Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere

Abstract Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168‐M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross‐talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1‐mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down‐regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.


26th Apr 2019 1st Editorial Decision
Dear Dr. Mayans Thank you for submitting your manuscript for consideration by EMBO Reports. I would like to apologize for this unusual delay in our decision process. Three referees agreed to review the manuscript, two of which returned their reports. The third referee, on the other hand, has not returned his/her report but given the present set of referee reports I can take a decision at this stage.
Please note that this is a preliminary decision made in the interest of time, and that it is subject to change should the third referee offer very strong and convincing reasons for this. If/When we receive the final report on your manuscript, we will forward it to you as well.
As you can see, both referees express interest in the analysis. However, they also raise concerns that need to be addressed in full before we can consider publication of the manuscript here. In particular, both referees require additional support into the functional relevance of titin ubiquitination and better evidence supporting that ubiquitination of titin recruits Nbr1 and p62, not just stretch induced exposure of the residues. Moreover, both referees would like to see the massspec data.
Should you be able to address these criticisms in full, we could consider a revised manuscript. I do realize that addressing all the referees' criticisms will require a lot of additional time and effort and be technically challenging. I would therefore understand if you wish to publish the manuscript rapidly and without any significant changes elsewhere, in which case please let us know so we can withdraw it from our system.
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Yours sincerely, Deniz Senyilmaz Tiebe ---Deniz Senyilmaz Tiebe, PhD Editor EMBO Reports Referee #1: Revision 2019-04-01; EMBO reports Titin ubiquitination causes the scaffolding of autophagosomal receptors onto the sarcomere Bogomolovas et al. performed a structural analysis of titin domains, which have been previously associated with Murf1, Nrb1 and p62 binding. This structural analysis is certainly very insightful and allows wide speculations about the functional behavior of these domain upon stretch/mechanical forces. Further, by in-vitro ubiquitylation assay, the authors were able to identify ubiquitylation sites on titin as targets for Murf1. Using SMDS, the authors in turn simulate folding of this titin domain upon stretching and characterize potential opening sites. These opening site is reported to contain a human SNP which has been linked to cardiac diseases. Mutagenesis were used to introduce the SNP variation in order to identify changes in titin domain ubiquitylation. Overall, the authors propose an altered mechanosensory mechanism of higher complexity as previously thought. While the structural data were quite convincing, other parts of the manuscript unfortunately displayed weaknesses that need to be addressed. The authors argue convincingly that titin is unlikely to be transported to protein degradation sites and that protein degradation is more likely to occur through the recruitment of ubiquitin binding protein. However, the motivation of the subsequent experiments is not clear as it only partially addresses the underlying scientific question. Based on the EMBO manuscript submission guidelines, proteomics data should be submitted to a publicly accessible database, typically PRIDE. In addition, all identified sites should be documented in a suppl. table with relevant MS data to judge the quality of the MS data and modified peptides. The authors claim an absence of ubiquitylation in the D24728V mutant at a specific site but do not provide experimental evidence for this. A central claim of this manuscript describes titin not only as a sequestering protein, but also as a target of further modification with other functions. While the modification of titin is rather obvious, it is unclear what these new functions may be and the manuscript in its current form do not shed light onto this question. Ubiquitylation of titin may just change sequestration targets by creating binding sites for different types of proteins, which is per se not new. Detailed characterization of the ubiquitylation site is advisable in particular with respect to protein stability. However, this might be a challenge for the giant protein titin and will be hard to address. The authors suggest that ubiquitylation recruits Nbr1 and p62 to titin and not stretch-induced exposure of binding sites. A supportive experiment would be useful to show that stretched titin (or equivalent construct) does not bind to Nbr1 or p62. Supporting evidence is lacking and colocalization studies with the unstretched A168-TK construct cannot exclude that Nbr1 and p62 are recruited to titin. It might be helpful to perform an immunoprecipitation experiment with ubiquitylation titin to identify the ubiquitylation-interaction of titin with Nbr1 and p62 via mass spectrometry. The authors propose that Nbr1 and p62 are recruited titin via their ubiquitin binding domain. Importantly, while the A168-TK showed diffuse expression, co-expression with Murf1 leads to A168-TK accumulation in large aggregates (Fig 3c) with remarkable shapes, suggesting for a change in localization of A168-TK itself. One obvious explanation is that A168-TK gets ubiquitylated and subsequently recruited to sites of protein degradation. Conversely, ubiquitin binders Nbr1 and p62 are recruited to these sites as well, independently of A168-TK. Colocalization is not sufficient to show binding of Nbr1 and p62 to A168-TK. The authors describe that the A170-TKD24728V sample was subjected to ubiquitylation assays and modified lysine residues were identified by MS. The author should show the results from three independent replicates with fold changes, statistical relevant values and MS data. The authors point out correctly, that endogenous titin is static and rather immobile. However, experiments are done with small constructs covering only one part of titin, which is acceptable given the size of full length titin. However, the authors should address experimentally or carefully discuss, why ubiquitylation of a diffusible protein construct has the same or a similar effect compared to the ubiquitylation of the entire titin. The authors propose a dynamic regulation of ubiquitylation upon stretch, supported by SMD simulation analysis. However, in-vitro ubiquitination data (Fig2) show that stretch is not required for efficient ubiquitylation. This point should be at least discussed. Minor points: For reader convenience, all abbreviations should be clarified. Fig.1A requires improvements. In principle, pieces of information that come up later in the manuscript should be displayed in this panel. This includes: (i) all motifs (ii) the localization of detected ubiquitylation sites (iii) an overview of full length titin, which certainly helps to better orient within this huge protein.
