Joint Public Review:

Summary of the work:

This manuscript defines the differential stress response signaling induced by nuclear and cytoplasmic protein misfolding. To accomplish this, the authors used superfolder GFP fused to a destabilized FKBP protein-bearing targeting signal for cytosolic or nuclear localization. When cells were grown in the presence of the ligand Shield-1, this protein was stable, allowing fluorescence of the GFP protein. Upon removal of Shield-1, the FKBP protein is unfolded targeting the entire fusion protein to proteasomal degradation. Using this approach, they performed RNAseq to probe similarities and differences in transcriptional responses to the accumulation of unfolded proteins in the cytosol or nucleus. As expected, many of the pathways upregulated in both datasets involved protein homeostasis pathways such as the proteasome and cytosolic chaperones. The increase in proteasome subunits correlated with the stabilization of Nrf1 under these conditions, suggesting that protein misfolding might induce proteasome subunits through an Nrf1-dependent mechanism, but this was not explicitly tested. In contrast, the authors report that the p53-dependent transcriptional response was selectively induced by protein misfolding stress in the nucleus, but not the cytosol. Deletion of p53 blocked this increase, indicating that this response is attributable to p53 stabilization. The increased p53 transcriptional activity corresponded with the stabilization of p53 and its target p21 in cells subjected to nuclear but not cytosolic protein misfolding stress. Using a reporter of nuclear proteasome activity, they show that nuclear proteasome activity is reduced in cells following protein misfolding stress in the nucleus, indicating that the stabilization of p53 (and other transcription factors such as NRF1) might be attributed to reduced proteasomal degradation. Additionally, the authors showed that nuclear misfolding stress also induces cell cycle arrest. However, this effect was not dependent on p53 deletion, indicating that this is mediated by other unknown mechanisms.

Major strengths and weaknesses of the methods and results:

The findings reported here define specific transcriptional outputs induced by targeted protein misfolding stress in the nucleus and cytosol, revealing new insights into the organelle-specific stress signaling. The approach is interesting and effective at revealing cellular responses induced by compartment-specific protein misfolding stress.

One major weakness of the study is the lack of mechanistic follow-up for the transcriptional study. For example, what is the mechanistic basis for p53 stabilization by nuclear-destabilized domain (Nuc DD)? Is this entirely caused by diminished nuclear degradation activity as shown in Figure 6 or are there additional factors to be considered? If limited proteasome degradation capacity is the main reason for p53 upregulation, wouldn't the authors also see stabilization of other short-lived transcription factors? The fact that Nrf1 and Nrf2 are also stabilized by Nuc DD is consistent with the authors' hypothesis. On the other hand, if Nuc DD also affects other short-lived transcription factors such as c-fos or c-myc via proteasome inhibition, why did the gene expression analysis only pick up the p53 pathway as the one differentially regulated by Nuc DD? Would this imply that only p53 is specifically targeted by the nuclear proteasome, whereas other short-lived transcription factors are degraded either by the cytosolic proteasome or by both nuclear and cytosolic proteasome like Nrf1? Is there any evidence in the literature that supports this speculation? Additionally, how does Nuc DD affect the UPS system in the nucleus? Does it clog the proteasome directly or affect other assisting factors like chaperones or ubiquitinating enzymes? Lastly, it isn't clear what the functional implications of p53 stabilization would be for cells subjected to nuclear protein misfolding stress, particularly as the small effect on cell cycle arrest is not dependent on p53. In the end, the lack of mechanistic and/or functional follow-up reduces the overall importance of this manuscript. While the reviewers do not expect the authors to answer all these questions by experiments, additional work/clarifications/discussions along these lines would significantly improve the paper (see the recommendations).

Another major weakness is the lack of statistical analysis (SA) to better support their conclusions. In fact, no SA was provided for many figures even though the authors tried to make many comparisons.

The failure of the DD reporter to mount a significant heat shock response was puzzling. The presence of non-native proteins is the primary trigger for the heat shock response, but the authors acknowledge that inducible chaperones such as Hspa1a/b and Hsp90aa1 were not significantly changed in their system (page 8). Could this suggest a problem with the approach? What exactly is the nature of the stress mounted by Nuc DD?

The cell cycle data presented in Figure 5 is less robust, particularly as the p53 data in panels C and D was collected only once.

The Western blot data shown in Figure 6 does not have quantification to show how representative the blot is and how robust the changes in protein levels are over time. Western blots are known to be variable with different replicates and therefore the authors need to mention the number of biological repeats represented by the blot.

Figures and data

Compartment-specific unfolded DD is degraded and competes with proteasome substrates for degradation.

Global transcriptional profiling reveals common and distinct features

Common response features protein quality control and oxidative stress response elements.

Nuclear unfolded protein robustly induces the p53 pathway.

Nuclear unfolded protein induces a transient p53-independent cell cycle delay.

Compartment-specific increase in proteasome demand stabilizes short-lived transcription factors.