CHIP ubiquitin ligase is involved in the nucleolar stress management
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Abstract
The nucleolus is a dynamic nuclear biomolecular condensate involved in cellular stress response. Under proteotoxic stress, the nucleolus can store damaged proteins for refolding or degradation. HSP70 chaperone is a well-documented player in the recovery process of proteins accumulated in the nucleolus after heat shock. However, little is known about the involvement of the ubiquitin-proteasome system in the turnover of its nucleolar clients. Here we show that HSP70, independently of its ATPase activity, promotes migration of the CHIP (carboxyl terminus of HSC70-interacting protein) ubiquitin ligase into the granular component of the nucleolus, specifically after heat stress. We show that while in the nucleolus, CHIP retains mobility that depends on its ubiquitination activity. Furthermore, after prolonged exposure to heat stress, CHIP self-organizes into large, intra-nucleolar droplet-like structures whose size is determined by CHIP ubiquitination capacity. Using a heat-sensitive nucleolar protein luciferase, we show that excess CHIP impairs its regeneration, probably through deregulation of HSP70. Our results demonstrate a novel role for CHIP in managing nucleolar proteostasis in response to stress.
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Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Sónia Gomes Pereira, Rachel Lau, Sam Lord, Sanjeev Sharma, Parijat Sil. Review synthesized by Richa Arya.
General comments
It may be helpful to elaborate on how it is established that CHIP mobility is dependent on activity. The conclusion in the paper has been primarily drawn from the catalytically inactive H260Q mutant which is less mobile. However the fact that the puncta of the mutant are brighter and larger than the wild type and that it recovers slowly also indicates the protein might be inherently more prone to aggregation upon heat shock.
Related to the above point, under conditions such as VER treatment and Act-D treatment, the nucleolar recruitment is unaltered but recovery is affected (which implies mobility may be …
Review coordinated via ASAPbio’s crowd preprint review
This review reflects comments and contributions by Sónia Gomes Pereira, Rachel Lau, Sam Lord, Sanjeev Sharma, Parijat Sil. Review synthesized by Richa Arya.
General comments
It may be helpful to elaborate on how it is established that CHIP mobility is dependent on activity. The conclusion in the paper has been primarily drawn from the catalytically inactive H260Q mutant which is less mobile. However the fact that the puncta of the mutant are brighter and larger than the wild type and that it recovers slowly also indicates the protein might be inherently more prone to aggregation upon heat shock.
Related to the above point, under conditions such as VER treatment and Act-D treatment, the nucleolar recruitment is unaltered but recovery is affected (which implies mobility may be affected). This leads to the accumulation of CHIP in the nucleus. In these scenarios, it may be relevant to report on the status of wild type CHIP activity? Conducting the ubiquitination assay as in Figure 5A with Act-D and Ver treatment would be informative. If no difference in ubiquitination is observed, it can be concluded that it is not the change in CHIP mobility that affects its activity, but rather it's activity that promotes CHIP mobility/dynamics (the conclusion from Figure 5).
o Figure 1: The question arises as to why the control and recovery show puncta in panel C, but not the HS condition. Also, to make it easier to appreciate the nucleolar localization of CHIP in the HS condition, zoomed in regions and overlay images would be useful.
Figure 1b: To support interpretation of the results, it would be helpful to highlight some examples of the nucleolar localization of CHIP. Additionally, it looks like there are specific dots (that could be like condensates) in the Control and Recovered, but not during the Heat Shock cells, not in panel B. Maybe some quantification such as number of dots per cell/ intensity/size could accompany the images. Similar parameters of the condensate structures in the nuclei in the transiently transfected cells could be quantified.
Figure 1: Quantifications such as 2B and 2C could also be done for Figure 1, for both Hsp70 and CHIP.