The authors are advised to consistently use the term "mass spectrometry" rather than "mass spectroscopy". In addition, the authors may discuss how many ubiquitylation sites are unknown and which of them have been observed in in-vivo studies using the given reference. An overlap to known sites from the phosphosites.org database could also be useful. The authors shifted several important findings to the supplement, which makes the manuscript difficult to follow. Along these lines, some experiments were obviously performed, but not described or discussed in the manuscript.
Referee #2: The study by Bogomolovas et al is well designed and executed, providing important new information on the role of titin's extreme COOH-terminus including a pseudokinase domain and the structural elements that flank it in sensing and transmitting mechanical signals via a multiprotein complex consisting of Nbr1/p62 and MURF1.
Major concerns: 1. Ubiquitination experiments: Given that these experiments were done in vitro, the authors need to comment on how confident they are about all the identified sites possibly undergoing ubiquitination in vivo. Also, were the common sites identified in both constructs by mass spec? This needs to be clarified/included.
2.Overexpression studies n COS9 cells: how do the findings from these studies translate to cardiac or skeletal muscle cells? The authors need to discuss this. Ideally, these studies should have been done in muscle cells, however this reviewer recognizes the technical difficulties of such experiments, although viral constructs can be effective. Moreover, coincident localization does not necessarily mean association. The authors should perform co-immunoprecipitation experiments of the combinations of overexpressed proteins described in Fig. 3B-D to validate the co-localization findings.
3. The SMDS observations will be more convincing if they are confirmed by atomic force microscopy studies of the TK construct(s).
We have submitted a revised version of our manuscript EMBOR-2019-48018V2. We thank you and the reviewers for the constructive comments to our original submission. We have carried out both additional experimentation as well as an in-depth revision of the text following these recommendations (modified text is shown in red in the revised manuscript to ease identification). In brief, we have performed pull-downs experiments from cell extracts, repeated the transfection work on a muscle-relevant cell line, deposited mass spectrometry data with a public repository and modified the written manuscript to better clarify the nature and novelty of our findings as well as to fit the journal format. I include below a detailed account of the changes implemented and answers to specific questions by the referees: [1] Referee #1: "The authors argue convincingly that titin is unlikely to be transported to protein degradation sites and that protein degradation is more likely to occur through the recruitment of ubiquitin binding protein. However, the motivation of the subsequent experiments is not clear as it only partially addresses the underlying scientific question." Our work focuses on the mechanism by which the sarcomere recruits its binding partners in a stretch dependent manner, thereby linking its content of functional and signaling factors to the mechanical demand on it exerted. While it is well established that the scaffolding properties of the sarcomere vary in function of mechanical activity, the molecular mechanisms by which such regulated scaffolding occurs remain largely unknown. A current hypothesis is that mechanical stretch induces conformational changes in sarcomere protein components that expose cryptic binding sites for functional factors. This mechanism was originally proposed to mediate the association of the autophagosomal receptors Nbr1 and p62 to M-line titin kinase in a stretchdependent manner (Lange et al, Science, 2005). Unrelated to the topic of sarcomere-based interactions, independent reports have shown that protein components of the sarcomere - . However, a connection between ubiquitination and sarcomere-based interactions had not been made until now. In this work, we propose for the first time that ubiquitination mediates sarcomere-based interactions and that the ubiquitination pattern of the sarcomere is sensitive to the mechanical state of the myofibril, in turn altering dynamically the protein associations that it supports. In this regard, we show that the recruitment of Nbr1 and p62 to titin kinase occurs in non-stretched forms of the kinase once ubiquitination has taken place, so that the binding is not necessarily dependent on stretch-exposed cryptic binding sites but on post-translational modification. We also propose a mechanism by which ubiquitination could be modulated by stretch in the titin kinase locus. Our work is not directed to reveal the functional consequences of the recruitment of Nbr1/p62 to M-line titin (which we anticipate is a step within the muscle atrophy induced by MuRF1), but to explore the molecular mechanisms by which such regulated recruitment is exerted. We have modified Abstract, Introduction and Conclusion to better clarify to the reader the focus of our work and the novelty of the findings.
