From the journal RSC Chemical Biology Peer review history

17-May-2021

Dear Prof Scheck:

Manuscript ID: CB-ART-04-2021-000088
TITLE: Members of the Legionella pneumophila Sde Family Target Tyrosine Residues for Phosphoribosyl-Linked Ubiquitination

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Associate Editor, RSC Chemical Biology

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Reviewer 1

The manuscript by Zhang et al. is overall a well-executed study that describes the activity of the Legionella pneumophila effector protein SdeC, which catalyzes phosphoribosyl-linked ubiquitination of acceptor substrates. The authors find that Sde family members are able to catalyze their reaction at tyrosine (Tyr) residues in addition to serine residues by demonstrating that the HA peptide tag on ubiquitin acts as an acceptor target via its Tyr4 residue. These results suggest that SdeC can modify both Tyr and Ser residues, which was previously overlooked by others.
Major:
1. While it is beyond the scope of this work, can the authors add a brief discussion about natural substrates that may be modified at Tyr vs Ser residues, and what this might imply for previous work that reported modification at Ser residues only?
Minor:
1. p5 line 55- pneumophila is misspelled
2. p5 line 60- amoebae is misspelled

Reviewer 2

The manuscript by Zhang et al. reports an unexpected modification of phosphoribosyl-linked ubiquitination (pr-UB) at the protein target catalyzed by a bacterial effector protein of the Sde family. This discovery is of major importance to better understand the role of this crucial bacterial effector proteins during infection and their deleterious effects to host cells.
The authors start by analysis of substrates produced by individual functional catalytic domains utilizing a clever immobilization strategy. In the course of these experiments the authors realized a regular band laddering on their SDS-gels which resulted from functional ART and NP domains. Surprisingly, a crosslink between pR-UB and the HA-tag was observed. This unusual modification was further investigated by MS fragmentation and mutational scanning in the tag by several analogs to reveal that SdeC is promiscuous for tyrosine residues at various positions. This is an interesting discovery and of high interest to a broad chemical biology audience. One question remains with regards to the substrate scope: the main study focus are HA-tag derived peptides. Is it possible to investigate the extent of tyrosine vs. serine modification on substrate proteins such as reticulon directly?

Reviewer #1
“The manuscript by Zhang et al. is overall a well-executed study that describes the activity of the Legionella pneumophila effector protein SdeC, which catalyzes phosphoribosyl-linked ubiquitination of acceptor substrates. The authors find that Sde family members are able to catalyze their reaction at tyrosine (Tyr) residues in addition to serine residues by demonstrating that the HA peptide tag on ubiquitin acts as an acceptor target via its Tyr4 residue. These results suggest that SdeC can modify both Tyr and Ser residues, which was previously overlooked by others.
Major:
1. While it is beyond the scope of this work, can the authors add a brief discussion about natural substrates that may be modified at Tyr vs Ser residues, and what this might imply for previous work that reported modification at Ser residues only?”
We greatly appreciate these comments from Reviewer 1. We certainly agree that the question of natural SdeC substrates modified at Tyr is beyond the scope of this manuscript. However, in an effort to at least begin to answer this question, we sought to interrogate a separate proteomics dataset on modification by SdeC that is part of another project that we are currently writing up. In the parallel project, we had set out to identify all the major substrates of SdeC in crude extracts from a cultured cell line. After exposure to SdeC, these extracts were subjected to MS/MS and candidates were identified based on their abundance relative to untreated controls. Among the proteins identified were 25 proteins (including Rtn4, a previously identified substrate) that showed high abundance in our MS/MS pools relative to control samples. Although it was an ambitious request, we thought we might get lucky by investigating this existing dataset. Unfortunately, we did not have sufficient information in that dataset to answer the query, and the steps we took are briefly detailed below.

Initial studies of SdeC-mediated pR-Ub could readily identify an [M+212] adduct at R42 of Ub, suggesting pR modification at that site. In a fortuitous turn of events, we were also able to identify an [M+212] adduct at Y2 of HA in our early studies. Based on the work reported herein, this adduct would be a diagnostic remnant of the pR crosslink between Ub R42 and Y2 on HA. Building on this, the MS/MS data from the 25 candidate protein hits were analyzed to identify peptides with [M+212] adducts. Unfortunately, the only unambiguous modification was on a peptide in Rtn4 that has a Ser, so this analysis clearly lacked power, and was unable to even identify all four modified peptides in Rtn4. We attribute this finding to the fact that crosslinks are extremely difficult to assess by unbiased MS/MS workflows like those used in a traditional proteomics experiment. This is because both peptides connected by the crosslink will fragment during MS/MS, leading to a variable mass change that can be vanishingly difficult to catch. Additionally, the crosslink itself is likely to be in low abundance relative to all of the other unmodified peptides, again making it challenging to detect. Therefore, we believe that to answer this query, we would have to do considerable work to try to figure out how to marry an MS/MS approach to analysis of proteins en masse. This analysis would have to go far beyond the study we submitted and is more appropriate for our other comprehensive analysis dealing with the nature of the non-Rtn4 substrates.

