Proteasome dysfunction disrupts adipogenesis and induces inflammation via ATF3
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RC-2022-01245 Willemsen et al., 2022
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary:
Willemsen et al studied the contribution of PSMB4 and PSMB8 on proteasomal activity in adipocyte tissue/cells. Mutations in PSMB4/8 have been associated with metabolic diseases that lead to inflammation and proteotoxicity. They used mice and murine cell lines to assess the abundance and activity of the proteasome as well as the stress response upon depletion of psmb4 and regulatory factors. The study is interesting and could provide new insight into the role of specific proteasomal subunits of the immunoproteasome on the metabolism and associated …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
RC-2022-01245 Willemsen et al., 2022
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Summary:
Willemsen et al studied the contribution of PSMB4 and PSMB8 on proteasomal activity in adipocyte tissue/cells. Mutations in PSMB4/8 have been associated with metabolic diseases that lead to inflammation and proteotoxicity. They used mice and murine cell lines to assess the abundance and activity of the proteasome as well as the stress response upon depletion of psmb4 and regulatory factors. The study is interesting and could provide new insight into the role of specific proteasomal subunits of the immunoproteasome on the metabolism and associated diseases. However, there are a number of issues on the presented data that should be addressed. See below.
RESPONSE: We thank the reviewer for her/his positive remarks.
Major comments:
- Most of the presented data are secondary data (graphs) and the differences between the experimental conditions (e.g. siRNA) are sometimes only minor and statistical analyses lacking or only indicated with a letter (a, b,..), but its meaning not explained. RESPONSE: We apologize if some of the statistics were difficult to see. Using letter designations for groups of indifference declutters the figure compared to using asterisks to indicate significant differences between groups. We have now made sure the statistical analyses are emphasized in the methods and figure legends.
The authors should introduce all tested marker genes e.g. the genes that were analyzed in figures 2H/I, 3D-F, 4A/D. What was the hypothesis and do they represent all or a selected set of genes of the integrated stress response?
RESPONSE: We apologize if the relevance of these markers was left unclear. We have now introduced the marker genes in the text.
Figure 1A: RNA levels were analyzed yet not protein levels. Why not?
RESPONSE: As we performed loss of function experiments later anyway, we decided not to venture into more descriptive analyses. The fact that Psmb4 and Psmb8 are robustly expressed in adipocytes was enough for us to justify studying their function further.
Figure 2C: This figure is problematic. Apart from the smear on the right side, a loading control is missing. This is essential to quantify signal intensities. Moreover, on the left side, the intensities of lanes 1 and 2 are very different, yet are both controls and were used for the conclusion that proteasomal activity is reduced upon siPsmb4 (lanes 3 and 4 - that do not differ from lane 2). In addition: from which day were the data collected? This information is missing, but important as Figure 2E shows opposing proteasomal activity on day 3 and day 5.
RESPONSE: We agree with the reviewer that the duplicate of the scrambled control cells showed variation. Therefore, we have now repeated the experiment and replaced the figure (now figure 2A). The outcome is not affected, as knockdown reduces proteasomal activity and leads to abnormal proteasome formation in the native PAGE. Regarding the internal loading, it is common practice to display both in-gel activity and immunoblot on the membrane as is. For recent examples in the literature please see VerPlank et al., PNAS 2019 (10.1073/pnas.1809254116) or Yazgili et al., Cell Press STAR Protocols 2021 (10.1016/j.xpro.2021.100526). Obviously, the same amount of protein was loaded, and this is also seen in the immunoblot. As this is a native PAGE, there is no beta-tubulin or other commonly used loading controls for immunoblots. Furthermore, we apologize for the missing information, this experiment was performed using day 5 mature cells. This information is now included in the figure legend.
Figure 2D: tubulin was used as loading control, yet the signal of tubulin in lane 1 is by far weaker compared to the other lanes. How does that affect the quantification (missing) of Nfe2I1?
RESPONSE: We have now included the quantifications, which do not affect the outcome of the experiments and the conclusions drawn.
Figure 3C and Fig 2E (control vs siPsmb4) contradict each other. Please clarify.
RESPONSE: We respectfully point out that the reviewer might have overlooked that 2E shows the time course and 3C only shows day 5 data – both in 2E and 3C, day 5 total proteasomal activity is (insignificantly) increased. Hence, the panels do not contradict each other.
Figure 3B: Issues with the loading control: tubulin signals are in first 3 lanes much weaker. Where is the quantification for that data set that takes the fluctuations of the tubulin signals into account?