Figure 1E.’K30A mutant exhibited impaired CHIP migration to nucleoli after heat shock (Fig. 1E)…’ How strong is this impairment? Could it be quantified either by fluorescence intensity or via Western blot of the different cellular fractions.
o Figure 2: It would be helpful to have additional clarification on what the different parameters such as -"% of cells with EGFP-CHIP in the nucleolus' or 'CHIP intensity in the nucleolus' represent, as well as clarification on the transition from measuring CHIP nucleolar-to-nucleus intensity ratios for immunostaining (as in Fig S1E) to measuring just nucleolar CHIP intensities in the main Figure for the EGFP-CHIP overexpression experiments. Perhaps a western blot showing HSP70 expression with VER might be helpful in demonstrating that total protein expression is not affected and that it is only its activity being affected.
‘a small molecule inhibitor of HSP70…’ Some suggestions alongside the loss of function assays such as knockdown and inhibitor treatment:
What happens to Hsp70 and thereby CHIP translocation to the nucleus in cells with high, medium versus low levels of HSP70 expression? Do the high-expressing cells show more enhanced CHIP recruitment to the nucleolus? Can it be quantified as to how correlated the efficiency of recruitment of CHIP is to the expression level of Hsp70? How does the nucleolar translocation of Hsp70 itself correlate with its expression level?
Figure 2a: It is clear that the HSP70 co-localises with CHIP upon heat shock. Overlaid images might be better to highlight this but the use of green and red is not ideal for colour-blind readers. May be changed for bar graphs too (2d,e).
Figure 2b,c: There is a question about the statement that mutant CHIP was unable to localise in the nucleoli due to lack of HSP70 binding in Fig 1E. In Fig 2B and 2C CHIP was able to migrate into the nucleoli (albeit at a lesser extent) with HSP70 knockdown? Maybe images corresponding to this experiment might help as well to allow the reader to see the difference in localisation? It is mentioned that CHIP auto-ubiquitination is important in its localisation in Fig 5 so does the CHIP K30A mutant necessarily verify that the lack of HSP70 binding is causing impaired migration to the nucleus in Fig 1E? Could K30A also affect its auto-ubiquitination? Suggest referencing supplementary figure 2 alongside Fig 2B and 2C, and changing the dots in this graph to red, to make it consistent with panel F.
Figure 2d,e: Bar plots could be replaced with scatter plots showing individual data points as done in Supp. Fig 1E. Adding t1/2 values with FRAP traces would support the changes observed for recovery times across conditions. Calculating mobile fraction and reporting would also be helpful.
Figure 2f,g: Suggest updating the figure legend to clearly distinguish both curves. Some additions may complement the FRAP analysis presented:
- How does the FRAP mobility of CHIP compare between absence and presence of heat shock?
- How does the FRAP mobility of CHIP compare in the recovery phase in presence and absence of heat Ver?
- Is the CHIP mobility different in nucleolus versus nucleus?
‘and HSP70 inhibition did not si+gnificantly reduce its dynamics (Fig. 2F)…’ Would there be any change in CHIP dynamics in siHSP70 cells? It would be helpful to mention this following Fig 2B/C. Maybe use 'mobility' instead of dynamics, to be more specific.
o Figure3: It will be helpful to include an overlay/merged image of the two channels, and to explain in the legend how the measured correlation coefficient is obtained. It would be nice to see what kind of sub-structures show the maximum colocalization.
Fig 3c: HS+Rec condition should show a loss of correlation between CHIP and NPM1 and is an important control in this figure. Comparison with Fibrilarin is good, demonstrating a loss of correlation between the NPM1 and CHIP themselves under different conditions and data for Ctrl only conditions would also add value.
o ‘it altered CHIP distribution, which more prominently overlapped with Act D-induced NPM1 ring formations (Fig. 4D)…’ Can this be quantified? Maybe it will show more pronounced colocalization compared to heatshock alone.
o 'this observation suggests that proper nucleolar assembly may be necessary for CHIP dynamics'. It may be worth specifying the reference to Dynamics here:
- Mobility measured via FRAP
- Translocation efficiency done via intensity measurement or ration of nucleolus/nucleus intensity. FRAP measurement of CHIP may be helpful to conclude about the mobility of CHIP in the nucleolus upon heat shock, in presence or absence of Act D pre-treatment. A change in mobility may support the lack of translocation during the recovery phase in presence of Act D.