To answer this query, and directly respond to the referee’s request, we note in the Discussion:

lines 292-294: “Previous studies using model peptides as acceptors for pR-Ub modification failed to include Tyr as a potential replacement for Ser, providing an explanation for why this target was overlooked.37”

In addition, we added (in blue):

lines 320-327: “Although we agree with previous studies37–39,41 that phosphoribosyl-linked ubiquitination of the serine-rich Rtn4 protein likely occurs exclusively at Ser, this does not preclude crosslinking at Tyr from occurring in other targets. In fact, Rtn4 appears to be an excellent model for Sde modification precisely because of its high serine content. Here we have found that the HA peptide, rich in tyrosines (3 out of 9 residues), is also an attractive target for pR-Ub crosslinking. This observation suggests that a search for short stretches within proteins that are similarly tyrosine-rich could reveal a number of new potential targets of the Sde family.”

- and-

lines 336-340: “Moreover, although Ser occurs in the human proteome with far greater frequency than Tyr, Tyr is particularly enriched at binding interfaces on protein surfaces, suggesting an important functional role in the binding of small molecules, nucleotides, or other proteins.45,46 Thus, phosphoribosyl-linked ubiquitination at Tyr could offer L. pneumophila an added benefit by wreaking maximal havoc on host cell signaling.”

“Minor:”
1. “p5 line 55- pneumophila is misspelled”

We thank Reviewer 1 for bringing this to our attention. This typo has been fixed in the revised manuscript.
2. “p5 line 60- amoebae is misspelled”
We thank Reviewer 1 for bringing this to our attention. This typo has been fixed in the revised manuscript.

Reviewer #2
“The manuscript by Zhang et al. reports an unexpected modification of phosphoribosyl-linked ubiquitination (pr-UB) at the protein target catalyzed by a bacterial effector protein of the Sde family. This discovery is of major importance to better understand the role of this crucial bacterial effector proteins during infection and their deleterious effects to host cells.
The authors start by analysis of substrates produced by individual functional catalytic domains utilizing a clever immobilization strategy. In the course of these experiments the authors realized a regular band laddering on their SDS-gels which resulted from functional ART and NP domains. Surprisingly, a crosslink between pR-UB and the HA-tag was observed. This unusual modification was further investigated by MS fragmentation and mutational scanning in the tag by several analogs to reveal that SdeC is promiscuous for tyrosine residues at various positions. This is an interesting discovery and of high interest to a broad chemical biology audience. One question remains with regards to the substrate scope: the main study focus are HA-tag derived peptides. Is it possible to investigate the extent of tyrosine vs. serine modification on substrate proteins such as reticulon directly?”

Thank you very much for the positive review and interest in the manuscript. As noted in the above response to Reviewer 1, our ongoing work is continuing to investigate the extent of tyrosine vs. serine modification on substrate proteins. As described above, our interrogation of an existing proteomics dataset was unsuccessful in revealing any useful information about the sites of crosslinking, though it was possible to identify 24 previously unidentified SdeC targets that will be reported separately. Due to the unique challenges of performing site identification on crosslinked proteins, we plan to follow up on this analysis using a more targeted approach now that we know the complete spectrum of proteins targeted by SdeC. Such an analysis will take considerable time and will go far beyond the scope of this study and is therefore more appropriate for our other comprehensive analysis dealing with the nature of the non-Rtn4 substrates.

Although we were not able to address this Reviewer feedback with new data, we have added new text to the Discussion that addresses why Rtn4 is such a good SdeC substrate and points to features that we would expect to find in SdeC substrates modified at Tyr, as seen in our response above.

28-Jul-2021

Dear Prof Scheck:

Manuscript ID: CB-ART-04-2021-000088.R1
TITLE: Members of the Legionella pneumophila Sde Family Target Tyrosine Residues for Phosphoribosyl-Linked Ubiquitination

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology

Reviewer 2

The manuscript is now ready for publication