Transglutaminase 2 mediates hypoxia-induced selective mRNA translation via polyamination of 4EBPs

This study highlights the role of transglutaminase 2 in selective mRNA translation of hypoxic cancer cells by polyamination-dependent modulation of 4EBPs, providing a target for cancer treatment.

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--An editable version of the final text (.DOC or .DOCX) is needed for copyediting (no PDFs).
--High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, http://www.life-sciencealliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.

B. MANUSCRIPT ORGANIZATION AND FORMATTING:
Full guidelines are available on our Instructions for Authors page, http://www.life-sciencealliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Sung-Yup Cho et al. showed that under hypoxia condition, Transglutaminase 2(TG2) promotes eIF4E associated 4E-BP1's polyamination. Polyaminated 4E-BP1 has an increased binding affinity with Raptor, leading to its phosphorylation by mTORC1, thus promoting the translation of a subset of mRNAs with GC-rich 5'UTR. In general, it is an interesting and novel mechanism showing that TG2 elicits4E-BPs polyamination in eIF4E dependent manner and promotes 4E-BP1 inactivation by mTORC1 under hypoxia condition. However, some issues and concerns must be addressed. Major concerns: 1. The authors only showed that polyaminated 4E-BP1 associated more strongly with Raptor in vitro (Fig. 4D). However, under hypoxia condition, mTORC1 is inactivated, thus, their results would be more convincing if they can show that purified Raptor-mTOR complex from hypoxic cells is able to phosphorylate polyaminated 4E-BP1 in vitro. 2. The authors showed that TG2 regulates eIF4E dependent mRNA translation by Cap binding assay (Fig. 4B). However, the m7GTP pull-down experiment only demonstrated a small difference between control and TG2 knockdown cells, which have been exposed to hypoxia for 48 hours (Fig. 4B). Since they showed that the TG2 is required for maintaining 4E-BP1 phosphorylation in cells, which have been exposed to hypoxia for 24 hours (Fig. 4A), they should use these cells to repeat their experiment. Moreover, by using a bicistronic translation reporter, the authors showed that overexpression of TG2 promoted cap-dependent translation under normoxic conditions in polyaminated 4E-BP1-dependent manner (Fig. 5A, B and C). However, it should be noted that TG2 overexpression may promote PI3K/mTOR activity Minor points: 1. Fig. 1A, the authors should indicate that pull-downed 4E-BP1 corresponds to the upper or lower band of 4E-BP1 in the input. 2. Fig. 1G, why in PP242 treated cells there is not a clearly increased polyaminated 4E-BP1? 3. Fig. 1H, why Myc tagged 4E-BP1 appears only as one band? Phospho-4E-BP1 antibody should be used to evaluate the phosphorylation state of 4E-BP1-MYC. 4. Fig. 3B, in eIF4E knockdown cells, 4E-BP1 seems to be more phosphorylated as there are clearly increased upper bands. According to the authors, phosphorylated 4E-BP1 will be less polyaminated. The authors thus should use the phospho-4E-BP1 antibody to examine the 4E-BP1 phosphorylation state in eIF4E knockdown cells. 5. Fig. 4C, the authors should explain why AKT and S6K dramatically decreased in cells that have been exposed to hypoxia for 24 and 48 hours? 6. Fig. 4D, why Spermine alone without TG2 can also clearly increase 4E-BP1 and raptor binding? A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival. Boroughs  Reviewer #2 (Comments to the Authors (Required)): The manuscript from Cho et al describes work to address the role of TG2 in polyamination of 4E-BP1 in response to hypoxia. Previous work has shown that 4E-BP1 is subject to phosphorylation, ubiquitination and cleavage as modes of regulating its activity. The work here shows that 4E-BP1 is also regulated by polyamination which enhances 4E-BP1 affinity for raptor, thereby enhancing phosphorylation by mTORC1. Furthermore, it is shown that translation of a subset of mRNAs, ric in GC content, is preferentially enhanced by polyamination of 4E-BP1. As such, the conclusions are supported by the data shown. For completeness, in my opinion, the authors need to address the following points: 1. p4; removed reference to 4E-BP3 here as it is not relevant to the argument made here and it not addressed during this study. 2. Fig 1; could the authors mentions that this is a high Km and comment of the physiological relevance. 3. Fig.4C; there is a decrease in total Akt and p70S6K at 24hrs with shTG2; this needs to be addressed in the text and the reasons discussed. What happens with Akt-S308 phosphorylation which is more of an indicator of PI3-K activity? The Western blot for Raptor is unclear; a better version needs to be presented. 4. Fig.4D; why use HeLa extracts here? What happens with Q28/93/113A here? 5. Fig.5A; what happens with CTA alone in this assay? Does CTA decreased TG2 expression? 6. Fig.5C; The authors need to provide a Western blot showing equal levels of protein expression between variables. Please find below our responses to each specific comment and suggestion raised by the two reviewers.
