Replicative aging impedes stress-induced assembly of a key human protein disaggregase
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Abstract
The collapse of protein homeostasis manifests itself in a toxic protein aggregation cascade, which is associated with degenerative diseases and aging. To solubilize aggregates, dedicated protein disaggregases exist in unicellular organisms, but these have no nuclear/cytosolic orthologs in metazoa. Alternative metazoan disaggregation machines have been described, but how these are operated and regulated in vivo remained unknown. We show that protein disaggregases are functionally diversified in human cells to efficiently target different types of stress-induced aggregates in sequential and temporally distinct phases. In particular, we show the selective assembly of an Hsp70-DNAJA1-DNAJB1 trimeric disaggregase that forms during late phase of stress recovery., i.e. , after VCP-dependent solubilization of non-native proteins that accumulate in cellular condensates such as nucleoli or stress granules. When activated, the trimeric disaggregase provides resistance to stress toxicity and contributes to amyloid disposal. Strikingly, this disaggregase collapses early in cells undergoing replicative aging with important underlining pathophysiological consequences.
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Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Richa Arya, Joseph Biggane, Luciana Gallo, Arthur Molines, Sónia Gomes Pereira. Review synthesized by Vasanthanarayan Murugesan.
In this preprint, the authors describe a novel pathway that maintains protein homeostasis in cells recovering from heat stress termed stress-induced protein disaggregase activation pathway (siDAP). siDAP induces the DNAJA1+DNAJB1-Hsp70 protein disaggregase and targets aggregates of tightly misfolded proteins. This pathway is distinct from more-known ubiquitin-dependent quality control and works in sequence with it. Further, the authors show that this pathway is compromised in aging cells. The authors have provided a wealth of convincing data to support the claims made.
The following items were raised:
Majo…
Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Richa Arya, Joseph Biggane, Luciana Gallo, Arthur Molines, Sónia Gomes Pereira. Review synthesized by Vasanthanarayan Murugesan.
In this preprint, the authors describe a novel pathway that maintains protein homeostasis in cells recovering from heat stress termed stress-induced protein disaggregase activation pathway (siDAP). siDAP induces the DNAJA1+DNAJB1-Hsp70 protein disaggregase and targets aggregates of tightly misfolded proteins. This pathway is distinct from more-known ubiquitin-dependent quality control and works in sequence with it. Further, the authors show that this pathway is compromised in aging cells. The authors have provided a wealth of convincing data to support the claims made.
The following items were raised:
Major comments
Manuscript:
- It is recommended to revise the manuscript to better integrate the data and the text. The paper provides extensive data to support the study claims, but further background material for the experiments in the introductory or results section would support interpretation e.g., concepts required to understand the final two figures are not discussed in the introduction.
- Reducing the number of supplementary figures may make the manuscript easier to follow and help in tightening the narrative.
Experiments:
- Results ‘Immediately after HS, DNAJA1 and DNAJB1 rapidly relocalized to nucleoli’ - It is unclear from the DAPI stain what happens to the nucleolus at 0h after HS. It seems to be present in some cells but not all. Could a marker of the nucleolus be used and/or some clarification included?
- Results ‘This suggests that predominantly newly synthesized DNAJA1 and DNAJB1 molecules drive the assemblage of the DNAJA1+DNAJB1-Hsp70 disaggregase in cells after HS’ - Fig S5D shows that B1 forms puncta after HS even in CHX treated cells, which suggests that protein synthesis is not needed. Can some clarification be added for this fragment.
- Results ‘diffuse GFP fluorescent signal (cyan) indicating that protein aggregates were largely absent’ - The presence of aggregates or puncta before HS cannot be ruled out, the puncta or aggregate could be too small to be resolved. Recommend commenting on this.
- Results ‘Blocking Hsp70 activity by VER-155008 also caused DNAJA1+DNAJB1 scaffolds to persist up to 24h after HS, presumably due to their continuous association with the aggregates (Figure 2D).’ - The HSP70 aggregates look different after treatment with VER, they look more like the A1/B1 puncta than in the DMSO condition, it may be worth commenting on this.
- In Figure 6, the distinction between biological aging and replicative aging could be stated more clearly. Cell lines derived from donors of different biological ages form siDAP puncta and recover from heat shock. However, the cells lose this ability when cultured in dishes at passage 12 or 18 irrespective of biological age. Hence it is not clear if passaging cells mimics biological aging with regard to protein homeostasis.
Minor comments
- Figure 1H: Recommend including some comments on why the size of HSF is more at 0 hr, and commenting on whether HSF-1 depletion changes HSP70 levels.
- Figure 2 (B-D) - The size of cells in U vs 0 hour appear different, the 0 hr cells look bigger. Suggest adding a scale bar and clarification on whether the magnification is the same.
- In Figure S4/S5, it is hard to infer the state of the nucleolus during stress with DAPI staining and subsequently the localization of DNAJA1 and DNAJB1 to the nucleolus is not clear.
- In Figure S4D, it is shown that CHX doesn’t affect the formation of puncta but the text states that newly synthesized DNAJA1 and DNAJB1 are required for the assembly of DNAJA1-DNAJB1-HSP70. Please provide some clarification for this contradiction.
- In Fig S8, statistical analysis of different siDAP induction is suggested.
- In Fig 3, please provide clarification for the choice of experiments in CHX-treated cells for testing the effect of VER-155008.
- In Fig 5, the caption mentions cells with/without VER-155008 treatment which cannot be seen in the figure.
- ‘In fact, we found that human cells can tune the activation of siDAP according to the level of protein damage sustained after HS’ - It may be informative to check if the cytotoxicity levels differ from HS at 39ºC and at 42ºC.
- In Fig 6, quantification of PLA^Dt, similar to Fig 1F is suggested. Please also report the conditions used for heat shock in these experiments, 42oC for 2 hrs?
- ‘Moreover, siDAP was fully active in all fibroblast lines tested (Figure 6A; Figure S22A and B). Similar to immortalized HeLa cells, primary dermal fibroblasts only induced the DNAJA1+DNAJB1 JDP scaffold after HS (Figure S22C)’ - May be worth mentioning that the apparently higher intensity of the fluorescence signal in the cells derived from aged subjects. The fluorescence signal per cell looks much greater in 70 yo (Fig. S22 only) and 75 yo (Figs. 6 and S22). The next few lines discuss the relevance of decreased fluorescence (representative of loss of siDAP induction) with serial passaging/replicative age. However, upon HS, siDAP signal seems to go up with chronological age, but then in the replicative aging experiments, siDAP is lost quickly.
- Discussion ‘There is some evidence to suggest that cellular surveillance systems that usually keep protein aggregation in check deteriorate during aging….’ - There may be some conflation of biological aging and "replicative aging". There seemed to be conflicting results when looking at differently biologically aged samples, which may affect interpretation of whether replicative aging in a dish recapitulates aging processes.
- Methods Cell culture - Please provide further information about the age and other details for the 6 primary fibroblast cell lines.
- Recommend increasing the size of the microscopic image panels in several figures to better highlight the features mentioned.
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