PA28γ promotes the malignant progression of tumor by elevating mitochondrial function via C1QBP
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eLife Assessment
This work attempts to demonstrate an ATP-independent non-canonical role of proteasomal component PA28y in the promotion of oral squamous cell carcinoma growth, migration, and invasion. The evidence remains incomplete and the work would benefit from further experimental work. The authors have not adequately addressed the reviewers' comments.
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Abstract
Proteasome activator 28γ (PA28γ) plays a critical role in malignant progression of various tumors, however, its role and regulation are not well understood. Here, using oral squamous cell carcinoma (OSCC) as main research model, we discovered that PA28γ interacted with complement 1q binding protein (C1QBP), which is dependent on the N-terminus of C1QBP rather than the known functional domain (amino acids 168-213). Notably, we found that PA28γ can enhance C1QBP protein stability in OSCC. Functionally, PA28γ contributes to the malignant progression of OSCC by affecting mitochondrial morphology and oxidative phosphorylation (OXPHOS) through C1QBP in vitro and vivo. Mechanically, PA28γ upregulate the expression of optic atrophy 1 (OPA1), mitofusin 2 (MFN1), mitofusin 2 (MFN2) and the mitochondrial respiratory complex by C1QBP. Moreover, in a clinical cohort of OSCC patients, PA28γ was positively correlated with C1QBP expression and negatively correlated with prognosis. Therefore, C1QBP is also a potential target for the treatment and prognosis of cancer.
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eLife Assessment
This work attempts to demonstrate an ATP-independent non-canonical role of proteasomal component PA28y in the promotion of oral squamous cell carcinoma growth, migration, and invasion. The evidence remains incomplete and the work would benefit from further experimental work. The authors have not adequately addressed the reviewers' comments.
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Reviewer #1 (Public review):
Summary:
The manuscript the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteosomal function in an ATP independent manner. Although there are multiple works that have highlighted the role of this protein in tumour, this study specifically tried to develop a correlate with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.
Strengths:
The observations of the authors hint that beyond PA28y association with proteasome, it might also stabilize certain proteins such as C1QBP which influences the energy metabolism.
Weaknesses:
The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.
In most of the …
Reviewer #1 (Public review):
Summary:
The manuscript the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteosomal function in an ATP independent manner. Although there are multiple works that have highlighted the role of this protein in tumour, this study specifically tried to develop a correlate with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.
Strengths:
The observations of the authors hint that beyond PA28y association with proteasome, it might also stabilize certain proteins such as C1QBP which influences the energy metabolism.
Weaknesses:
The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.
In most of the experiments the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex which is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in more convincing manner.
In all of the assays C1QBP has been detected as doublet. However, the expression pattern of the two bands vary depending on the experiment. In some cases the upper band is intensely stained and in some the lower bands. Does C1QBP isoforms exist and whether they are differentially regulated depending on experiment conditions/tissue types?
Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of literatures that have dealt with metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, significance of this study and how it will extend our understanding of OSCC needs to be elaborated.
Review of Revised Version:
Although the authors have partly corrected the manuscript by removing the mislabeling in their Co-IP experiments, my primary concern on the actual functional connotations and direct interaction between PA28y and C1QBP still remains unaddressed. As already mentioned in my previous review, since the core idea of the work is PA28y's direct interaction with C1QBP, stabilizing it, the same should be demonstrated in a more convincing manner.
My other observation on the detection of C1QBP as a doublet has been addressed by usage of anti-C1QBP Monoclonal antibody against the polyclonal one used before. C1QBP doublets have not been observed in the present case.
The authors have also worked on the presentation of the background by suitably modifying the statements and incorporating appropriate citations.
However, the authors are requested to follow the recommendations provided to them by the reviewers to address the major concerns.
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Reviewer #2 (Public review):
Summary:
The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.
Strengths:
They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria and the effect on mitochondrial …
Reviewer #2 (Public review):
Summary:
The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.
Strengths:
They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria and the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.
Weaknesses:
Many data sets are shown in figures that cannot be understood without more descriptions either in the text or the legend, e.g., Fig. 1A. Similarly, many abbreviations are not defined.
