For proteins to accurately carry out their role in the cell, they must first be precisely folded into specific 3D shapes. Stress, aging or disease can interfere with this delicate process, leading to misfolded proteins clumping together and causing damage. In response, the cell can deploy ‘chaperones’ which disentangle these aggregates and ensure that proteins recover their proper structure. Chaperones from the Hsp70 protein family, for example, are crucial for cell survival, especially under biologically stressful conditions. Yet Hsp70 proteins cannot perform their role without the assistance of co-chaperones such as Hsp110; why this is the case, however, has remained unclear.

To investigate this question, Sztangierska et al. used a variety of biochemical assays to test how purified human and yeast Hsp70, Hsp110 and other co-chaperones could bind aggregates and recover misfolded proteins. The role of each protein was examined at every stage of the disaggregation process – from the initial aggregate binding, through chaperone-driven changes in aggregate structure to the final protein folding.

The experiments revealed that Hsp110 helps draw Hsp70 to the aggregate surface, breaking down the protein ‘clump’ into smaller pieces which are more easily processed by other chaperones. The results also showed that the various co-chaperones compete for Hsp70 binding; too much of one might interfere with another, emphasizing the need for balance between chaperones for optimal disaggregation.

Overall, these results clarify the role of Hsp110 in the Hsp70 system and reveal several mechanistic details of the protein rescue process. Further experiments will be needed to fully understand these dynamics and identify how they may be relevant to conditions in which harmful protein aggregates are observed, such as Parkinson’s or Alzheimer’s disease.

During stress, protein homeostasis is perturbed by protein misfolding and aggregation. Accumulation of protein aggregates disrupts cellular functions and contributes to ageing and is a hallmark of many neurodegenerative diseases (Hartl et al., 2011; Morimoto, 2008). A protein quality control system, relying on a network of cooperating chaperones, has evolved to moderate stress-induced proteotoxicity and rebalance proteostasis.

To refold proteins from aggregates into their native state, eukaryotes use a system comprising Hsp70, an ATP-dependent chaperone, and its co-chaperones: an Hsp110 nucleotide exchange factor (NEF) and a J-domain protein (JDP/Hsp40), together with an Hsp100 disaggregase (Glover and Lindquist, 1998). The process is initiated by a JDP, which delivers Hsp70 to an aggregated substrate and whose J-domain induces ATP hydrolysis in Hsp70, resulting in conformational changes that stabilise the interaction with aggregates (Rohland et al., 2022). Next, Hsp100 interacts with the Hsp70-aggregate complex and the aggregate-trapped polypeptides are translocated through the Hsp100 hexamer. The disentangled and released polypeptides can fold back into their native conformation, alone or with further aid of chaperones (Lum et al., 2004; Schaupp et al., 2007; Seyffer et al., 2012; Weibezahn et al., 2004; Zietkiewicz et al., 2004).

Hsp110 co-chaperone boosts the Hsp70 activity by stimulating nucleotide exchange and substrate release (Dragovic et al., 2006; Raviol et al., 2006; Shaner et al., 2005). It belongs to the Hsp70 superfamily, with identical domain organisation to Hsp70 but distinct size and arrangement of the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) (Easton et al., 2000; Liu and Hendrickson, 2007). In contrast to Hsp70, Hsp110 is unable to refold denatured proteins, yet it may bind to and prevent aggregation of certain misfolding substrates (Garcia et al., 2017; Glover and Lindquist, 1998; Goeckeler et al., 2002; Oh et al., 1997; Oh et al., 1999; Polier et al., 2010; Xu et al., 2012). In its well-established NEF function, the NBD of Hsp110 interacts with the ADP form of Hsp70, which mediates the release of the nucleotide. Subsequent ATP binding to the NBD of Hsp70 prompts the opening of the SBD and substrate dissociation, resetting Hsp70 for another round of the cycle (Andréasson et al., 2008a; Laufen et al., 1999; Liberek et al., 1991; Mayer and Bukau, 2005; Mayer and Gierasch, 2019).

