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Cited by 0RNA and protein synthesis mechanismsUbiquitin and proteasome pathwaysChemical Synthesis and Analysis

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Non-hydrolyzable acetyllysine analogs to study protein acetylation in vitro and in cells

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Kienle, Simon Maria, Sigg, Matthias, Schneider, Tobias, Stuber, Katrin, Lehmann, Jan, Jansen, Jasmin, Stengel, Florian, Marx, Andreas, Kovermann, Michael, Scheffner, Martin·2026·Nature Communications
DOI 10.1038/s41467-026-69782-6PMC PMC12932645

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Abstract

Lysine acetylation plays a prominent regulatory role in eukaryotic cells. Yet, determining the functional consequences of acetylation for a given protein represents a considerable challenge. For instance, lysine residues are subject to various posttranslational modifications, rendering interpretation of mutational studies difficult. The genetic code expansion technology enables site-specific incorporation of acetyllysine (AcK) into proteins, but the applicability of AcK is limited, as within cells, the acetyl group is removed by deacetylases. Here, we show that site-specific incorporation of the non-hydrolyzable AcK analog ketolysine (KeK) into ubiquitin closely resembles the structural and functional effects of AcK incorporation. Furthermore, AcK and KeK can be efficiently incorporated into the tumor suppressor p53 in cells. However, whereas AcK becomes deacetylated, KeK remains stable. Accordingly, incorporation of KeK, but not AcK, affects p53-mediated transcription. Thus, we propose that KeK is a well-suited AcK surrogate for studying acetylation of a given protein in cells.

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Research works printed but not linked: 57