The revision addressed these issues.
Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Fig. 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Fig. 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Fig. 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.
The revision addressed these problems.
The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation, and analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.
The revision corrected this method.
To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and also show whether a mutation that disrupt the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Fig. 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Fig. 1G is a quantification of a Western blot data that should be shown.
The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Fig. 1I, more Flag-C1QBP 1-167 was pull-down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?
The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect.
The revision added AlphaFold models for the protein interaction. However, the models were not analyzed and potential mutations that would disrupt the interact were not predicted, made and tested. The revision did not addressed the request for the protease inhibitor.
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteasomal function in an ATP-independent manner. Although there are multiple works that have highlighted the role of this protein in tumours, this study specifically tried to develop a correlation with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.
Strengths:
The observations of the authors hint that beyond PA28y's association with the proteasome, it might also stabilize certain proteins such as C1QBP which influences energy metabolism.
Weaknesses:
The strength of the work also becomes its main drawback. That is, how …
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteasomal function in an ATP-independent manner. Although there are multiple works that have highlighted the role of this protein in tumours, this study specifically tried to develop a correlation with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.
Strengths:
The observations of the authors hint that beyond PA28y's association with the proteasome, it might also stabilize certain proteins such as C1QBP which influences energy metabolism.
Weaknesses:
The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.
In most of the experiments, the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex that is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in a more convincing manner.Thank you very much for the important comments. Using AlphaFold 3, we found that interaction between PA28γ and C1QBP may depend on amino acids 1-167 and 1-213 (Revised Appendix Figure 1D-H), which was confirmed by our immunoprecipitation (Revised Figure 1I). In the future, we will use nuclear magnetic resonance spectroscopy to analyze protein-protein interaction between PA28γ and C1QBP and demonstrate it by GST pull down in vitro experiments.
In all of the assays, C1QBP has been detected as doublet. However, the expression pattern of the two bands varies depending on the experiment. In some cases, the upper band is intensely stained and in some the lower bands. Do C1QBP isoforms exist and are they differentially regulated depending on experiment conditions/tissue types?
Thank you very much for the important comments. We have rechecked the experimental results with two bands, which may have been caused by using polyclonal antibody of C1QBP (Abcam: ab101267). Therefore, we conducted the experiment with monoclonal antibody of C1QBP (Cell Signaling Technology: #6502) and replaced the corresponding images in revised figure (Revised Figure 1E and Revised Appendix Figure 3D).
Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of works of literature that have dealt with the metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond the estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, the significance of this study and how it will extend our understanding of OSCC needs to be elaborated.
Thank you very much for the important comments. Based on your suggestion, we have revised the content and updated the references (“Introduction”, Paragraph 2, Line 13-17 and Paragraph 4, Line 5-8). In addition, we plan to conduct experiments to investigate the regulation of metabolism by PA28γ and C1QBP and update our data in the future.
The modified content is as follows:
“Current research on metabolic reprogramming in OSCC primarily focused on mechanism of glycolytic metabolism and metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) of oral squamous cell carcinoma, which lays the groundwork for novel therapeutic interventions to counteract OSCC (Chen et al., 2024; Zhang et al., 2020).”
“It is the first study to describe the undiscovered role of PA28γ in promoting the malignant progression of OSCC by elevating mitochondrial function, providing new clinical insights for the treatment of OSCC.”
Reviewer #2 (Public review):
Summary:
The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.
Strengths:
They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria, the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.
Weaknesses:
Many data sets are shown in figures that cannot be understood without more descriptions, either in the text or the legend, e.g., Figure 1A. Similarly, many abbreviations are not defined.
Thank you very much for the important comments. We have revised the descriptions in the legend to make it easier to understand.
Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Figure 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Figure 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Figure 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.
Thank you very much for the meticulous review. We have rechecked the experimental results, and we made a mistake in the labeling of the image. Therefore, we have corrected it in the revised figure (Revised Appendix Figure 1B, C). In addition, we have conducted a quantitative analysis of gray values to confirm the results of western blot data are accurate by Image J software.