In vivo studies on yeast chaperones demonstrated that the Hsp110 co-chaperone Sse1 plays a key role in Hsp70 recruitment to aggregates and is essential for protein disaggregation (Kaimal et al., 2017). Hsp110 is a major cytoplasmic NEF of Hsp70 and the deletion of its both paralogs, SSE1 and SSE2, is lethal and the growth can only be supported, albeit with less efficacy, by overexpression of FES1, another cytoplasmic NEF from an unrelated armadillo family (Abrams et al., 2014; Raviol et al., 2006).

The importance of Hsp110 is also manifested in vitro in the low disaggregation activity of the yeast Hsp70 system without Hsp110 and the complete interdependence between their human orthologs (Glover and Lindquist, 1998; Mattoo et al., 2013; Nillegoda et al., 2015; Rampelt et al., 2012). Recent studies on the human system revealed that Hsp110 function in disaggregation of amyloid fibrils is not limited to its NEF activity but it may also affect the architecture of chaperone complexes, inducing Hsp70 clustering, although it is not clear whether similar effects occur during disaggregation of non-fibrillar, stress-associated aggregates. The effect is not well understood, but it presumably increases entropic pulling of aggregated polypeptides, ultimately leading to their disentanglement by the Hsp70 system, efficient even without the Hsp100 disaggregase. Developing an Hsp70 system that is self-sufficient in disaggregation could have compensated for the loss of the Hsp100 disaggregase in a common metazoan ancestor (Mattoo et al., 2013; Shorter, 2011).

Another factor that potentiates the Hsp70 system is the diversity of JDP paralogs, assigned to cytoplasmic classes A and B, which differently regulate Hsp70 (Lu and Cyr, 1998; Nillegoda et al., 2015). The main distinction between class A and B JDPs is an auxiliary interaction site between CTD1 domain of class B JDPs and the C-terminal EEVD motif of Hsp70 (Yu et al., 2015). Class B JDPs are additionally regulated by an autoinhibitory mechanism, in which Hsp70 binding by the J-domain is restricted by a neighbouring helix. Upon binding to the C-terminal EEVD motif of Hsp70, the J-domain is released and it can interact with the NBD of Hsp70 (Faust et al., 2020; Wentink et al., 2020). We have recently shown that the yeast class A JDP Ydj1 and class B JDP Sis1 exhibit diverse mechanisms during Hsp70 binding to aggregated substrates. Ydj1, in accordance with the classical model of the Hsp70 ATPase cycle, binds misfolded polypeptides and loads Hsp70 onto aggregates. Unlike that, Sis1 only weakly binds protein substrates but due to the more complex interaction with Hsp70, it loads more Hsp70 molecules onto aggregates, which results in more efficient disaggregation (Wyszkowski et al., 2021).

Despite the vast knowledge on how Hsp110 serves as a regulator of the ATPase cycle of Hsp70 (Andréasson et al., 2008a; Andréasson et al., 2008b; Dragovic et al., 2006; Raviol et al., 2006; Shaner et al., 2006), little is known about the function of Hsp110 considering the mechanism of Hsp70 interaction with different JDP classes.

Here, we demonstrate that the interplay between Hsp110 and Hsp70 in disaggregation strictly relies on the class of a JDP and unravel the critical role of the B-class-specific interaction with the EEVD motif of Hsp70 for the Hsp110-dependent stimulation. Furthermore, we elucidate differential contribution of the NEF across different phases of protein recovery from aggregates. We employ biolayer interferometry (BLI) to investigate the Hsp110 impact on the formation of chaperone complexes at the aggregate surface and we assess changes in aggregates’ properties associated with the abundant chaperone binding. Finally, we address a question of the competition between the NEF and JDP co-chaperones. Our findings shed new light on the mechanisms behind the potentiation and inhibition of the disaggregation activity of Hsp70 by its co-chaperones.