o Figure 4: (a) It may be worth commenting on why the Hoechst staining looks different between the Control and the Act-D conditions. Fig4d: It could be helpful to add images of NPM1 localization in cells treated with Act D, but not under heat shock. In other words, are these NPM1 rings specific to the heat shock response? The size of the cells and the nucleus are different for HS versus Act-D+HS panels. If the scale bar is consistent and this is a normally observed morphological change upon Act-D treatment, it might be helpful to note this size difference in the legend.
o ‘We found that the activity of CHIP is not indispensable for heat shock-induced migration to the nucleolus (Fig. 5B). However, FRAP analysis of the nucleolar CHIP H260Q mutant showed a decrease in its dynamics compared to CHIP WT…’ Maybe the fragment could be rewritten for clarity (e.g. is dispensable). What happens to the mutant CHIPH260Q localization upon recovery? Is it slower than wt? Is more mutant CHIP retained in the nucleolus upon recovery?
o Figure 5: Suggest showing a wt image as comparison, in panel B. An alternate interpretation for the observations with H260Q mutant could be that the mutation leads to instability and misfolding of CHIP (as suggested in the paper) which leads to increased aggregation (larger and brighter droplets, low mobility) upon heat shock with itself and other interacting proteins. This interpretation does not need to invoke a loss of ubiquitination activity as a cause, it could be another consequence of misfolded CHIP.
Figure 5c: How do the mobility of wild type CHIP compare with the H260Q mutant in the nucleus or in absence of heat shock? If the mobility is the same during pre-heat shock/pre-translocation to the nucleolus, the wild type and mutant protein have inherently similar dynamics. And if this gets altered only in the nucleolus of heat shocked cells, it would support the conclusion that it is the activity of CHIP that helps retain its mobility in the nucleolus and possibly prevent its aggregation in this compartment.
Figure 5f: If there were two independent experiments, can both be represented? Or was the data pooled from the two experiments?? Suggest representing the data as two points for CHIP wild type and mutant each, from two independent experiments.
Figure 5g,h,i: Dot plot overlay on the boxplot might be nice to see the spread of datapoints.
o ‘Interestingly, sizeable intra-nucleolar CHIP droplet-like structures could be observed after overnight heat shock in cells expressing the CHIP H260Q mutant, outnumbering their WT protein counterparts (Fig. 5E-I)…’ In Figure 1C some bright foci are also observed in control and recovered cells. Are these similar to the "droplet-like structures" described here?
o ‘These differences between CHIP WT and mutant assemblies may stem from the alterations in CHIP H260Q dynamics within the nucleolus (Fig. 5C and D)’. Similar measurement as in Fig 5C could be done upon overnight heatshock to support this statement.
o ‘Surprisingly, we found comparable redistribution of all CHIP variants to nucleoli during heat shock, suggesting an…'. Is this a cell line-specific difference, or could it be due to differences in approach, i.e. stable cell line vs. transient overexpression? Similar transient expressions in HeLa may help clarify this.
o Based on Fig S1E, it appears there might be both an HSP70 activity-dependent (smaller) and HSP70 activity-independent (larger) contributions to CHIP localization. VER treatment reduces CHIP relocalization to the nucleus by a small but significant amount both in control and HS-treated cells.
o Cell transfection - Suggest reporting the confluency of the cells before transfection (or at which they were seeded).
Methods - In Figs 3C and 5G-I, there is a concern about the statistical approach to calculate p-values based on multiple measurements (nuclei) within each sample. The t-test and ANOVA assume that each measurement is independent, and multiple nuclei within the same sample are not independent. Recommend to either not report p-values or to average together the values from each sample and calculate the p-value using those sample-level means. For more information, see https://doi.org/10.1371/journal.pbio.2005282 and https://doi.org/10.1083/jcb.202001064.
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