Balanced Interplay Between Hsp110, Hsp70 and Class B J-Domain Protein Improves Aggregate Disassembly

Reviewer #1 (Public Review):

Summary:

The manuscript by Sztangierska et al explores how the Hsp70 chaperone together with its JDP-NEF cofactors and Hsp104 disentangle aggregated proteins. Specifically, the study provides mechanistic findings that explain what role the NEF class Hsp110 has in protein disaggregation. The results explain several previous observations related to Hsp110 in protein disaggregation. Importantly, the study provides compelling evidence that Hsp110 acts early in the disaggregation process.

Strengths:
(1) This is a very well-performed study with multiple in vitro experiments that provide convincing support for the claims.

(2) An important finding is that the study places the Hsp110 function early in the disaggregation process.

(3) The study has an important value in that it picks up on a number of observations in the field that have not been explored or directly tested by experiment. The presented results settle questions and controversy regarding Hsp110 function in disaggregation.

Weaknesses:

(1) While the key finding of this manuscript is that it places Hsp110 early in the disaggregation process, the other findings are advancing the field less.

(2) A claim in the paper is that Hsp110 NEFs improve disaggregation by Hsp70 in a manner dependent on the class of JDP (class A vs class B). However, it rather appears that in the experiments class B JDPs support robust disaggregation, while class A JDPs are not as effective. This simple fact may very well underly the differences and questions if class specificity should be in focus in the interpretation of the data.

(3) The experiments differ somewhat in regard to the aggregated protein used. For example, in Figure 1A, FFL is used with only limited reactivation (10% reactivated at the last timepoint and the curve is flattening), while in Figure 2B FFL-EGFP is used to monitor microscopically what appears to be complete disaggregation. Does FFL-EGFP behave the same as FFL in assays such as the one in Figure 1A or are there major differences that may impact how the data should be interpreted?

Reviewer #2 (Public Review):

Sztangierska et al. have investigated the impact of the nucleotide exchange (NEF) factor Hsp110 on the Hsp70-dependent dissolution of amorphous aggregates in the presence of representative members of two classes of J-domain protein.

The authors find that the nucleotide exchange factor of the Hsp110 family, sse1, stimulates the disaggregation activity of yeast Hsp70, ssa1, in particular in the presence of the J-domain protein sis1. Linking chaperone-substrate interactions as determined by biolayer interferometry (BLI) to activity assays, they show that sse1 facilitates the loading of more ssa1 onto the aggregate substrate and propose that this is due to active remodeling of the protein aggregate which exposes more chaperone binding sites and thus facilitates reactivation. This study highlights two important facets of Hsp70 biology: different Hsp70 functions rely on the functional cooperation of specific co-chaperone combinations and the stoichiometry of the different players of the Hsp70 system is an important parameter in tuning Hsp70 chaperone activity.

Strengths:

The manuscript presents a systematic analysis of the functional cooperation of sse1 with a class B J-domain protein sis1 in the disaggregation of two different model aggregate substrates, allowing the authors to draw more general conclusions about Hsp70 disaggregation activity.

The authors can pinpoint the role of sse1 to the initial remodeling of aggregates, rather than the later stages of refolding, highlighting the functional specificity of Hsp70 co-chaperones.

They demonstrate the competitive nature of binding to ssa1 between sse1 and sis1 which can explain the poisoning of Hsp70 chaperone activities observed at high NEF concentrations.

Weaknesses:

Experimental data concerning the class A JDPs should be interpreted with caution. These experiments show very small reactivation activities for luciferase in the range of 0-1% without the addition of Hsp104 and 0-15% with the addition of Hsp104. Moreover, since the assay is based on the recovery of luciferase activity, it conflates two chaperone activities, namely disaggregation and refolding. It is possible that the small degree of reactivation observed for the class A JDP reflects a minor subpopulation of the aggregated species that is particularly easy to disaggregate/refold and may thus not be representative of bulk behaviour.

While structural requirements have been identified that allow sse1, in cooperation with sis1, to facilitate the loading of Hsp70 on the amorphous aggregate substrate, how this is achieved on a mechanistic level remains an open question.

Reviewer #3 (Public Review):

Summary:

The authors studied the function of Hsp110 co-chaperones (e.g. yeast Sse1) in Hsp70-dependent protein disaggregation reactions. The study builds on former work by the authors (Wyszkowski et al., 2021, PNAS), analyzing the binding of Hsp70 and J-domain protein (JDP) cochaperones to protein aggregates using bio-layer interferometry (BLI). It was shown before by other groups that Hsp110 enhances Hsp70 disaggregation activity. The mechanism of Hsp110-stimulated disaggregation activity, however, remained poorly defined. Here, the authors show that yeast Hsp110 increases Hsp70 recruitment to the surface of protein aggregates. The effect is largely dependent on J-domain protein (JDP) identity and is particularly pronounced for class B JDPs (e.g. yeast Sis1), which are also more effective in disaggregation reactions. The authors also confirm former results, showing inhibition by increased Hsp110 levels, and provide novel evidence that the inhibitory effect is caused by competition between Hsp110 and JDPs for Hsp70 binding.

Strengths:

The work represents a very thoroughly executed study, which provides novel insights into the mechanism of Hsp70-mediated protein disaggregation. Key findings established for yeast chaperones are also documented for human counterparts. The observation that Hsp110 might displace JDPs from Hsp70 during the disaggregation reaction is very appealing. It will now become important to validate this initial finding and dissect how it propels the disaggregation reaction.

Weaknesses:

How exactly the interplay between JDPs and Hsp110 orchestrates protein disaggregation remains largely speculative and further analysis is required for a deeper mechanistic understanding. Enhanced recruitment of Hsp70 in the presence of Hsp110 was shown for amyloid fibrils before (Beton et al., EMBO J 2022) and should be acknowledged. The assay reporting on the refolding activity of Hsp70 seems problematic due to the high spontaneous refolding of the substrate Luciferase and should be modified.