Our results uncover a comprehensive picture of the role of Hsp110 co-chaperones in aggregate processing by the Hsp70 system. Potentiation of the Hsp70 activity by the NEF strongly depends on the class of the JDP and the preference for class B, conserved in yeast and human orthologs, relies on Hsp70 binding through the EEVD motif. Furthermore, we demonstrate that the Hsp110-dependent stimulation of disaggregation is limited to its initial stages: chaperone recruitment to aggregates and their disassembly, but not final protein folding (Figure 5). The initial Hsp110-dependent loading of more Hsp70 onto aggregates correlates with their remodelling into smaller aggregated species, which improves their recognition by the Hsp104 disaggregase. We also gained insight into the biphasic impact of Hsp110: with increasing Sse1 level, the stimulation is overshadowed by inhibition and the contribution of each trend depends on the phase of disaggregation and the composition of the disaggregating system, with a crucial role of the NEF’s affinity for Hsp70. Finally, we show that the disaggregation inhibition by Hsp110 involves disruption of the Hsp70-JDP interaction, suggesting competition between JDP and Hsp110 co-chaperones.

Figure 5

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The balance between the partners within the Hsp70 system is key for efficient disaggregation, as an individual co-chaperone with a critical function at one step may inhibit another. To investigate this differential contribution, we used biochemical assays reflecting individual stages of protein disaggregation. The results of the BLI experiments show that Hsp110 with class B JDP (JDP^B) greatly increases Hsp70 aggregate binding (Figure 1B and C, Figure 1—figure supplements 1E, F and 2A), which correlates with the final reactivation yield (Figure 1A, Figure 1—figure supplement 1H), implying that the initiation phase determines the overall disaggregation efficiency. On the other hand, the final folding of a soluble substrate by Ssa1 relies much more on the class A JDP (JDP^A) Ydj1 than on Sis1 (Figure 1—figure supplement 1D; Lu and Cyr, 1998), and not on Sse1 altogether (Figure 1—figure supplement 1D), which is in line with previous reports that its human ortholog, Hsp105 inhibits luciferase folding (Rauch and Gestwicki, 2014).

The higher degree of stimulation with class B JDP has been previously observed, albeit without broader insight, for Hsp110 and other NEF BAG (Rampelt et al., 2012; Rauch and Gestwicki, 2014). We show that the distinguishing feature fundamental for this specificity is the auxiliary interaction between the CTD domain of Sis1 and the EEVD motif of Hsp70. The Sis1 E50A variant, featuring derepressed J-domain (Yu et al., 2015), together with Ssa1 ΔEEVD exhibited a similar aggregate binding pattern with or without Sse1 to the system with Ydj1 (Figure 1B, Figure 1—figure supplement 1E, F, and J). However, while Ydj1 can bind misfolded proteins and prevent aggregation, Sis1 cannot (Lu and Cyr, 1998). When Sis1 is deprived of Ssa1 binding through EEVD, which has a major contribution to the stability of their interaction (Wyszkowski et al., 2021), the effect of Sse1 on disaggregation is detrimental (Figure 1—figure supplement 1I). This underlines the importance of the Hsp70-JDP complex stability for the stimulation of substrate binding and disaggregation by Sse1.

In our previous work, we reported sigmoidal aggregate binding kinetics characteristic for the Ssa1-Sis1 system, suggesting that the binding gradually generates more chaperone binding sites (Wyszkowski et al., 2021). Now, we show that this apparent cooperativity is conserved in yeast and human, implying mechanistic similarities between the systems (Figure 1B and C). While the s-shaped Ssa1-Sis1 binding to the aggregate results in the association of more Ssa1 than with Ydj1 (Figure 1—figure supplement 2A), Hsp110 shortens the initial lag phase and boosts the binding efficacy, loading a thicker layer of Hsp70 molecules onto aggregates (Figure 1B and C, Figure 1—figure supplement 2A), similarly as has been shown for amyloid fibrils (Beton et al., 2022). This corresponds with Hsc70 clustering on amyloid in the presence of Hsp110, reported by Wentink et al., 2020. Clustered, densely packed Hsp70 has been proposed to generate entropic pulling effect that leads to fibril fragmentation (Goloubinoff and De Los Rios, 2007; Sousa and Lafer, 2019; Wentink et al., 2020). Likewise, we observed remodelling of amorphous aggregates into smaller aggregated species, which resembles amyloid disassembly in terms of the dependence on the NEF activity of Hsp110, the class B JDP and its interaction with the Hsp70 EEVD motif (Figure 2B, Figure 2—figure supplement 1B; Beton et al., 2022). We propose that the local extraction of polypeptides by Hsp70-JDP^B-Hsp110, which penetrates the surface and takes apart amorphous aggregates, although not enough for full protein recovery, could successfully generate more manageable substrates for chaperones, as we demonstrated by applying the derepressed Hsp104 variant (Figures 2A and 5). This Hsp70 activity, albeit yielding very low protein reactivation (Figure 2—figure supplement 1A), might be relevant for the fragmentation of a thinner, linear amyloid structure.

