DNAJB chaperones suppress destabilised protein aggregation via a region distinct from that used to inhibit amyloidogenesis

Abstract

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.

Factors Affecting Protein Recovery During Hsp40 Affinity Profiling

“…The small change in client affinity for DNAJB8 WT , as opposed to DNAJB8 H31Q , could be due to the enhanced proteostasis activity following stress. It was recently reported that DNAJB8 interacts with Hsp70 to promote proteasomal degradation of destabilized, aggregation-prone clients 48 . If increased protein association with DNAJB8 WT during heat shock also increases the flux of hand-off to Hsp70, then the steady state association of client proteins will only modestly change.…”

Flow cytometry allows rapid detection of protein aggregates in cellular and zebrafish models of spinocerebellar ataxia 3

“…Next, we aimed to determine whether we could employ the high-throughput flow cytometric analysis approach FloIT ( Whiten et al, 2016 ) to quantify the number and size of detergent-insoluble ataxin-3 particles in SCA3 cells. This method has been previously used to quantify TDP-43, SOD1 and huntingtin + inclusions ( Whiten et al, 2016 ; McAlary et al, 2016 ; Zeineddine et al, 2017a , b ), and other cellular processes associated with protein aggregation, including heat shock response induction ( San Gil et al, 2020 ), the role of molecular chaperones in suppressing protein aggregation ( McMahon et al, 2021 ) and chaperone-assisted selective autophagy ( Adriaenssens et al, 2020 ). Here, we provide the first adaption of FloIT to examine the presence of detergent-insoluble protein species in a model of spinocerebellar ataxia.…”

“…This approach has since been used to rapidly quantify the number of detergent insoluble inclusions across many in vitro models expressing aggregation-prone proteins, including mutant huntingtin (linked with Huntington's disease) ( Whiten et al, 2016 ) and motor neuron disease-linked proteins SOD1 ( Whiten et al, 2016 ; McAlary et al, 2016 ) and TDP-43 ( Zeineddine et al, 2017a , b ). The application of FloIT has also been expanded to investigate other cellular processes associated with protein aggregation, including induction of a heat shock response ( San Gil et al, 2020 ), the role of molecular chaperones in suppressing protein aggregation ( McMahon et al, 2021 ) and chaperone-assisted selective autophagy ( Adriaenssens et al, 2020 ). Considering that the formation of detergent-insoluble protein aggregates is a disease mechanism common to a wide variety of diseases, including Alzheimer's disease, Parkinson's disease, polyglutamine diseases, such as Huntington's disease and spinocerebellar ataxias, and even type 2 diabetes and dilated cardiomyopathy ( Adriaenssens et al, 2020 ; Chiti and Dobson, 2017 ; Ross and Poirier, 2004 ; Taylor et al, 2002 ; Gidalevitz et al, 2006 ; Mukherjee et al, 2015 ), it is important to determine whether FloIT can be widely adopted to study other proteinopathy disease models.…”

Exploiting flow cytometry for the unbiased quantification of protein inclusions inCaenorhabditis elegans

“…Recently developed flow cytometry approaches for quantifying protein aggregation in cellular model systems have shown great promise in providing a simple and rapid alternative to traditional methods (Hidalgo et al 2016; McMahon et al 2021; Ramdzan et al 2012; Shiber et al 2014; Whiten et al 2016). In this study, we describe a method that enables fluorescent inclusions to be enumerated and characterised in C. elegans by exploiting a standard flow cytometry setup.…”

“…This technique also has the advantage of allowing for the quantification of nuclear flux of fluorescently-tagged proteins. The application of flow cytometric analyses to quantify inclusions has been demonstrated to be a powerful tool for cell-based studies as they are unbiased and can be applied to high-throughput screens (McMahon et al 2021; Whiten et al 2016). However, a method exploiting flow cytometry to quantify inclusions formed in C. elegans has not yet been described.…”