Protein SUMOylation and Its Functional Role in Nuclear Receptor Control

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review concerns the post-translational modification SUMOylation and more precisely its role in the control of cellular receptors. It first describes the SUMO conjugation process, with a detailed presentation of the conjugating/deconjugating enzymes. It then focuses on the SUMOylation of receptors with a large part on nuclear receptors. It then describes interaction between post-tranlational modifications in the control of receptor function. It ends with a part on the implication of receptor SUMOylation in pathologies. The topic of this review is interesting. The first part on SUMO enzyme is rather exhaustive, maybe too much considering that many review already exist on the subject. The part on receptor is more original and relevant. However, this part is rather confusing, the references are not always appropriate and many statements and conclusions are not in agreement with the cited literature. This part needs a major reorganization and a much more careful analysis of the bibliography. 

 

Specific comments: 

-        The notion of receptor should be introduced in the beginning of the review. Here, it mixes both plasma membrane receptors and nuclear receptors, which are two very different situations. While Nuclear Receptor SUMOylation is well described, these are transcription factors/co-regulator and their SUMOylation occurs in the nucleus. The SUMOylation of plasma membrane receptors, in particular its occurrence at the plasma membrane is a much less common process and is somehow disputed. A clear distinction should be made between these types of receptors and they should be addressed in two separate parts instead of dividing according to their role in transcription, trafficking or interplay with other PTMs. Moreover, in these parts, most of the examples given are not concerning receptors but proteins downstream of those receptors.

-        To start the part on Receptor SUMOylation, it would be interesting to give an idea of the percentage of receptors, which have been found SUMOylated in various large-scale mass spectrometry approaches. Among them, a classification should be made between cell-surface receptors and nuclear receptors.

-        Line 341-347: SUMOylation of IGF-1R doesn’t affect PI3K and MAPK. This paragraph is thus unrelated to SUMOylation and not very relevant here. 

-        Part 4.3: the SUMO-dependant phosphorylation motif should be described. Ref 140 concerns mostly plants. There are probably more relevant reviews on cross-talks between SUMOylation and PTMs to cite. Ref 140-141 are also not highly relevant as they are not concerning SUMOylation. 

-        Part 5: the cited publications are generally not refering to receptor SUMOylation but of general SUMOylation. As in other parts of the manuscript (see previous comments), the references cited are often not appropriate. For example, they state that aberrant SUMOylation of HER2 has be observed in cancers (ref 167). This reference, which is a review, makes not mention at all of SUMOylation of HER2. In the same sentence they also mention VEGFR but give a reference for a review on EGFR, which also do not mention SUMO at all. I cannot check all references in the manuscript but these two examples in just one sentence, as well as those previously cited, cast a strong doubt on the accuracy of the cited litterature.

-        Some other examples of wrong citations: Ref 144 is about Delta-lactoferrin, a transcription factor and not a receptor, Ref 146 about IRF1, which is also a transcription factor, Ref 148/149 on IKBa, which is also not a receptor.          

-        Ref 162/164 : are about SUMO and cancer resistance but not linked to receptors. 

-        Line 452, it is indicated that TLR and cytokine receptor are SUMOylated but the references they indicate are reviews and not primary publication showing this. To my knowledge, there is no direct evidence that TLR are SUMOylated. If so, the primary work showing this SUMOylation should be cited .

Comments on the Quality of English Language

The review is well written and the quality of english language appropriate

Author Response

Dear Reviewer 1,

Thanks a lot for reviewing our manuscript. We addressed all raised concerns as you will see in the attached manuscript (all changes are highlighted in yellow). Please find below the point to point rebuttal letter.

This review concerns the post-translational modification SUMOylation and more precisely its role in the control of cellular receptors. It first describes the SUMO conjugation process, with a detailed presentation of the conjugating/deconjugating enzymes. It then focuses on the SUMOylation of receptors with a large part on nuclear receptors. It then describes interaction between post-tranlational modifications in the control of receptor function. It ends with a part on the implication of receptor SUMOylation in pathologies. The topic of this review is interesting. The first part on SUMO enzyme is rather exhaustive, maybe too much considering that many review already exist on the subject. The part on receptor is more original and relevant. However, this part is rather confusing, the references are not always appropriate and many statements and conclusions are not in agreement with the cited literature. This part needs a major reorganization and a much more careful analysis of the bibliography.

Thanks for the general comments, we followed your advice and shortened the introduction. Further - as suggested by reviewer 2 - we restricted the focus of the manuscript to SUMOylation of nuclear receptors and adapted the title accordingly. Further, we also verified the literature and corrected the citation throughout the text.
 

