Regulation of the 26S Proteasome: From Homeostasis to Stress and Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is timely and highly relevant. It provides a comprehensive overview of the emerging concepts regulating proteasome function beyond its classical role as a passive degradation machine. The discussion of proteasome dynamics, compartmentalization, proteaphagy, and disease relevance is particularly strong and reflects important recent advances in the field. The figures are informative and help readers navigate complex concepts. The review is also well written and balanced.

I have only a few minor suggestions:

1. Correcting several typographical and grammatical errors throughout the text is needed
2. A section discussing proteasome condensates and amino-acid-dependent regulation of proteasome localization, could benefit from a brief concluding paragraph highlighting remaining open questions in the field.

 

Author Response

Summary:

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Point-by-point response to Comments and Suggestions for Authors

1. Comment:
   Correcting several typographical and grammatical errors throughout the text is needed
   
   Response:
   We have carefully revised the manuscript and corrected typographical and grammatical errors throughout the text to improve clarity and readability.
   
   
2. Comment:
   A section discussing proteasome condensates and amino-acid-dependent regulation of proteasome localization, could benefit from a brief concluding paragraph highlighting remaining open questions in the field.
   ResponseWe thank the reviewer for this helpful suggestion. In response, we have added a brief concluding paragraph at the end of the relevant section (new lines – 419- 432) highlighting key open questions in the field. The added text reads as follows:

“While these observations underscore the importance of spatial proteasome regulation during cellular stress, several key questions remain unresolved. The molecular mechanisms that coordinate nutrient sensing with proteasome translocation are still incompletely understood, and it remains unclear whether additional nutrient sensors or signaling pathways cooperate with the Sestrin3–mTORC1 axis to regulate proteasome localization. Likewise, the mechanisms governing proteasome recruitment to distinct biomolecular condensates, the determinants of condensate specificity, and the interplay between condensate-mediated proteolysis and proteaphagy require further investigation. Also, as numerous target substrates are ubiquitinated, an open question relates to their recognition.  Are they recognized by their ‘individual’ ligases, or there is a ‘universal’ ligase that recognizes them all, and the question here is the identity of the common signal that this putative ligase recognizes. Addressing these questions will provide a more comprehensive understanding of how spatial organization contributes to proteasome regulation during cellular adaptation to stress.”

 

Reviewer 2 Report

Comments and Suggestions for Authors

The Review entitled "Regulation of the 26 Proteasome: From Homeostasis to Stress 2 
and Disease" by Cohen-Kaplan and Co-workers is nice, well written and fit the topics of the Special Issue.

Here are reported my revisions:

1. The title should report 26S.
2. This Manuscript needs a Multi-panel Figure 1 (or two Figures) with Three-dimensional Structures of Proteasome, Proteasome particles and modulators that helps the readers for the p1.1 "Dynamic Assembly and Composition of Proteasome Complexes". 
3. During the description of the assembly a sentence dedicated to UMP1/POMP-related factors should be added and not only reported at the end in p3.3.
4. Please re-write the sentence lines 469-472 avoiding to repeat "under conditions".
5. Re-think the sentence in Table 1 "Growth support, suppression of catabolic 
   response".
6. Move Ref [38] to the end of the sentence [38, 39] line 117.

Author Response

Summary:

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Point-by-point response to Comments and Suggestions for Authors

1. Comment:
The title should report 26S.

Response:
We have corrected the title and the entire text, so the proteasome now shows as the 26S proteasome as suggested.

2. Comment:
This Manuscript needs a Multi-panel Figure 1 (or two Figures) with Three-dimensional Structures of Proteasome, Proteasome particles and modulators that helps the readers for the p1.1 "Dynamic Assembly and Composition of Proteasome Complexes". 

Response:
We have addressed this point by adding two new Figures, 1 and 2. Figure 1 describes the different modes of plasticity of the 26S complex, whereas Figure 2 shows the 3D structures of the proteasome, proteasome particles, and relevant modulators, to better support the section “Dynamic Assembly and Composition of Proteasome Complexes” (p.1.1) and improve clarity.

 

3. Comment:
During the description of the assembly a sentence dedicated to UMP1/POMP-related factors should be added and not only reported at the end in p3.3.

Response:
We have added a dedicated paragraph on UMP1/POMP-related factors in Section 1.1 “Dynamic Assembly and Composition of Proteasome Complexes” (new lines - 128–131) to better integrate this important aspect of proteasome biogenesis into the assembly description. The added text reads as follows:

“Proteasome assembly and maturation are coordinated by dedicated chaperones, including UMP1/POMP, which are essential for 20S core particle biogenesis and ensure proper maturation of functional proteasomes by preventing the accumulation of incomplete or inactive assembly intermediates (doi:10.1242/jcs.259622).”