[2] Referee #1: "Based on the EMBO manuscript submission guidelines, proteomics data should be submitted to a publicly accessible database, typically PRIDE. In addition, all identified sites should be documented in a suppl. We thank the reviewer for pointing our attention to the PRIDE database. For each sample type (i.e. wild-type and D24728V), three independent biological replicates were generated and two samples from each were analysed (amounting to a redundancy of 6 measurements per sample type). The mass spectrometry data have been deposited with PRIDE under accession codes PXD015339 and PXD015340 (wild-type and D24728V samples, respectively). PRIDE provides access to those entries for review purposes, as follows: To our knowledge, ubiquitination has not been proposed previously as a mechanism of sarcomere-based scaffolding and sarcomere ubiquitination has also not been proposed to be regulated by stretch. While sarcomere ubiquitination is dependent on the population of E3 ubiquitin ligases present in the cell at a given time, to our knowledge no previous proposal has been made that correlates the mechanical activity of the sarcomere with its targetability by such E3 Ub ligases. Our model proposes that the ubiquitination state of the sarcomere is the combined result of cell proteome (physiological cues) and mechanical cues derived from exercise. Unraveling the functional outcomes of sarcomere ubiquitination and its promoted interactions is highly challenging and beyond the scope of this work. A possibility is that sarcomere compartmentation could cause the sequestration of Nbr1/p62 away from their place of action in the cell, thereby reducing cytoplasmic pools and down-regulating their activity. Alternatively, the targeting of the sarcomere M-line by Nbr1/p62 (possibly combined with the action of proteases) might contribute to sarcomere degradation. In line with that potential functional outcome, it is known that titin M-line is required for sarcomere integrity ( [4] Referee #1: "Detailed characterization of the ubiquitylation site is advisable in particular with respect to protein stability. However, this might be a challenge for the giant protein titin and will be hard to address." We agree with the reviewer that measuring titin stability in the sarcomere as a function of the ubiquitination sites identified in the M-line is a staggering challenge. The huge size of the titin molecule (>3 MDa) and the fact that it is an intrasarcomeric protein makes the generation of mutagenized transgenic muscle samples and their subsequent analysis extraordinarily demanding. In addition, ubiquitination sites have been found in titin regions other than the Mline kinase. Thus, measuring the contribution of M-line ubiquitination to titin stability in a cellular or myofibrillar context is extremely difficult.
[5] Conceivably, these two distinct modes of Nbr1/p62 association with titin -in active and atrophic muscle, respectively -are not mutually exclusive but lead to different functional outcomes. It is tantalizing to envision that distint association modes might regulate the functionality of Nbr1/p62, as to differentiate sequestration from targeting for degradation outcomes. We have modified the Conclusion section to clarify this question to the reader.