RESPONSE: We have now included the quantification, which does not affect the outcome of the experiments and the conclusions drawn. The quantifications can be found in figure S3.
Minor comments:
Why did the authors not use human adipocyte cells and performed all experiments in murine cells?
RESPONSE: The advantage of the cell lines used lies in the ability to study both brown and white features as well studying aspects of adipogenesis and thermogenesis simultaneously. Based on this comment and the comment of reviewer #2, we have reproduced our findings in 3T3-L1 adipocytes, in the hope of strengthening our study. These data are shown in the new Supplementary figure 4.
In which cell/tissue is Psmb4 expressed?
RESPONSE: Thank you for this question, we have now measured Psmb4 expression in a panel of mouse tissues. As shown in figure S1, Psmb4 is ubiquitously expressed in all tissues measured with the highest levels in kidney and liver, followed by brown fat.
Figure 4G: information on the different colors is missing.
RESPONSE: Thank you for bringing this to our attention. We have now included a legend.
The result section appears to have been restructured as sections do not build up on each other well. This should be corrected.
RESPONSE: We appreciate this critical feedback. We have now improved the flow for an enhanced reading experience.
There are a number of grammatical errors or doubling of words/phrases e.g. bottom of page 1: "In addition, PRAAS patients display suffer from..." or on page 2: "Adapting proteasomal activity to the needs of the UPS..." This statement does not make sense. Maybe the authors mean "proteolytic demands"?
RESPONSE: Thank you, we have fixed the remaining typos.
Although, the UPS is a proteostasis node, the authors should avoid statements such as "We show that proteostasis and lipid metabolism are intricately linked..." Better is "UPS activity and lipid metabolism..." Or the authors should expand their analysis to protein synthesis, folding and additional clearance pathways.
RESPONSE: Thank you, we have specified our statement regarding proteostasis and UPS.
Reviewer #1 (Significance (Required)):
This study links clinical research with basic science and if the authors address the above mentioned issues this work will provide new insight into the role of the UPS in the lipid metabolism.
target audience: clinical scientist on lipid metabolism and basic researchers on the UPS and associated pathologies
my expertise: UPS
RESPONSE: We are very thankful for these positive concluding remarks.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The molecular mechanism by which proteasome mutations cause lipodystrophy in PRAAS, which is caused by proteasome dysfunctions, has not been well understood. However, it was shown that Psmb4 (β4), a component without enzymatic activity, is required for the formation and maintenance of adipocyte function. In the proteasome dysfunction state of Psmb4 deficiency, the expression of Nfe2l1 was enhanced for proteostasis, but it could not complement the adipocyte formation defect. We showed that repression of Arf3, which is associated with stress response and is markedly expressed in this situation, resulted in the recovery of inflammation and adipogenesis.
Major comments
- The Graphical abstract seems to indicate that Loss of PSMB4 activates NFE2L1 and ATF3, resulting in the suppression of proteotoxicity and Inflammation. In the case of NFE2L1, this is correct, but in the case of ATF3, as shown in Fig. 4D, ATF3 acts in a promotive manner on Inflammation, causing misleading to the reader. RESPONSE: Thank you, we agree that this aspect of the graphical abstract was partially misleading. We hope the new version now makes more sense.
Fig. 2 shows the rise of Nfe2l1 and the restoration of proteasomal capacity on Day 5 (Fig. 2E). [Nfe2l1 is cleaved, and initiates the transcription of proteasome subunits, which results in restoration or heightening of proteasomal capacity (16,17).] It is known that the brown adipocyte mount an adaptive response to overcome UPS dysfunction, and the transcription of proteasome subunits was increased in this experimental system. However, there are no results showing that the transcription of proteasome subunits is actually increased in this experimental system.
RESPONSE: Thank you for pointing this out. In Psmb4 KD cells, we see an increase in Psmd2 protein levels (Fig. 3B). In addition, we see a small increase in expression levels of various proteasome subunits. We have now included a graph showing these expression levels (Fig 3C).
Are Psmb4KO mice available? If yes, are there any symptoms? Is there any change in proteasome activity, etc.?
RESPONSE: We do not have a Psmb4 KO mouse model, yet, and to the best of our knowledge, none is available. We agree with the reviewer that it would be insightful to study Psmb4 in an in vivo model, but in this project, we have used a cell model to study the cell intrinsic mechanisms of Psmb4.
4A. In PRAAS patients, most of the lipodystrophy occurs in white adipocytes, but if Psmb4 deficiency is induced in white adipocytes, do they show the same dynamics?