Specific comments:

-        The notion of receptor should be introduced in the beginning of the review. Here, it mixes both plasma membrane receptors and nuclear receptors, which are two very different situations. While Nuclear Receptor SUMOylation is well described, these are transcription factors/co-regulator and their SUMOylation occurs in the nucleus. The SUMOylation of plasma membrane receptors, in particular its occurrence at the plasma membrane is a much less common process and is somehow disputed. A clear distinction should be made between these types of receptors and they should be addressed in two separate parts instead of dividing according to their role in transcription, trafficking or interplay with other PTMs. Moreover, in these parts, most of the examples given are not concerning receptors but proteins downstream of those receptors.

-        To start the part on Receptor SUMOylation, it would be interesting to give an idea of the percentage of receptors, which have been found SUMOylated in various large-scale mass spectrometry approaches. Among them, a classification should be made between cell-surface receptors and nuclear receptors.

As stated above focus is given to nuclear receptors now.

-        Line 341-347: SUMOylation of IGF-1R doesn’t affect PI3K and MAPK. This paragraph is thus unrelated to SUMOylation and not very relevant here.

The paragraph has been removed.

-        Part 4.3: the SUMO-dependant phosphorylation motif should be described. Ref 140 concerns mostly plants. There are probably more relevant reviews on cross-talks between SUMOylation and PTMs to cite. Ref 140-141 are also not highly relevant as they are not concerning SUMOylation.

Thanks to this point, the references have been adapted.

-        Part 5: the cited publications are generally not refering to receptor SUMOylation but of general SUMOylation. As in other parts of the manuscript (see previous comments), the references cited are often not appropriate. For example, they state that aberrant SUMOylation of HER2 has be observed in cancers (ref 167). This reference, which is a review, makes not mention at all of SUMOylation of HER2. In the same sentence they also mention VEGFR but give a reference for a review on EGFR, which also do not mention SUMO at all. I cannot check all references in the manuscript but these two examples in just one sentence, as well as those previously cited, cast a strong doubt on the accuracy of the cited litterature.

-        Some other examples of wrong citations: Ref 144 is about Delta-lactoferrin, a transcription factor and not a receptor, Ref 146 about IRF1, which is also a transcription factor, Ref 148/149 on IKBa, which is also not a receptor.         

Sorry for the references, as stated above the references have been verified and re-organised.

 

-        Ref 162/164 : are about SUMO and cancer resistance but not linked to receptors.

This paragraph has been removed.

-        Line 452, it is indicated that TLR and cytokine receptor are SUMOylated but the references they indicate are reviews and not primary publication showing this. To my knowledge, there is no direct evidence that TLR are SUMOylated. If so, the primary work showing this SUMOylation should be cited .

This paragraph has been removed.

Comments on the Quality of English Language

The review is well written and the quality of english language appropriate

Thanks for the comment.

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, this has the potential to be an interesting and relevant review for the field. I feel substantial editing is required before this review should be published. Reading this review, I could not tell if it was describing nuclear receptors or receptors in general. Table 1 exclusively details nuclear receptors, but later other receptor types are included. Including all receptor types in a single review I feel is too ambitious and the authors should instead narrow their focus.

The description of the SUMO enzymology is overly detailed. There are many other review articles detailing this aspect of SUMO biology that could be cited. Reducing this section would give the authors more scope to discuss the receptor SUMOylation biology in more detail.

There is no description of SUMO interacting motifs (SIMs) this is a significant omission on any SUMO focused review.

In trying to describe so many examples of receptor SUMOylation very little detail is given on any. 

One example being ER-alpha which is a well described SUMOylation substrate, but it takes the authors 10 lines to convey just that ER-alpha SUMOylation reduces its transcriptional activity. 

The authors include a section of SUMO-Ubiquitin crosstalk but there is no mention of the StUbl RNF4 which regulates several receptors.

It is not clear why TRAF6 is included in this review - its not a receptor. There are actual examples of RNF4 - ubiquitin-SUMO crosstalk on the LXR receptor which would have been much more relevant to this review but were not included.

There are also several incorrect statements in the introduction section.

·        “Moreover, SUMO1 is constitutively conjugated to its specific substrates under physiological conditions” This statement is technically incorrect. SUMO1ylation is also dynamic.

·        Line 122 “Protein SUMOylation is critical for … a similar statement has already been made earlier in the manuscript and does not need repeating.