The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation and be analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.
Thank you very much for the careful review. We have corrected it in the revised appendix file (“Supplemental Materials and Methods”, Part“Immunoprecipitation assay”, Line 4-6).
The modified content is as follows:
The sample was shaken on a horizontal shaker for 4 h, after which the deposit was collected for western blotting.
To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and show whether a mutation that disrupts the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Figure 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Figure 1G is a quantification of Western blot data that should be shown.
Thank you very much for the meticulous review. We have rechecked the experimental results, and we made a mistake in the labeling of the image. Therefore, we have corrected it in the revised figure. Compared with the control group, the presence of Myc-PA28γ significantly increased the expression level of Flag-C1QBP (Revised Figure 1F). Gray value analysis showed that in cells transfected with Myc-PA28γ, the decay rate of Flag-C1QBP was significantly slower than that of the control group (Revised Figure 1G), suggesting that PA28γ can delay the protein degradation of C1QBP and stabilize its protein level. This indicates that an increase in the level of PA28γ protein can significantly enhance the expression level of C1QBP protein, while PA28γ can slow down the degradation rate of C1QBP and improve its stability. In addition, we plan to conduct experiments to investigate the effects of protease inhibitors and PA28γ mutants on the stability of C1QBP and update our data in the future.
The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Figure 1I, more Flag-C1QBP 1-167 was pulled down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?
Thank you very much for the important comments. Immunoprecipitation is a qualitative experiment. Using AlphaFold 3, we found that interaction between PA28γ and C1QBP may depend on amino acids 1-167 and 1-213 (Revised Appendix Figure 1D-H), which was confirmed by our immunoprecipitation (Revised Figure 1I).
The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect.
Thank you very much for the important comments. Based on your suggestion, we have added relevant content to the revised appendix figure. (Revised Appendix Figure 1D-H).
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) There are a lot of typos in the figure and manuscript that need to be addressed.
Thank you very much for the important comments. We have corrected the typos in the revised figure and manuscript.
(2) Figure 1A: The amount of protein that has been immunoprecipitated is more than the actual amount present in the lysate. The authors should calculate the efficiency of the precipitation to support their results.
Thank you very much for the important comments. Immunoprecipitation is a qualitative experiment. Moreover, it can enrich specific proteins and their binding partners, increase their concentration in the sample, and thus improve the sensitivity of detection.
(3) Figure 1D: The relative expression levels of C1QBP look similar in almost all cell lines except for HN12. It seems that the relation of PA28y with C1QBP is more of a cell type-specific effect. It would be better if the blots were quantified, and the differences were statistically determined.
Thank you very much for the important comments. We have conducted a quantitative analysis of gray values to confirm the results of western blot data are accurate by Image J software.
(4) Figure 1E: How do the authors quantify the expression of the protein in absolute terms? From the methods, it is understood that the flag-tagged construct is stably expressed. Under such conditions, how the authors observed the variable expression of the protein should be elaborated.
Thank you very much for the important comments. We transfected Flag-PA28γ plasmids at 0ug, 0.5ug, 1ug, and 2ug in 293T cells. After collecting the protein for Western Blot, we found that the protein expression of Flag-PA28γ gradually increased. Moreover, the increased protein expression of C1QBP is consistent with the expression of Flag-PA28γ, which indicated a dose-dependent relationship between the two proteins.
(5) Figures 1F, G: The data does not correlate with the arguments presented in the text. The authors propose that interaction with PA28y increases the stability of C1QBP. However, the experiment lacks appropriate controls. Ideally, the expression of C1QBP should be tested in the presence and absence of PA28y. Moreover, the observed difference in expression between lanes 1-4 and 5-8 for myc-PA28y needs to be explained. Are the samples from different sources with variable PA28y expression? Figure 1G quantification for C1QBP does not correlate with the figure presented in F since the expression of the protein in the first four lanes is undetectable.
