Disturbances to protein homeostasis (proteostasis) can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. DNAJBs are molecular chaperones which have been identified as potent suppressors of disease-related protein aggregation. In this work, a destabilised isoform of firefly luciferase (R188Q/R261Q Fluc; FlucDM) was overexpressed in cells to assess the capacity of DNAJBs to inhibit inclusion formation. Co-expression of all DNAJBs tested significantly inhibited the intracellular aggregation of FlucDM. Moreover, we show that DNAJBs suppress aggregation by supporting the Hsp70-dependent degradation of FlucDM via the proteasome. The serine-rich stretch in DNAJB6 and DNAJB8, essential for preventing fibrillar aggregation, is not involved in the suppression of FlucDM inclusion formation. Conversely, deletion of the C-terminal TTK-LKS motif in DNAJB6 and DNAJB8, a region not required to suppress polyQ aggregation, abolished its ability to inhibit inclusion formation by FlucDM. Thus, our data suggest that DNAJB6 and DNAJB8 possess two distinct regions for binding substrates, one that is responsible for binding β-hairpins that form during amyloid formation and another that interacts with exposed hydrophobic patches in aggregation-prone clients.

Disturbances to protein homeostasis (proteostasis) can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. DNAJBs are molecular chaperones previously identified as potent suppressors of disease-related protein aggregation. In this work, we over-expressed a destabilised isoform of firefly luciferase (R188Q/R261Q Fluc; FlucDM) in cells to assess the capacity of DNAJBs to inhibit inclusion formation. Co-expression of all DNAJBs tested significantly inhibited the intracellular aggregation of FlucDM. Moreover, we show that DNAJBs suppress aggregation by supporting the Hsp70-dependent degradation of FlucDM via the proteasome. The serine-rich stretch in DNAJB6 and DNAJB8, essential for preventing fibrillar aggregation, is not involved in the suppression of FlucDM inclusion formation. Conversely, deletion of the C-terminal TTK-LKS region in DNAJB8, a region not required to suppress polyQ aggregation, abolished its ability to inhibit inclusion formation by FlucDM. Thus, our data suggest that DNAJB6 and DNAJB8 possess two distinct domains involved in the inhibition of protein aggregation, one responsible for binding to β-hairpins that form during amyloid formation and another that mediates the degradation of destabilised client proteins via the proteasome.Summary statementSpecialised DNAJB molecular chaperones are potent suppressors of protein aggregation and interact with different types of client proteins via distinct C-terminal regions