 

·        “For all known SUMO paralogs, the conjugation cascade is functionally equal” also not correct, as endogenous SUMO4 conjugates have never been identified and SUMO5 may still be a pseudogene. This should be corrected to SUMO1-3.

·        “A special amino acid motif that is consensus in all target proteins is then recognized” The authors should be more specific here, are they discussing the SUMO consensus motif? If they are they need to state that. The all target proteins part of this statement is also not technically correct. Only ~50% of SUMOylation occurs on consensus motifs.

 

·        In Figure 1 the SENP is labelled as SNEP

 

Comments on the Quality of English Language

I find the writing very wordy with a lot of repetition throughout. It often takes the authors 10+ lines to make a simple point. This makes the review challenging to read.

There are a lot of typos and poor phrasing, including in the abstract (e.g. "The today best understood PTM of this type...") SNEP instead of SENP also.

Author Response

Dear Reviewer 2,

Thanks a lot for reviewing our manuscript. We addressed all raised concerns as you will see in the attached manuscript (all changes are highlighted in yellow). Please find below the point to point rebuttal letter.

Overall, this has the potential to be an interesting and relevant review for the field. I feel substantial editing is required before this review should be published. Reading this review, I could not tell if it was describing nuclear receptors or receptors in general. Table 1 exclusively details nuclear receptors, but later other receptor types are included. Including all receptor types in a single review I feel is too ambitious and the authors should instead narrow their focus.

Thanks to the comment, we agree that the scope of this review might be to broad and therefore we narrowed the focus to nuclear receptors and adapted also the title.

The description of the SUMO enzymology is overly detailed. There are many other review articles detailing this aspect of SUMO biology that could be cited. Reducing this section would give the authors more scope to discuss the receptor SUMOylation biology in more detail.

The initial descriptions has been shorten according the the reviewer's comments.

There is no description of SUMO interacting motifs (SIMs) this is a significant omission on any SUMO focused review.

Thanks to this observation, we included a paragraph on SUMO interacting motifs.

In trying to describe so many examples of receptor SUMOylation very little detail is given on any.

One example being ER-alpha which is a well described SUMOylation substrate, but it takes the authors 10 lines to convey just that ER-alpha SUMOylation reduces its transcriptional activity.

The text has been modified furthermore - as outlined above - focus is given on nuclear receptors during SUMOylation therefore the paragraphs have been condensed.

The authors include a section of SUMO-Ubiquitin crosstalk but there is no mention of the StUbl RNF4 which regulates several receptors.

A respective paragraph has been included.

It is not clear why TRAF6 is included in this review - its not a receptor. There are actual examples of RNF4 - ubiquitin-SUMO crosstalk on the LXR receptor which would have been much more relevant to this review but were not included.

A section on crosstalk on the LXR receptor has been added.

There are also several incorrect statements in the introduction section.

·        “Moreover, SUMO1 is constitutively conjugated to its specific substrates under physiological conditions” This statement is technically incorrect. SUMO1ylation is also dynamic.

·        Line 122 “Protein SUMOylation is critical for … a similar statement has already been made earlier in the manuscript and does not need repeating.

·        “For all known SUMO paralogs, the conjugation cascade is functionally equal” also not correct, as endogenous SUMO4 conjugates have never been identified and SUMO5 may still be a pseudogene. This should be corrected to SUMO1-3.

 Thanks to these observations, the text has been corrected.

·        “A special amino acid motif that is consensus in all target proteins is then recognized” The authors should be more specific here, are they discussing the SUMO consensus motif? If they are they need to state that. The all target proteins part of this statement is also not technically correct. Only ~50% of SUMOylation occurs on consensus motifs.

The text has been adapted accordingly.
 

·        In Figure 1 the SENP is labelled as SNEP

The text has been modified.
 

Comments on the Quality of English Language

I find the writing very wordy with a lot of repetition throughout. It often takes the authors 10+ lines to make a simple point. This makes the review challenging to read.

There are a lot of typos and poor phrasing, including in the abstract (e.g. "The today best understood PTM of this type...") SNEP instead of SENP also.

The entire manuscript has been re-reviewed and typos have been removed.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have nicely improved the manuscript. I just have a minor comment concerning SUMO-E3, which should not be named SUMO-E3 ligase or enzymes as "ligase" or "enzyme" implies an enzymatic activity and SUMO-E3 generally don't have such activity as they function as bridges between the E2 and the target

Author Response

REF 1:

The authors have nicely improved the manuscript. I just have a minor comment concerning SUMO-E3, which should not be named SUMO-E3 ligase or enzymes as "ligase" or "enzyme" implies an enzymatic activity and SUMO-E3 generally don't have such activity as they function as bridges between the E2 and the target

Thanks to the reviewer to see the manuscript again and for the comment. We changed the the text throughout the entire manuscript according to the reviewer's suggestion.