Thank you very much for the meticulous review. We have rechecked the experimental results, and we made a mistake in the labeling of the image. Therefore, we have corrected it in the revised figure. Compared with the control group, the presence of Myc-PA28γ significantly increased the expression level of Flag-C1QBP (Revised Figure 1F). Gray value analysis showed that in cells transfected with Myc-PA28γ, the decay rate of Flag-C1QBP was significantly slower than that of the control group (Revised Figure 1G), suggesting that PA28γ can delay the protein degradation of C1QBP and stabilize its protein level. This indicates that an increase in the level of PA28γ protein can significantly enhance the expression level of C1QBP protein, while PA28γ can slow down the degradation rate of C1QBP and improve its stability. In addition, we plan to conduct experiments to investigate the effects of protease inhibitors and PA28γ mutants on the stability of C1QBP and update our data in the future.
(6) Appendix Figure 1B: Lane 1 does not express Myc-tagged protein but pull-down has been performed using Myc beads. Then how come flag-C1qbp is getting pulled down in lane 1 if there is no PA28y? This indicates a non-specific interaction of C1qbp with the substrata under the experimental conditions used. Similarly, in Figure 1C SFB-PA28y is expressed in both lanes but is reflected only in lane 2 and not in lane 1 even when pull-down is being performed using SFB beads, again reflecting the non-specificity of the interactions shown through immunoprecipitated.
Thank you very much for the meticulous review. We have rechecked the experimental results, and we made a mistake in the labeling of the image. Therefore, we have corrected it in the revised figure (Revised Appendix Figure 1B, C).
(7) Figure 2A: Figure 2A the co-localization of P28y with C1QBP in mitochondria is not very convincing. The authors are urged to provide high-resolution images for the same along with quantification of co-localization coefficients.
Thank you very much for the important comments. We plan to obtain high-resolution images of co-localization of PA28γ with C1QBP in mitochondria and add the quantification analysis. We will update our data in the future.
(8) Figure 2C: Mitochondria dynamics is an interplay of multiple factors. From the images, it seems that PA28y OE elevates mitochondria biogenesis in general which is having an umbrella effect on mitochondria fusion/fission and OCR. Images also do not convincingly indicate changes in mitochondrial length. The role of PA28y on mitochondria dynamics requires further justification. However, the presented data does not underline whether the changes in mitochondria behaviour are a consequence of PA28y and C1QBP interaction. Correlating higher mitochondria respiration with ROS generation is a far-fetched conclusion since, at present, there are multiple reports that suggest otherwise.
Thank you very much for the important comments. We plan to knock out the interaction regions between PA28γ and C1QBP (like amino acids 1-167 and 1-213) to confirm whether PA28γ affects mitochondrial function through C1QBP and update our data in the future.
(9) Line 157: The presented data does not substantiate the claims made that Pa28y regulates mitochondrial function through C1QBP.
Thank you very much for the important comments. Based on your suggestion, we have made some modifications to make it more accurate (“Results”, Part “PA28γ and C1QBP colocalize in mitochondria and affect mitochondrial functions”, Paragraph 3, Line 1-2).
The modified content is as follows:
“Collectively, these data suggest that PA28γ, which co-localizes with C1QBP in mitochondria, may involve in regulating mitochondrial morphology and function.”
(10) Line 159: From the past data it is not very clear how PA28y upregulates C1QBP, hence the statement is not well supported. The presented data indicates the presence of a functional association between the two proteins.
Thank you very much for the important comments. We detected the expression of C1QBP in two PA28γ-overexpressing OSCC cells (UM1 and 4MOSC2) and found an increase in C1QBP expression (Revised Figure 4B). Based on the results of the protein levels of the mitochondrial respiratory chain complex and other mitochondrial functional proteins, we believe that PA28γ regulates mitochondrial function by upregulating C1QBP.
(11) Figure 4A, B: Given the mitochondrial role of C1QBP, the lesser levels of mitochondrial proteins upon C1QBP silencing are expected. Does it get phenocopied upon PA28y silencing? Similarly, all the subsequent mitochondrial phenotypes in D should be seen in a PA28y-depleted background.
Thank you very much for the important comments. We plan to detect the mitochondrial protein expressions and OCRs of PA28γ-silenced OSCC cells. We will update our data in the future.