RESPONSE: Thank you for your stimulating question. We have repeated our Psmb4 KD experiments in 3T3-L1 cells, to study the dynamics in a white adipocyte model. We found that also in 3T3-L1 cells, Psmb4 knockdown disrupts adipogenesis. The results can be found in Supplemental figure 4.
4B. The first mention of heat production was made, but it was not clear how much the patient's cyclic fever symptoms were related to changes in brown adipocyte function.
RESPONSE: Our data suggest that aberrant brown adipose tissue does not contribute to cyclic fevers in PRAAS patients. We elaborate on this in the Discussion.
minor points
fig2; The numbering of the figure is not correct.
RESPONSE: Thank you, we have corrected the error.
Fig. 2: (day 5) in the figure legend of (E) is unnecessary.
RESPONSE: Thank you but based on the other reviewers’ questions we think it is important to indicate the stage of the differentiation.
Fig. 4 The figure legend in (A) and (B) are switched.
RESPONSE: Thank you, we have corrected the error.
In Fig. 4 (G), there are n = 8 and n = 6 in the figure legend, which is difficult to understand.
RESPONSE: Thank you, we have corrected the error.
Reviewer #2 (Significance (Required)):
It has been thought that the accumulation of defective proteins caused by proteasome dysfunction stresses cell metabolism and leads to lipodystrophy, but the detailed mechanism has not been understood. In this paper, we have clarified a part of the mechanism that links the accumulation of ubiquitinated proteins caused by proteasome dysfunction to the disruption of proteostasis, inflammation and adipogenesis. The results of the study, which showed the relationship between intracellular proteostasis, inflammation and lipid metabolism, will help us understand not only PRAAS patients but also abnormal lipid metabolism, obesity, induction of inflammation, and chronic inflammation with persistent inflammation.
RESPONSE: We are very thankful for these positive concluding remarks.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this study Willemsen et al. investigated the role of proteasome subunit beta 4 and 8 (PSMB4/8) in immortalized brown (pre)adipocytes regarding adipogenesis ability, inflammation, function, and proteostasis. The group showed that Psmb4/8 are expressed in brown adipose tissue and adipocytes but that they are differently regulated. The loss of PSMB8 had no effect on brown adipose tissue/adipocyte function. In contrast, knock-down of PSMB4 altered proteostasis, which was partially compensated by NFE2L1, as well as reduced adipocyte differentiation, lipid accumulation, and beta adrenergic-stimulated glycerol release in immortalized brown adipocytes. The group further demonstrated that the effect of PSMB4 knock-down on impaired adipogenesis was mediated via Atf3 activation.
The manuscript is well-written with clearly structured text and figures. The data and methods are presented in a way that makes it easy to reproduce the experiments. The statistical analysis is adequate.
Some suggestions:
- Since you stated in 3.1. Result section that Psmb4/8 are robustly expressed in BAT, it would be interesting to directly compare the expression of Psmb4/8 in BAT. RESPONSE: We thank the reviewer for this interesting suggestion. The comparison is now included in figure S3.
Please normalize the glycerol release to protein content (Fig 2J, 4F). It would be also interesting to show whether the reduced glycerol release is due to reduced TG content and/or lipolytic activity. Therefore, you should determine the expression of lipases (e.g. Atgl, Hsl) in adipocytes.
RESPONSE: We thank the reviewer for this interesting suggestion. We have looked at the expression of lipases. Psmb4 knockdown did not alter the expression of lipases, which indicates that the reduced glycerol release is rather due to reduced TG content than due to the absence of lipases. The comparison is now included as Figure 4G. For the glycerol assays, we have normalized glycerol release to protein content. Comparing an undifferentiated (in this case the cells with silencing of Psmb4) vs differentiated cells (in this case the scrambled siRNA control cells) will result in many fundamental differences. Specifically, the lipid to protein ratio is very different, much higher in mature adipocytes, obviously. This obscures some if the differences in lipolysis when glycerol release is normalized to protein levels. Therefore, we have included a figure, in which we show the fold change. Interestingly, this way in the Psmb4 knockdown cells, it is evident that they become refractory to norepinephrine stimulation, and this is rescued when Atf3 is silencing, too.
Please define early/late transfection - on which day of differentiation was Psmb8 silenced? (Fig S2)
RESPONSE: Psmb8 was silenced on day(-1). We have now added this information.
Reviewer #3 (Significance (Required)):
This study clearly demonstrated that proteasome dysfunction via impaired PSMB4 action modulates brown adipocytes differentiation, function, and health. In this study a novel link between dysfunctional proteostasis and impaired lipid metabolism was identified.