Reviewer 2 Report

Comments and Suggestions for Authors

This article is much improved. I have made some minor comments below that should improve the text further. 

 

with an equal three-dimensional ubiquitin like structure

 This is not technically correct as there are subtle differences in the 3D structures of SUMO1-3 I suggest removing this line.

 

Approximately half of the identified SUMO substrates directly interact with the SUMO-E2 conjugating enzyme through the consensus motif ψKxE.

 

We do not know for sure if this is true as it has never been directly experimentally shown. It would be more accurate to simply state that ~ 50% of SUMOylation sites are found at this consensus and include a citation for one of the many SUMO proteomic screens published (such as Hendriks et al. 2017).

 

Lysin à Lysine

 

and therefore a SIM, a short stretch of hydrophobic amino acids in the sequence of the interacting protein.

 

Also we do not know this for sure, it may be true in some cases. I recommend removing this sentence.

 

he deSUMOylation is initiated by the SUMO protease recognizing SUMO which is conjugated to the target protein. After the protease has bound, the substrate undergoes a conformation change which enables the protease to catalyze a sequence of biochemical reactions finally leading to the separation of SUMO from the target protein.

 

This is a very long statement that could simply be shortened to something along the lines of SUMO proteases deconjugate SUMOs from target proteins.

 

Since the occurrence of SUMOylation of the receptors at the plasma membrane is still disputed as a much less common process, this review will focus on the SUMOylation of nuclear receptors.

I recommend amending this statement, there is a fair amount of evidence from multiple groups to conclude membrane bound receptors are SUMOylated. I wouldn’t want the authors to offend any of these research groups.

 

 

Comments on the Quality of English Language

I also recommend before publication that the text is carefully checked for English language usage because there are some errors throughout.

Author Response

REVIEWER 2:

This article is much improved. I have made some minor comments below that should improve the text further.

Thanks to the reviewer to see the manuscript again and for the comments. As recommended we also revised the English language throughout the entire text.
 

with an equal three-dimensional ubiquitin like structure
This is not technically correct as there are subtle differences in the 3D structures of SUMO1-3 I suggest removing this line.

We changed the sentence accordingly.

Approximately half of the identified SUMO substrates directly interact with the SUMO-E2 conjugating enzyme through the consensus motif ψKxE.
We do not know for sure if this is true as it has never been directly experimentally shown. It would be more accurate to simply state that ~ 50% of SUMOylation sites are found at this consensus and include a citation for one of the many SUMO proteomic screens published (such as Hendriks et al. 2017).

We changed the sentence to: As shown by SUMO proteomic screens, approximately 50% of SUMOylation sites are found at the consensus motif ψKxE [13].

Lysin à Lysine

Thanks for spotting this mistake, we corrected the spelling

and therefore a SIM, a short stretch of hydrophobic amino acids in the sequence of the interacting protein.
Also we do not know this for sure, it may be true in some cases. I recommend removing this sentence.

We changed the sentence to: This specific sequence is composed of a hydrophobic amino acid (ψ), a lysine residue to which SUMO is attached (K), any amino acid (x), and an acidic residue (E) [34, 43–45]. Other SUMO substrates require SUMO-E3 proteins for the interaction with the target protein.

the deSUMOylation is initiated by the SUMO protease recognizing SUMO which is conjugated to the target protein. After the protease has bound, the substrate undergoes a conformation change which enables the protease to catalyze a sequence of biochemical reactions finally leading to the separation of SUMO from the target protein.
This is a very long statement that could simply be shortened to something along the lines of SUMO proteases deconjugate SUMOs from target proteins.

We changed the sentence to: This deSUMOylation is enzymatically catalyzed by SUMO proteases. In a mechanistically similar way, these proteases also function during the initial SUMO processing.

Since the occurrence of SUMOylation of the receptors at the plasma membrane is still disputed as a much less common process, this review will focus on the SUMOylation of nuclear receptors.
I recommend amending this statement, there is a fair amount of evidence from multiple groups to conclude membrane bound receptors are SUMOylated. I wouldn’t want the authors to offend any of these research groups.

Thanks for the advice, we changed the sentence accordingly.