(12) Line 198: The presented data do indicate a functional association between these two proteins but it does not provide a solid evidence for the same.
Thank you very much for the important comments. Based on your suggestion, we have made some modifications to make it more accurate (“Discussion”, Paragraph 1, Line 9-10).
The modified content is as follows:
“Excitingly, we found the evidence that PA28γ interacts with and stabilizes C1QBP.”
(13) Line 218-220: In this work, the authors highlight the non-degradome role of PA28y and hence, this fact should be treated appropriately in discussion in line with the presented data.
Thank you very much for the important comments. Based on your suggestion, we have added relevant content to the revised manuscript (“Discussion”, Paragraph 2, Line 16-19).
The modified content is as follows:
“In addition, PA28γ can also play as a non-degradome role on tumor angiogenesis. For example, PA28γ can regulate the activation of NF-κB to promote the secretion of IL-6 and CCL2 in OSCC cells, thus promoting the angiogenesis of endothelial cells ( S. Liu et al., 2018).”
(14) Line 236-240: Although the authors' statement on organ heterogeneity being the cause for getting the contrasting result is justifiable but here there is no direct evidence of PA28y involvement in regulation of OXPHOS and its impact on cellular metabolism (glycolysis, metabolic signalling, etc).
Thank you very much for the important comments. Based on your suggestion, we have made some modifications to make it more accurate (“Discussion”, Paragraph 3, Line 7-9).
The modified content is as follows:
“Therefore, PA28γ's regulation of OXPHOS may impact cellular energy metabolism.”
(15) Line 249: No conclusive data supporting this statement.
Thank you very much for the important comments. Based on your suggestion, we have made some modifications to make it more accurate (“Discussion”, Paragraph 5, Line 1-3).
The modified content is as follows:
“Furthermore, our study reveals that PA28γ can regulate C1QBP and influence mitochondrial morphology and function by enhancing the expression of OPA1, MFN1, MFN2 and the mitochondrial respiratory complex.”
Reviewer #2 (Recommendations for the authors):
(1) The images shown in Figure 2A need to be quantified before the conclusion about the mitochondrial colocalization of the two proteins can be drawn. In Figure 2B and Appendix Figure 2A, the mitochondrial vacuoles and ridge should be indicated for general readers, and quantification should be performed before the conclusion is drawn.
Thank you very much for the important comments. We will update our data in the future.
(2) The OCR data from two cell lines are shown in Figure 2E and F. Which is which? The sentence, "The results indicated ... compared to control cells" in lines 130-132, was confusing; perhaps, it would be clear if "were significantly greater" could be deleted.
Thank you very much for the important comments. We have re-labeled the Figure 2E and F to make it clearly (Revised Figure 2E, F). Based on your suggestion, we have deleted the words in revised manuscript. (“Results”, Part “PA28γ and C1QBP colocalize in mitochondria and affect mitochondrial functions”, Paragraph 1, Line 9-11).
The modified content is as follows:
“The results indicated significantly higher basal respiration, maximal OCRs and ATP production in PA28γ-overexpressing cells compared to control cells (Fig. 2G-I and Appendix Fig. 2B-D).”
(3) Figures 4E-H show the migration, invasive, and proliferation capabilities of the cells. Which for which?
Thank you very much for the important comments. We have re-labeled the Figure 4F-H to make it clearly (Revised Figure 4F-H).
(4) In the Discussion, lines 198-201, it states that "C1QBP enhances ... function of OPA1, MNF1, MFN2..." What is the evidence? In lines 222-224, it says that "the binding sites ... may mask the specific ... modification sites". Please justify. In lines 253-254, "fuse" and fuses" are misleading, Did the authors mean "localize" and "localizes"?
Thank you very much for the important comments. Based on your suggestion, we have made some modifications to make it more accurate (“Discussion”, Paragraph 1, Line 9-13, Paragraph 2, Line 20-23, and Paragraph 5, Line 3-6).