RESPONSE: We are very thankful for these positive concluding remarks.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
In this study Willemsen et al. investigated the role of proteasome subunit beta 4 and 8 (PSMB4/8) in immortalized brown (pre)adipocytes regarding adipogenesis ability, inflammation, function, and proteostasis. The group showed that Psmb4/8 are expressed in brown adipose tissue and adipocytes but that they are differently regulated. The loss of PSMB8 had no effect on brown adipose tissue/adipocyte function. In contrast, knock-down of PSMB4 altered proteostasis, which was partially compensated by NFE2L1, as well as reduced adipocyte differentiation, lipid accumulation, and beta adrenergic-stimulated glycerol release in immortalized …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
In this study Willemsen et al. investigated the role of proteasome subunit beta 4 and 8 (PSMB4/8) in immortalized brown (pre)adipocytes regarding adipogenesis ability, inflammation, function, and proteostasis. The group showed that Psmb4/8 are expressed in brown adipose tissue and adipocytes but that they are differently regulated. The loss of PSMB8 had no effect on brown adipose tissue/adipocyte function. In contrast, knock-down of PSMB4 altered proteostasis, which was partially compensated by NFE2L1, as well as reduced adipocyte differentiation, lipid accumulation, and beta adrenergic-stimulated glycerol release in immortalized brown adipocytes. The group further demonstrated that the effect of PSMB4 knock-down on impaired adipogenesis was mediated via Atf3 activation.
The manuscript is well-written with clearly structured text and figures. The data and methods are presented in a way that makes it easy to reproduce the experiments. The statistical analysis is adequate.
Some suggestions:
Since you stated in 3.1. Result section that Psmb4/8 are robustly expressed in BAT, it would be interesting to directly compare the expression of Psmb4/8 in BAT. Please normalize the glycerol release to protein content (Fig 2J, 4F). It would be also interesting to show whether the reduced glycerol release is due to reduced TG content and/or lipolytic activity. Therefore, you should determine the expression of lipases (e.g. Atgl, Hsl) in adipocytes.
Please define early/late transfection - on which day of differentiation was Psmb8 silenced? (Fig S2)
Significance
This study clearly demonstrated that proteasome dysfunction via impaired PSMB4 action modulates brown adipocytes differentiation, function, and health. In this study a novel link between dysfunctional proteostasis and impaired lipid metabolism was identified.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The molecular mechanism by which proteasome mutations cause lipodystrophy in PRAAS, which is caused by proteasome dysfunctions, has not been well understood. However, it was shown that Psmb4 (β4), a component without enzymatic activity, is required for the formation and maintenance of adipocyte function. In the proteasome dysfunction state of Psmb4 deficiency, the expression of Nfe2l1 was enhanced for proteostasis, but it could not complement the adipocyte formation defect. We showed that repression of Arf3, which is associated with stress response and is markedly expressed in this situation, resulted in the recovery of inflammation …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
The molecular mechanism by which proteasome mutations cause lipodystrophy in PRAAS, which is caused by proteasome dysfunctions, has not been well understood. However, it was shown that Psmb4 (β4), a component without enzymatic activity, is required for the formation and maintenance of adipocyte function. In the proteasome dysfunction state of Psmb4 deficiency, the expression of Nfe2l1 was enhanced for proteostasis, but it could not complement the adipocyte formation defect. We showed that repression of Arf3, which is associated with stress response and is markedly expressed in this situation, resulted in the recovery of inflammation and adipogenesis.
Major comments
- The Graphical abstract seems to indicate that Loss of PSMB4 activates NFE2L1 and ATF3, resulting in the suppression of proteotoxicity and Inflammation. In the case of NFE2L1, this is correct, but in the case of ATF3, as shown in Fig. 4D, ATF3 acts in a promotive manner on Inflammation, causing misleading to the reader.
- Fig. 2 shows the rise of Nfe2l1 and the restoration of proteasomal capacity on Day 5 (Fig. 2E). [Nfe2l1 is cleaved・・・・, and initiates the transcription of proteasome subunits, which results in restoration or heightening of proteasomal capacity (16,17).] It is known that the brown adipocyte mount an adaptive response to overcome UPS dysfunction, and the transcription of proteasome subunits was increased in this experimental system. However, there are no results showing that the transcription of proteasome subunits is actually increased in this experimental system.
- Are Psmb4KO mice available? If yes, are there any symptoms? Is there any change in proteasome activity, etc.? In PRAAS patients, most of the lipodystrophy occurs in white adipocytes, but if Psmb4 deficiency is induced in white adipocytes, do they show the same dynamics? The first mention of heat production was made, but it was not clear how much the patient's cyclic fever symptoms were related to changes in brown adipocyte function.