[6] Referee #1: "The authors propose that Nbr1 and p62 are recruited titin via their ubiquitin binding domain. Importantly, while the A168-TK showed diffuse expression, co-expression with Murf1 leads to A168-TK accumulation in large aggregates (Fig 3c) with remarkable shapes, suggesting for a change in localization of A168-TK itself. One obvious explanation is that A168-TK gets ubiquitylated and subsequently recruited to sites of protein degradation. Conversely, ubiquitin binders Nbr1 and p62 are recruited to these sites as well, independently of A168-TK. Co-localization is not sufficient to show binding of Nbr1 and p62 to A168-TK." We thank the reviewer for calling our attention to this possibility. In our opinion, existent data do not suggest a coincidental, independent localization of samples in the cell. It can be predicted that the recruited A168-TK is ubiquitinated by the active MuRF1 in those formations (as observed in in vitro assays) and, furthermore, that even MuRF1 expressed in isolation is heavily auto-ubiquitinated (in vitro assays show that MuRF1 undergoes strong self-ubiquitination as it is characteristic of E3 ubiquitin ligases). Yet, neither the pattern of MuRF1 nor that of the MuRF1/A168-TK co-expressions resembles that observed upon co-expression with Nbr1 or p62, which adopts the form of puncta. If MuRF1/A168-TK were recruited to puncta formations independently from Nbr1 and p62, the phenotypes would coincide, which is not the case. To address this point, we have expanded Figure 3 to display cell localization phenotypes for all proteins expressed in isolation as well as the A168-TK/MuRF1 complex in the absence of Nbr1/p62.
[7] Referee #1: "It might be helpful to perform an immunoprecipitation experiment with ubiquitylation titin to identify the ubiquitylation-interaction of titin with Nbr1 and p62 via mass spectrometry." Referee #2: "Coincident localization does not necessarily mean association. The authors should perform co-immunoprecipitation experiments of the combinations of overexpressed proteins described in Fig. 3B-D Fig EV3). Sadly, despite multiple attempts, good quality data could not be obtained for Nbr1. Those experiments were troubled by continued solubility problems and a persistent unspecific association of Nbr1 to affinity beads. Because of the technical difficulties posed by these samples, in this work we had constructed instead truncated Nbr1 and p62 that lacked ubiquitin binding domains (Nbr1-DUBA; p62-DUBA) and tested this in cell co-transfection experiments (Fig 3). Those results showed that UBA domain deficiency abrogated the colocalization of Nbr1 and p62 with titin A168-TK, indicating that the association is mediated by ubiquitin-binding. As expected, titin and MuRF1 continued to associate mutually undisturbed in the presence of the UBA-truncated Nbr1/p62 samples (Fig 3). Taken together, pull-down results on p62 and cell transfections with -DUBA Nbr1/p62 variants support the view that the interaction of the TK-MuRF1 complex with autophagy adaptors p62 and Nbr1 is ubiquitin-mediated. This is in good agreement with existing knowledge of the interaction of p62/Nbr1 with poly-ubiquitinated proteins (e.g. Zaffagnini et al, EMBO J, 2018 As the reviewer correctly states, our work shows that the ubiquitination of titin kinase by MuRF1 occurs in the absence of stretch. Our postulate is that stretch would decrease the levels of ubiquitination in this titin region by distancing ubiquitination target sites from the docked MuRF1 protein. The distancing of the sites is based on SMD simulations (and in agreement with previous experimental AFM data; point [15]) that reveal the unraveling of the NL sequence (i.e. the linker between the MuRF1 docking site and the kinase fraction) as the first unfolding event.
We have clarified better this point to the reader in the Results Section, pg10.  Fig 1C and Fig 2). Thus, we kindly request that Fig  1A is considered as provided in this updated version.
[11] Referee #1: "The authors are advised to consistently use the term "mass spectrometry" rather than "mass spectroscopy". In addition, the authors may discuss how many ubiquitylation sites are unknown and which of them have been observed in in-vivo studies using the given reference. An overlap to known sites from the phosphosites.org database could also be useful." We thank the referee for calling our attention to the inconsistent usage of the terms spectrometry/spectroscopy. We now use "mass spectrometry" throughout.
In an attempt to focus our work on the ubiquitination mediated by MuRF1, we originally narrowed the comparison of our in vitro identified sites to those found in vivo by [12] Referee #1: "The authors shifted several important findings to the supplement, which makes the manuscript difficult to follow. Along these lines, some experiments were obviously performed, but not described or discussed in the manuscript." We thank the reviewer for making us aware of this problem. To facilitate the information flow and better fit the journal's format, we have now promoted 5 Appendix Figures to the Expanded View (Fig EV1-EV5). We hope that this eases the access to that information by the readers.