The modified content is as follows:
“Excitingly, we found the evidence that PA28γ interacts with and stabilizes C1QBP. We speculate that aberrantly accumulated C1QBP enhances the function of mitochondrial OXPHOS and leads to the production of additional ATP and ROS by activating the expression and function of OPA1, MNF1, MFN2 and mitochondrial respiratory chain complex proteins.”
“Our study reveals that PA28γ interacts with C1QBP and stabilizes C1QBP at the protein level. Therefore, we speculate that the binding sites of PA28γ and C1QBP may mask the specific post-translational modification sites of C1QBP and inhibit its degradation.”
“Mitochondrial fusion, crucial for oxidative metabolism and cell proliferation, is regulated by MFN1, MFN2, and OPA1. The first two fuse with the outer mitochondrial membrane, while the last fuses with the inner mitochondrial membrane (Westermann, 2010).”
(5) Figure 6 was not referred to in the text. In this figure, PA28g and C1QBP are located in the inner membrane and matrix. Has this been determined? What is the blue ovals that are intermediaries of PA28g/C1QBP and OPA1/MFN1/MFN2?
Thank you very much for the important comments. According to our immunofluorescence assay (Figure 2A), PA28γ is in both the nucleus and cytoplasm. A recent study has demonstrated that PA28γ can shuttle between the nucleus and cytoplasm, participating in various cellular processes. Furthermore, GeneCard information indicates that the subcellular localization of PA28γ includes the nucleus, cytoplasm and mitochondria (Author response image 1). In this article, we mainly focus on the functions of PA28γ and C1QBP located in the cytoplasm. Therefore, figure 6 mainly displays PA28γ and C1QBP in the cytoplasm. Based on your suggestion, we have made some modifications to make it more accurate in revised figure (Revised Figure 6).
Author response image 1.
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eLife Assessment
This work attempts to demonstrate an ATP-independent non-canonical role of proteasomal component PA28y in the promotion of oral squamous cell carcinoma growth, migration, and invasion. The evidence around the following two areas remains incomplete and would benefit from further experimental work: 1) the stabilisation of the complement C1q binding protein (C1QBp) by PA28y, and 2) the impact of the PA28y-C1QBp interaction on mitochondrial function.
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Reviewer #1 (Public review):
Summary:
In this manuscript, the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteasomal function in an ATP-independent manner. Although there are multiple works that have highlighted the role of this protein in tumours, this study specifically tried to develop a correlation with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.
Strengths:
The observations of the authors hint that beyond PA28y's association with the proteasome, it might also stabilize certain proteins such as C1QBP which influences energy metabolism.
Weaknesses:
The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.
In most of …Reviewer #1 (Public review):
Summary:
In this manuscript, the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteasomal function in an ATP-independent manner. Although there are multiple works that have highlighted the role of this protein in tumours, this study specifically tried to develop a correlation with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.
Strengths:
The observations of the authors hint that beyond PA28y's association with the proteasome, it might also stabilize certain proteins such as C1QBP which influences energy metabolism.
Weaknesses:
The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.
In most of the experiments, the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex that is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in a more convincing manner.In all of the assays, C1QBP has been detected as doublet. However, the expression pattern of the two bands varies depending on the experiment. In some cases, the upper band is intensely stained and in some the lower bands. Do C1QBP isoforms exist and are they differentially regulated depending on experiment conditions/tissue types?
Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of works of literature that have dealt with the metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond the estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, the significance of this study and how it will extend our understanding of OSCC needs to be elaborated.
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Reviewer #2 (Public review):
Summary:
The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.
Strengths:
They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria, the effect on mitochondrial form …
Reviewer #2 (Public review):
Summary:
The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.
Strengths:
They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria, the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.
Weaknesses:
Many data sets are shown in figures that cannot be understood without more descriptions, either in the text or the legend, e.g., Figure 1A. Similarly, many abbreviations are not defined.
Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Figure 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Figure 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Figure 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.
The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation and be analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.
To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and also show whether a mutation that disrupts the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Figure 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Figure 1G is a quantification of Western blot data that should be shown.
The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Figure 1I, more Flag-C1QBP 1-167 was pulled down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?
The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect
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