Minor points
fig2; The numbering of the figure is not correct.
Fig. 2: (day 5) in the figure legend of (E) is unnecessary.
Fig. 4 The figure legend in (A) and (B) are switched.
In Fig. 4 (G), there are n = 8 and n = 6 in the figure legend, which is difficult to understand.
Significance
It has been thought that the accumulation of defective proteins caused by proteasome dysfunction stresses cell metabolism and leads to lipodystrophy, but the detailed mechanism has not been understood. In this paper, we have clarified a part of the mechanism that links the accumulation of ubiquitinated proteins caused by proteasome dysfunction to the disruption of proteostasis, inflammation and adipogenesis. The results of the study, which showed the relationship between intracellular proteostasis, inflammation and lipid metabolism, will help us understand not only PRAAS patients but also abnormal lipid metabolism, obesity, induction of inflammation, and chronic inflammation with persistent inflammation.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
Willemsen et al studied the contribution of PSMB4 and PSMB8 on proteasomal activity in adipocyte tissue/cells. Mutations in PSMB4/8 have been associated with metabolic diseases that lead to inflammation and proteotoxicity. They used mice and murine cell lines to assess the abundance and activity of the proteasome as well as the stress response upon depletion of psmb4 and regulatory factors. The study is interesting and could provide new insight into the role of specific proteasomal subunits of the immunoproteasome on the metabolism and associated diseases. However, there are a number of issues on the presented data that …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
Willemsen et al studied the contribution of PSMB4 and PSMB8 on proteasomal activity in adipocyte tissue/cells. Mutations in PSMB4/8 have been associated with metabolic diseases that lead to inflammation and proteotoxicity. They used mice and murine cell lines to assess the abundance and activity of the proteasome as well as the stress response upon depletion of psmb4 and regulatory factors. The study is interesting and could provide new insight into the role of specific proteasomal subunits of the immunoproteasome on the metabolism and associated diseases. However, there are a number of issues on the presented data that should be addressed. See below.
Major comments:
- Most of the presented data are secondary data (graphs) and the differences between the experimental conditions (e.g. siRNA) are sometimes only minor and statistical analyses lacking or only indicated with a letter (a, b,..), but its meaning not explained.
- The authors should introduce all tested marker genes e.g. the genes that were analyzed in figures 2H/I, 3D-F, 4A/D. What was the hypothesis and do they represent all or a selected set of genes of the integrated stress response?
- Figure 1A: RNA levels were analyzed yet not protein levels. Why not?
- Figure 2C: This figure is problematic. Apart from the smear on the right side, a loading control is missing. This is essential to quantify signal intensities. Moreover, on the left side, the intensities of lanes 1 and 2 are very different, yet are both controls and were used for the conclusion that proteasomal activity is reduced upon siPsmb4 (lanes 3 and 4 - that do not differ from lane 2). In addition: from which day were the data collected? This information is missing, but important as Figure 2E shows opposing proteasomal activity on day 3 and day 5.
- Figure 2D: tubulin was used as loading control, yet the signal of tubulin in lane 1 is by far weaker compared to the other lanes. How does that affect the quantification (missing) of Nfe2I1?
- Figure 3C and Fig 2E (control vs siPsmb4) contradict each other. Please clarify.
- Figure 3B: Issues with the loading control: tubulin signals are in first 3 lanes much weaker. Where is the quantification for that data set that takes the fluctuations of the tubulin signals into account?
Minor comments:
- Why did the authors not use human adipocyte cells and performed all experiments in murine cells?
- In which cell/tissue is Psmb4 expressed?
- Figure 4G: information on the different colors is missing.
- The result section appears to have been restructured as sections do not build up on each other well. This should be corrected.
- There are a number of grammatical errors or doubling of words/phrases e.g. bottom of page 1: "In addition, PRAAS patients display suffer from..." or on page 2: "Adapting proteasomal activity to the needs of the UPS..." This statement does not make sense. Maybe the authors mean "proteolytic demands"?
- Although, the UPS is a proteostasis node, the authors should avoid statements such as "We show that proteostasis and lipid metabolism are intricately linked..." Better is "UPS activity and lipid metabolism..." Or the authors should expand their analysis to protein synthesis, folding and additional clearance pathways.
Significance
This study links clinical research with basic science and if the authors address the above mentioned issues this work will provide new insight into the role of the UPS in the lipid metabolism.
target audience: clinical scientist on lipid metabolism and basic researchers on the UPS and associated pathologies
my expertise: UPS
-