[ We are most thankful to the reviewer for acknowledging the substantial technical difficulty of carrying such transfection studies in muscle cell lines. Nevertheless, we do recognize the importance of the question and, thus, have repeated the in cellula work using the H9C2 myoblast cell line. This cell line was originally isolated from BDIX rat ventricular tissue and is widely established as an in vitro model for skeletal and cardiac muscle. Although H9C2 cells do not have structured sarcomeres, they retain the fundamental biochemical, morphological, electrophysiological and molecular patterns of striated muscle. These include, but are not limited to, a muscle-like creatine phosphokinase isoenzyme profile, sensitivity to acetylcholine, cardiac L-type calcium current sensitive to isoproterenol, an overall muscle-like transcriptome and expression of cardiac muscle specific proteins ( Consistently, the AFM data revealed a first low-force unfolding event (hierarchically corresponding to the unfolding of the mechanically weakest element) that corresponds to the unravelling of a sequence of ~9.1 nm length. At the time of those studies, only an incomplete crystal structure of titin kinase (TK) was available, containing the C-terminal tail extension (CRD), but lacking the N-terminal segment (NL). Thus, that initial unfolding event could not be attributed to specific titin molecular features and it remained unexplained, although Puchner et al 2008 speculated that it could correspond to the unfolding of a sequence N-terminal to TK. The authors then placed the focus on studying the higher-force response of the CRD tail, which could be studied based on structural data available at that time. The SMDS calculations in our study use the complete structure of TK (reported in this manuscript) containing N-and C-terminal flanking tails, and permit now attributing the first force peak in experimental AFM traces to the dissolution of the NYD 'knot' motif. Thus, our simulations are in excellent agreement with AFM observations and explain now their molecular basis. We provide now an explanation of this point in pg 9.
In addition to addressing the specific referee points, we have also performed an overall revision of the manuscript format to adapt it to journal guidelines.
Finally, we would like to express our gratitude to the referees for their comments, which have undoubtedly contributed to the improvement of this manuscript. We hope to have addressed the issues raised satisfactorily in this letter and in the revised version of the text and, thus, that EMBO Reports is willing to consider our work further. In this hope, we remain expectant. Thank you for submitting your revised manuscript. It has now been seen by both of the original referees.
As you can see, the referees find that the study is significantly improved during revision and recommend publication. However, I need you to address the editorial points below before I can accept the manuscript: • Please make the PXD015339 and PXD015340 datasets public. • As per our format requirements, in the reference list, citations should be listed in alphabetical order and then chronologically, with the authors' surnames and initials inverted; where there are more than 10 authors on a paper, 10 will be listed, followed by 'et al. '. Please see https://www.embopress.org/page/journal/14693178/authorguide#referencesformat • Please upload the figures in one of the following formats: TIFF, PDF or EPS (please see https://www.embopress.org/page/journal/14693178/authorguide#figureformat for more information).
• Please upload the EV figures as individual files.
• As per our format requirements, the title length cannot exceed 100 characters (including spaces). The title is currently too long.
• We note the following about the figure callouts: Fig 4C+D callouts, Fig EV5 panel  We have been absolutely delighted to learn that the journal has given consideration to our revised manuscript and that this has been well received by the reviewers.
We apologize for the shortcomings of the documents we provided and that these did not fulfil the journal's requirements. We supply now amended versions of text and figures following the editorial points, as requested: • Please make the PXD015339 and PXD015340 datasets public We confirm that these entries are now publicly available in the PRIDE database.
• As per our format requirements, in the reference list, citations should be listed in alphabetical order and then chronologically, with the authors' surnames and initials inverted; where there are more than 10 authors on a paper, 10 will be listed, followed by 'et al. ' • As per our format requirements, the title length cannot exceed 100 characters (including spaces). The title is currently too long.
The title has been changed to "Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere" (95 characters, incl. spaces) • We note the following about the figure callouts: Fig 4C+D callouts, Fig EV5 panel  • The movies need to be renamed as Movie EV#. Please update the callouts in the text accordingly. The corrections have been implemented.
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This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript. (2-1) (ATCC® CRL-1446™) and COS-7 cells (ATCC® CRL-1651TM) cell lines were obtained from ATCC, autenthicated and certified to be mycoplasma free. Cells were grown in our laboratory for not more than 5 passages in medium supplemented with Normocin™ a formulation of three antibiotics active against mycoplasma, bacteria and fungi. NA SQSTM1/p62 Rabbit mAb #5114S (Cell Signalling Technology); NBR1 (D2E6) Rabbit mAb #9891 (Cell Signalling Technology); Anti-rabbit IgG, HRP-linked Antibody #7074 (Cell Signalling Technology)