Such clustering-augmented aggregate remodelling and fragmentation, possibly through expanding the total effective aggregate surface, leads to the much-improved protein reactivation by Hsp104 (Figure 1—figure supplement 1B and C, Figure 2—figure supplement 1A). This would explain the previous findings that although Sse1 is not necessary for Hsp104 recruitment to aggregates, it is essential for Hsp104-dependent aggregate clearance in the cell (Kaimal et al., 2017). Apparently, despite the more heterogenic and dynamic nature of cellular aggregates than of those generated in vitro, their disaggregation by Hsp104 largely relies on the Hsp110-boosted processing by the Hsp70 system.

Above a certain Hsp110 level, however, the disaggregation is inhibited. The susceptibility to the NEF depends on the phase of the process and the composition of the Hsp70 system (Figure 3A–D, Figure 3—figure supplement 1A), which may explain the discrepancies between different studies of Hsp110’s impact on disaggregation (Dragovic et al., 2006; Polier et al., 2008; Raviol et al., 2006; Shorter, 2011) As well established, the most effective is sub-stoichiometric Hsp110 proportion to Hsp70 (Gao et al., 2015; Polier et al., 2008; Wentink et al., 2020) which corresponds with the physiological conditions, where the ratio between all cytoplasmic Hsp70 and Hsp110 paralogs involved in disaggregation ranges from approximately 10:1.7 in yeast (Brownridge et al., 2013) to 10:2.4 in humans (Finka and Goloubinoff, 2013). According to our data, the optimum, at which the stimulation and inhibition curves intersect, is shifted to higher levels: (a) during the initiation versus final protein folding (Figure 3A and C), (b) with class B JDP, in comparison with class A (Figure 3A, B, and D), (c) with Hsp104, comparing with the system without the disaggregase (Figure 3A, Figure 3—figure supplement 1A), and (d) for the human Hsp70 system, comparing with the yeast chaperones (Figure 3A and B). The latter trend might result from the stronger dependence of the human Hsp70 chaperone on the nucleotide exchange activity, possibly associated with the lower basal rate of nucleotide dissociation than that reported for Ssa1 (Dragovic et al., 2006). Possibly, due to these problems with ADP release, the positive impact of Hsp110 on Hsc70 is manifested both in the presence of class A and B JDPs and the inhibition occurs at much higher Hsp110 levels. Accordingly, it is tempting to speculate that the human system, lacking Hsp104, has evolved towards better tolerance for Hsp110 to boost the disaggregation activity of the Hsp70 system alone, e.g., to counteract amyloid toxicity.

Excessive dissociation of Hsp70 from a substrate, promoted by nucleotide exchange, may explain the system’s susceptibility to Hsp110. Here, we propose an additional mechanism behind this inhibition. The fact that the NEF and JDP binding occurs at mutually exclusive nucleotide states of Hsp70 has raised a question of competition between the two co-chaperones (Sousa and Lafer, 2019), which, to our knowledge, has never been addressed before. On the other hand, class B JDP goes beyond the classical model of the Hsp70 cycle due to the auxiliary interaction with the EEVD motif, which is not nucleotide-sensitive and theoretically might not be exclusive with Hsp110 binding (Wyszkowski et al., 2021; Yu et al., 2015). Nonetheless, our results clearly demonstrate that Hsp110 disrupts the JDP’s complex with Hsp70 (Figure 4A, Figure 4—figure supplement 1C). This way, Hsp110 may cause the release of Hsp70 from an aggregate, as well as of a JDP, unless it remains tethered via another interaction, e.g., through the other JDP subunit in complex with an aggregate-bound Hsp70 molecule (Figure 5). The fate of the displaced proteins is yet to be established, presumably at low Hsp110 levels, they may re-bind at another site on the substrate or to the aggregate-bound chaperones. At high Hsp110 levels, the JDP^B-Hsp70 interaction is very strongly affected, similarly as aggregate binding and disaggregation (Figures 3A, B, D, 4A and B), suggesting that the proteins released by Hsp110 do not re-bind or that any subsequent binding is non-productive.