4. Comment:
Please re-write the sentence lines 469-472 avoiding to repeat "under conditions".

4. Response:
We have revised the sentence (new lines – 502-505) to avoid repetition of the wording “under conditions” which improves readability. The revised sentence reads as following:
"This cytoplasmic enrichment under conditions where the proteasome is fully active supports the maintenance of proteostasis during high anabolic demand and complements transcriptional programs governing proteasome biogenesis, thereby adding an additional layer of regulation to proteasome activity in tumors."

5. Comment:
Re-think the sentence in Table 1 "Growth support, suppression of catabolic 
response".

Response:
We have revised this sentence in Table 1 to improve clarity and accuracy. The revised wording is as follows: “Regulation of metabolism, cell growth and survival”. 

6. Comment:
Move Ref [38] to the end of the sentence [38, 39] line 117.

Response:
We have corrected the reference formatting and moved Ref. [38] to the end of the sentence as recommended.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Cohen-Kaplan et al have reviewed the topic in relation to the regulation of the 26 proteasome in terms of homeostasis and stress and disease. The topic is interesting, but there are several major and minor comments and concerns should be addressed before potential publication.

The authors have emphasized the “26” proteasome, but there is no information about this in the Abstract. The reviewer recommends the authors should add the rationale about why the regulation of 26 proteasome should be highlighted. Furthermore, please address why this review is needed.

In the Introduction section, the authors only showed the generational concept of degradation and cellular homeostasis. They should address why the regulation of the 26 proteasome is reviewed.

In the main text section like 1. Mechanisms Regulating…, please add tables and figures reflecting the contents, which will encourage the readers understand.

Please ensure if the authors have the permission of data reproduction of Figure 2.

It is not easy to understand Figure 2i. Please make it easy to understand.

The authors reviewed the proteasome dynamics in disease from cancer to aging, but there is no rationale why this topic should be integrated in this review.

Please add each reference in Table 1.

Overall, the topic is interesting, but it is not easy to follow to understand. If the author provide the rational in each section, it would be better to follow what the authors wanted to review.

Author Response

Summary:

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

 

Point-by-point response to Comments and Suggestions for Authors

 

Comment:
The authors have emphasized the “26” proteasome, but there is no information about this in the Abstract. The reviewer recommends the authors should add the rationale about why the regulation of 26 proteasome should be highlighted. Furthermore, please address why this review is needed.

Response:

The Abstract has been revised and information about the 26S proteasome was added in new line 12 as follows: " The 26S proteasome is the catalytic arm of the system that acts downstream to the conjugation machinery. For a long time, it has been considered as a constitutive multi-subunit proteolytic complex that recognizes in a non-discriminatory manner ubiquitin-marked target substrates with less than a handful of exceptions."

To clarify the rationale for focusing on the 26S proteasome, we highlight in this review the newly emerging recent findings showing that it is a dynamically regulated component of the UPS. The following statement stresses the current need for this review (new lines – 22-24): "Given the rapid expansion of these findings and their impact on our understanding of proteasome biology, an integrated overview of these regulatory mechanisms is timely."

 

Comment:
In the Introduction section, the authors only showed the generational concept of degradation and cellular homeostasis. They should address why the regulation of the 26 proteasome is reviewed.

Response:
We have revised the Introduction to clarify the rationale for focusing on regulation of the 26S proteasome. The following two paragraphs were added at the end of the Introduction: "While the original thought was that the proteasome is a protease that recognizes specifically ubiquitinated targets but has no additional layers of regulation, this picture has changed dramatically in recent years.  It has been shown that different modes of post-translational modifications affect different proteasomal subunits, which along with changes in subunit composition and even sub-complexes, and more recently in its subcellular compartmentalization - regulate its activity. Importantly, these regulatory mechanisms are not static but are continuously remodeled in response to physiological cues and are frequently disrupted in disease. Consequently, proteasome dysregulation emerges as a central determinant of proteostasis failure across a variety of pathological conditions.

Disease states provide a functional context in which the complexity of proteasome regulation becomes particularly evident, revealing how disruptions in proteasome dynamics contribute to the loss of proteostasis and disease pathogenesis. The main aim of this review is to summarize recent advances in our understanding of how structural, post-translational, and spatial regulation govern proteasome function in health, stress adaptation, and disease."

Comment:
In the main text section like 1. Mechanisms Regulating…, please add tables and figures reflecting the contents, which will encourage the readers understand.

Response:
We have added two new figures (1 and 2; Figure 2 combines graphics and a Table-like summary) in the main text which summarize the key mechanisms regulating the 26S proteasome.

Comment:
Please ensure if the authors have permission of data reproduction of Figure 2.

