undergoes condensation mediated by its C-terminal low-complexity domain (TDP-43^LCD). TDP-43 aggregation and condensation are associated with neurodegenerative disease. However, the proteostasis mechanisms that regulate these processes remain elusive. Some evidence has shown that the molecular chaperone small heat shock protein HspB1 binds to and regulates the cytoplasmic phase separation of TDP-43, indicating that other small heat shock proteins may have similar effects. Here, we demonstrate divergent behaviors for HspB1 and its homolog HspB5 on TDP-43^LCD condensation and aggregation. In addition to inhibiting TDP-43^LCD aggregation, HspB1 partitions into TDP-43^LCD condensates and increases the dynamic exchange of TDP-43^LCD within condensates and with the surrounding solution. Phosphorylation-mimicking mutations within HspB1 enhance these effects. HspB5 inhibits TDP-43^LCD aggregation more effectively than HspB1 and partitions into TDP-43^LCD condensates, where it delays the pathological transition of the condensate to a gel/solid. We identify the N- and C-terminal regions of HspB1 and HspB5 to be crucial for the chaperone effects, and highlight the role of sequence diversity within these regions in defining small heat shock protein function. These findings demonstrate that HspB1 and HspB5 are regulators of TDP-43 phase separation and aggregation and may be potential therapeutic targets in mitigating toxic TDP-43 aggregation in neurodegenerative disease.