Reviewer #1: 1. The authors only showed that polyaminated 4E-BP1 associated more strongly with Raptor in vitro (Fig. 4D). However, under hypoxia condition, mTORC1 is inactivated, thus, their results would be more convincing if they can show that purified Raptor-mTOR complex from hypoxic cells is able to phosphorylate polyaminated 4E-BP1 in vitro.  2. The authors showed that TG2 regulates eIF4E dependent mRNA translation by Cap binding assay (Fig. 4B). However, the m 7 GTP pull-down experiment only demonstrated a small difference between control and TG2 knockdown cells, which have been exposed to hypoxia for 48 hours (Fig. 4B). Since they showed that the TG2 is required for maintaining 4E-BP1 phosphorylation in cells, which have been exposed to hypoxia for 24 hours (Fig. 4A), they should use these cells to repeat their experiment.
: We performed the m 7 GTP pull-down experiment for cells which have been exposed to hypoxia for 24 hours as reviewer suggested. We found that knock-down of TG2 significantly increased the binding of 4EBP1 to eIF4E in hypoxic condition for 24 hours (Fig. 4B). These results are now included in the Results sections of the revised manuscript (p. 9) and in Figure   4B, and the data from cells, which have been exposed to hypoxia for 48 hours, was demonstrated in Supplemental Fig. S3. : When we performed the experiment again, we found that the migration of wild-type 4EBP1-myc was a little slower than T37,46A-mutant 4EBP1-myc (Fig. 1H, input), suggesting that the phosphorylation status of wild-type 4EBP1-myc was higher than that of mutant 4EBP1myc. Consistent to this result, the phosphorylation of the Myc-immunoprecipitated 4EBP1 was detected only in wild-type 4EBP1 ( Fig. 1H; Myc-IP). These results are now included in Fig.   1H and legend of Fig. 1H. 8. Fig. 3B, in eIF4E knockdown cells, 4E-BP1 seems to be more phosphorylated as there are clearly increased upper bands. According to the authors, phosphorylated 4E-BP1 will be less polyaminated. The authors thus should use the phospho-4E-BP1 antibody to examine the 4E-BP1 phosphorylation state in eIF4E knockdown cells.
: As the reviewer suggested, knock-down of eIF4E slightly increased the phosphorylated 4E-BP1 (Fig. 3B). This effect was probably due to the feedback effect to increase the capdependent translation, because the knockdown of eIF4E readily inhibited the cap-dependent translation. However, the decrease of 4EBP1 polyamination was significant compared to the increase of 4EBP1 phosphorylation (Fig. 3B). Therefore, we suggest that eIF4E is required for the polyamination of 4EBP1. These results are now included in the Results sections of the revised manuscript (p. 8) and Fig. 3B.
9. Fig. 4C, the authors should explain why AKT and S6K dramatically decreased in cells that have been exposed to hypoxia for 24 and 48 hours?
: When we performed the experiment again, we found that the total AKT and S6K levels slightly decrease in hypoxia differently with our previous results, and these results have been  (2):131-9). In our experiment, spermine increased the interaction between 4EBP1 and Raptor. However, the exact molecular mechanisms of enhancing the interaction by spermine need to be further studied. And, incorporation of spermine to 4EBP1 by TG2 still enhanced the interaction between 4EBP1 and Raptor ( Fig. 4E and Supplemental Fig. S4). These are now included in the Discussion sections of the revised manuscript (p. 16).
Reviewer #2: 1. p4; removed reference to 4E-BP3 here as it is not relevant to the argument made here and it not addressed during this study.
: We showed that 4EBP1, -2, -3 are substrates of TG2 and identified the target glutamine site for polyamination (Fig. 2). We investigated the functional change of TG2-mediated modification using 4EBP1. 3. Fig.4C; there is a decrease in total Akt and p70S6K at 24hrs with shTG2; this needs to be addressed in the text and the reasons discussed. What happens with Akt-S308 phosphorylation which is more of an indicator of PI3-K activity? The Western blot for Raptor is unclear; a better version needs to be presented.
: When we performed the experiment again, we found that the total AKT and S6K levels slightly decrease in hypoxia differently with our previous results, and these results have been possibly cleaved by caspase activation in hypoxic conditions resulting in the decreased expression levels, but it seems to be context dependent and need to be further studied. The explanation of the decrease of total AKT and S6K levels in hypoxia was included in the Discussion sections of the revised manuscript (p. 16). We also checked Akt-S308 phosphorylation and found little significant differences between wild-type and TG2knockdown cells (Fig. 4C). These are now included in the Result sections of the revised manuscript (p. 10) and Fig. 4C. We replaced the blots for AKT, S6K, mTOR and Raptor in Fig   4C with the more reproducible results in our experimental setting.
: We performed the experiment again using hypoxic A549 cell lysate, and showed similar results (Fig. 4E). For consistence of manuscript, we replaced the Fig. 4E with results from A549 cells, and moved the data from HeLa cells to Supplemental Fig. S4. When we adopted the 4EBP1 Q28/93/113A mutant in this experimental setting, we found that modification of mutant by TG2 did not increase the interaction between 4EBP1 and Raptor (Fig. 4F). These . We adopted these cell lines to assay the protein synthesis in stably TG2-overexpressing system. We added the western blot showing the overexpression of TG2 in HEK293 cells (Fig. 5F). The incorporation of S35methionine was depicted compared to control HEK293 cells at 0 h. We clearly indicated the If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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