The apparent competition between the co-chaperones is reflected in the fact that the inhibition by Hsp110 is moderated at increasing Sis1 level (Figure 4C). Interestingly, the class B JDP also exerts a biphasic effect on disaggregation, and optimal concentrations of the two co-chaperones are strongly interdependent (Figure 4B and C, Figure 4—figure supplement 1D and E). The highest disaggregation yield occurs at Ssa1:Sis1:Sse1 ratio closest to the average reported in the cytosol, 10:0.3:1.7 (Figure 4C; Brownridge et al., 2013), pointing to a possibility that the proportions between the co-chaperones have undergone evolutionary fine-tuning to develop higher tolerance to stress.

The sub-stoichiometric optimum is unique to Hsp110 (Gao et al., 2015; Wentink et al., 2020), while other NEF, Fes1, requires 10 times higher level to reach the same degree of stimulation, similarly as the Sse1-2 variant with reduced affinity for Hsp70. Fes1’s ability to improve Hsp70 loading onto aggregate is surprising, regarding findings of Wentink et al., 2020, who demonstrated, using Hsp110 and BAG1 variants, that Hsp70 clustering on amyloid requires a bulky NEF. Although Fes1 is only 33 kDa, the stretch of its armadillo repeats and different mode of interaction with NBD could possibly lead to similar excluded volume effects as Hsp110. Nevertheless, Fes1 is present in a cell at approximately one-fifth the level of Sse1, and even its 8.4-fold overexpression from two plasmids, shown by Kaimal et al., was insufficient to complement the thermosensitive phenotype of sse1-200 sse2Δ (Kaimal et al., 2017), as it was six times too low to reach the level effective in disaggregation in vitro (Figure 1—figure supplement 3A). It is worth noting that Fes1 has been reported to target substrates to degradation (Gowda et al., 2018; Gowda et al., 2013) and apparently its role in Hsp70 recruitment to disaggregation is minor.

An abundant association of Hsp70-JDP^B with aggregate surface, although generating strong pulling effect to disentangle polypeptides, could also mask access of chaperones to the newly emerging sites adjacent to and buried beneath the complex. We demonstrate that Hsp110 could theoretically uncover such sites not only through Hsp70 dissociation from the substrate due to the nucleotide exchange (Dragovic et al., 2006), but also by disrupting the JDP^B-Hsp70 interaction (Figure 5). Four Hsp70-binding sites in a JDP^B dimer allow to form an extensive network of interactions (Wyszkowski et al., 2021). We speculate that Hsp110 introduces plasticity into this network, enabling the chaperone complex to infiltrate cavities emerging within the aggregate in a fluid-like manner, binding to the uncovered chaperone-binding sites and pulling up to the aggregate fragmentation (Figure 5). Such behaviour would require only a small destabilising effect of the NEF that would not dissociate the complex completely (Figure 4A), similarly as observed at the Hsp110 concentration that supports the most effective aggregate binding and disaggregation (Figure 3A and D). Further experimental verification of this scenario and future studies of the dynamics within the Hsp70 chaperone system will be critical to understand and combat the stress- and disease-related protein aggregation.

All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for Figures 1-4 and all Figure Supplements.

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Author details

1. Wiktoria Sztangierska

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

2. Hubert Wyszkowski

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

3. Maria Pokornowska

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3233-7500

4. Klaudia Kochanowicz

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Michal Rychłowski

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Methodology

   Competing interests

   No competing interests declared

6. Krzysztof Liberek

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review and editing

   For correspondence

   krzysztof.liberek@ug.edu.pl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7532-9279

7. Agnieszka Kłosowska

   Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdańsk, University of Gdańsk, Gdańsk, Poland

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   For correspondence

   agnieszka.klosowska@ug.edu.pl

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5295-9473

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