Response:
As for the legend to Figure 2 - part 2i is original was newly drawn by us whereas for part 2ii we indicated specifically that it is replicated with permission form our own previously published article. 

Comment:
It is not easy to understand Figure 2i. Please make it easy to understand.

Response:
We have revised Figure 2i to improve its clarity and make it easier to understand.

Comment:
The authors reviewed the proteasome dynamics in disease from cancer to aging, but there is no rationale why this topic should be integrated in this review.

Response:
The rationale for including proteasome dynamics in disease contexts is that proteasome regulation is highly dynamic rather than static, and it is continuously remodeled in response to physiological demands. Importantly, these regulatory mechanisms are frequently disrupted in disease, leading to impaired proteostasis. Therefore, proteasome dysregulation represents a central and unifying mechanism underlying proteostasis failure across diverse pathological conditions, including cancer and aging.

To clarify this point, we have revised the last part of the Introduction to explicitly and clearly state this rationale as following (new lines 66-69):
“Importantly, these regulatory mechanisms are not static but are continuously remodeled in response to physiological cues and are frequently disrupted in disease. Consequently, proteasome dysregulation emerges as a central determinant of proteostasis failure across a variety of pathological conditions.”

Comment:
Please add each reference in Table 1.

Response:
As requested, we added the corresponding references to Table 1.

Comment:
Overall, the topic is interesting, but it is not easy to follow to understand. If the author provide the rational in each section, it would be better to follow what the authors wanted to review.

Response:

We thank the reviewer for this helpful suggestion. In order to improve the clarity and readability of the manuscript, we have added a short rationale at the beginning of each section to better guide the reader and highlight the conceptual flow of the review.

For Section 1: Structural Diversity and Post-Translational Modifications Affecting Proteasomal Function:
"Proteasome regulation is underpinned by its structural plasticity and compositional diversity (Figures 1 and 2), which enable the formation of functionally distinct assemblies adapted to different cellular demands. Post-translational modifications further refine proteasome activity by modulating its stability, assembly, and catalytic function. Together, these mechanisms define the core regulatory principles underlying proteasome adaptability under physiological conditions."

 

For Section 2: Spatial Regulation of Proteasome Function: Localization, Translocation, and Compartmentalization:
"Proteasome activity is also governed by its spatial organization within the cell. Dynamic redistribution between nuclear and cytosolic compartments is driven by nutrient and stress signaling, generating functionally specialized proteasome pools. This spatial regulation is further integrated with biomolecular condensates and proteaphagy, linking localization to adaptive control of protein homeostasis."

 

For Section 3: 26S Proteasome Dynamics in Disease: From Cancer to Aging:
"Proteasome regulation is profoundly reshaped in disease, where alterations across structural, post-translational, and spatial levels can disrupt cellular proteostasis. In cancer, proteasome capacity is adaptively enhanced to sustain survival under chronic proteotoxic and therapeutic stress, whereas in neurodegeneration, proteostasis imbalance arises in a context-dependent manner driven by aberrant protein species and cellular stress. In aging, progressive decline in proteasome function further limits protein quality control. Together, these disease contexts illustrate how dysregulated proteasome dynamics contribute to the collapse of proteostasis in pathologic states."

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have addressed most of the concerns and comments that were previously generated. Please ensure the copyright permission and source for Figure 2ii, which should be clearly documented. In addition, Figure 2i should be sufficiently clear to read. Because, it seems a little bit vague. Please add the exact information on X-axis, like wet weight? Minor grammatical editing is also recommended.

Overall, the manuscript has been substantially improved and is acceptable after minor revision.

Author Response

Summary:

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

 

Point-by-point response to Comments and Suggestions for Authors

1. Comment:
   Please ensure the copyright permission and source for Figure 2ii, which should be clearly documented.
   
   1. Response:
   We addressed this point in the previous round of revisions and clarified that both the copyright permission and the source of Figure 2ii are clearly indicated in both the previous and the latest versions of the manuscript. This information appears in the figure legend, specifically in the last sentence: "Reprinted from Livneh et al., Cell Death and Differentiation (2024), with a CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/)."

Therefore, we believe this comment has already been fully addressed.

1. Comment:
   Figure 2i should be sufficiently clear to read. Because, it seems a little bit vague. 
   
   

2. Response:
   We have clarified this figure. Please refer to the latest version of the manuscript, where it is presented as Figure 4ii.

 

Comment:
Please add the exact information on X-axis, like wet weight? Minor grammatical editing is also recommended.

3. Response:
The original figure, which was reproduced from a published paper, did not include any labels on the horizontal axis. Furthermore, the horizontal axis simply indicates the experimental groups of treated mice (control and YWF) and does not represent a quantitative variable requiring additional labeling