  1. Ramazi, S. & Zahiri, J. Post-translational modifications in proteins: Resources, tools and prediction methods. Database (Oxf.) 2021, baab012 (2021). · DOI
  2. Aebersold, R. & Mann, M. Mass-spectrometric exploration of proteome structure and function. Nature 537, 347–355 (2016). · DOI · Paper page
  3. Conibear, A. C. Deciphering protein post-translational modifications using chemical biology tools. Nat. Rev. Chem. 4, 674–695 (2020). · DOI
  4. Peng, T., Das, T., Ding, K. & Hang, H. C. Functional analysis of protein post-translational modifications using genetic codon expansion. Protein Sci. 32, e4618 (2023). · DOI
  5. Barber, K. W. & Rinehart, J. The abcs of ptms. Nat. Chem. Biol. 14, 188–192 (2018). · DOI
  6. Narita, T., Weinert, B. T. & Choudhary, C. Functions and mechanisms of non-histone protein acetylation. Nat. Rev. Mol. Cell Biol. 20, 156–174 (2018). · DOI · Paper page
  7. Wang, Z. A. & Cole, P. A. The chemical biology of reversible lysine post-translational modifications. Cell Chem. Biol. 27, 953–969 (2020). · DOI
  8. Knyphausen, P., Lang, F., Baldus, L., Extra, A. & Lammers, M. Insights into k-ras 4b regulation by post-translational lysine acetylation. Biol. Chem. 397, 1071–1085 (2016). · DOI
  9. Okada, A. K. et al. Lysine acetylation regulates the interaction between proteins and membranes. Nat. Commun. 12, 6466 (2021). · DOI
  10. Kienle, S. M. et al. Electrostatic and steric effects underlie acetylation-induced changes in ubiquitin structure and function. Nat. Commun. 13, 5435 (2022). · DOI
  11. Liu, C. C. & Schultz, P. G. Adding new chemistries to the genetic code. Annu Rev. Biochem 79, 413–444 (2010). · DOI · Paper page
  12. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding n(epsilon)-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234 (2008). · DOI
  13. Miller, G. M., Flynn, E. M., Tom, J., Song, A. & Cochran, A. G. Trifluoroacetyl lysine as a bromodomain binding mimic of lysine acetylation. ACS Chem. Biol. 17, 1022–1029 (2022). · DOI
  14. Huang, Y. et al. Genetic incorporation of an aliphatic keto-containing amino acid into proteins for their site-specific modifications. Bioorg. Med Chem. Lett. 20, 878–880 (2010). · DOI
  15. Reed, S. M. & Quelle, D. E. P53 acetylation: regulation and consequences. Cancers (Basel) 7, 30–69 (2014). · DOI
  16. Wen, J. & Wang, D. Deciphering the PTM codes of the tumor suppressor p53. J. Mol. Cell Biol. 13, 774–785 (2022). · DOI
  17. Xia, Z., Kon, N., Gu, A. P., Tavana, O. & Gu, W. Deciphering the acetylation code of p53 in transcription regulation and tumor suppression. Oncogene 41, 3039–3050 (2022). · DOI · Paper page
  18. Swatek, K. N. & Komander, D. Ubiquitin modifications. Cell Res 26, 399–422 (2016). · DOI
  19. Dikic, I. & Schulman, B. A. An expanded lexicon for the ubiquitin code. Nat. Rev. Mol. Cell Biol. 24, 273–287 (2023). · DOI
  20. Zhang, F. et al. A genetically encoded (19)f nmr probe for lysine acetylation. Chem. Commun. (Camb.) 54, 3879–3882 (2018). · DOI
  21. Kastenhuber, E. R. & Lowe, S. W. Putting p53 in context. Cell 170, 1062–1078 (2017). · DOI · Paper page
  22. Hafner, A., Bulyk, M. L., Jambhekar, A. & Lahav, G. The multiple mechanisms that regulate p53 activity and cell fate. Nat. Rev. Mol. Cell Biol. 20, 199–210 (2019). · DOI · Paper page
  23. Liu, Y. & Gu, W. The complexity of p53-mediated metabolic regulation in tumor suppression. Semin Cancer Biol. 85, 4–32 (2022). · DOI
  24. Elsässer, S. J. Generation of stable amber suppression cell lines. Methods Mol. Biol. 1728, 237–245 (2018). · DOI
  25. Schmied, W. H., Elsässer, S. J., Uttamapinant, C. & Chin, J. W. Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered erf1. J. Am. Chem. Soc. 136, 15577–15583 (2014). · DOI
  26. Mitsudomi, T. et al. p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene 7, 171–180 (1992).
  27. el-Deiry, W. S. et al. Waf1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993). · DOI · Paper page
  28. Arbely, E. et al. Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration. Proc. Natl. Acad. Sci. USA 108, 8251–8256 (2011). · DOI
  29. Bullock, A. N., Henckel, J. & Fersht, A. R. Quantitative analysis of residual folding and DNA binding in mutant p53 core domain: Definition of mutant states for rescue in cancer therapy. Oncogene 19, 1245–1256 (2000). · DOI
  30. Peterson, A. C., Russell, J. D., Bailey, D. J., Westphall, M. S. & Coon, J. J. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol. Cell Proteom. 11, 1475–1488 (2012). · DOI · Paper page
  31. Julier, A., Radtke, V., Marx, A. & Scheffner, M. Generation and characterization of site-specifically mono-ubiquitylated p53. Chembiochem 23, e202100659 (2022). · DOI
  32. Glozak, M. A., Sengupta, N., Zhang, X. & Seto, E. Acetylation and deacetylation of non-histone proteins. Gene 363, 15–23 (2005). · DOI · Paper page
  33. Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E. & Mann, M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 15, 536–550 (2014). · DOI · Paper page
  34. Dancy, B. M. & Cole, P. A. Protein lysine acetylation by p300/CBP. Chem. Rev. 115, 2419–2452 (2015). · DOI
  35. Drazic, A., Myklebust, L. M., Ree, R. & Arnesen, T. The world of protein acetylation. Biochim Biophys. Acta 1864, 1372–1401 (2016). · DOI
  36. Seto, E. & Yoshida, M. Erasers of histone acetylation: The histone deacetylase enzymes. Cold Spring Harb. Perspect. Biol. 6, a018713 (2014). · DOI
  37. Smith, B. C. & Denu, J. M. Mechanism-based inhibition of Sir2 deacetylases by thioacetyl-lysine peptide. Biochemistry 46, 14478–14486 (2007). · DOI
  38. Farkas, M. et al. Distinct mechanisms control genome recognition by p53 at its target genes linked to different cell fates. Nat. Commun. 12, 484 (2021). · DOI
  39. Vainer, R., Cohen, S., Shahar, A., Zarivach, R. & Arbely, E. Structural basis for p53 lys120-acetylation-dependent DNA-binding mode. J. Mol. Biol. 428, 3013–3025 (2016). · DOI
  40. Tang, Y., Luo, J., Zhang, W. & Gu, W. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis. Mol. Cell 24, 827–839 (2006). · DOI
  41. Mujtaba, S., Zeng, L. & Zhou, M. M. Structure and acetyl-lysine recognition of the bromodomain. Oncogene 26, 5521–5527 (2007). · DOI
  42. Höllmuller, E. et al. Interactome of site-specifically acetylated linker histone H1. J. Proteome Res 20, 4443–4451 (2021). · DOI
  43. Nie, L., Sasaki, M. & Maki, C. G. Regulation of p53 nuclear export through sequential changes in conformation and ubiquitination. J. Biol. Chem. 282, 14616–14625 (2007). · DOI
  44. Zong, Z. et al. Alanyl-tRNA synthetase, aars1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis. Cell 187, 2375–2392 e2333 (2024). · DOI
  45. Stuber, K. et al. Structural and functional consequences of NEDD8 phosphorylation. Nat. Commun. 12, 5939 (2021). · DOI
  46. Hammler, D. et al. Fluorescently labelled ATP analogues for direct monitoring of ubiquitin activation. Chemistry 26, 6279–6284 (2020). · DOI
  47. Götz, K. H. et al. Formation of the alarmones diadenosine triphosphate and tetraphosphate by ubiquitin- and ubiquitin-like-activating enzymes. Cell Chem. Biol. 26, 1535–1543.e1535 (2019). · DOI
  48. Delaglio, F. et al. Nmrpipe: A multidimensional spectral processing system based on Unix pipes. J. Biomol. NMR 6, 277–293 (1995). · DOI
  49. Johnson, B. A. & Blevins, R. A. Nmr view: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994). · DOI
  50. Grzesiek, S., Stahl, S. J., Wingfield, P. T. & Bax, A. The cd4 determinant for downregulation by HIV-1 Nef directly binds to nef. Mapping of the Nef binding surface by NMR. Biochemistry 35, 10256–10261 (1996). · DOI
  51. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006). · DOI
  52. Tyanova, S., Temu, T. & Cox, J. The maxquant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016). · DOI
  53. Tyanova, S. et al. The perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016). · DOI · Paper page
  54. Krüger, L. et al. Chemical proteomic profiling reveals protein interactors of the alarmones diadenosine triphosphate and tetraphosphate. Nat. Commun. 12, 5808 (2021). · DOI
  55. Feoktistova, M., Geserick, P. & Leverkus, M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb. Protoc. 2016, pdb.prot087379 (2016). · DOI
  56. Perez-Riverol, Y. et al. The pride database at 20 years: 2025 update. Nucleic Acids Res 53, D543–D553 (2025). · DOI
  57. Cornilescu, G., Marquardt, J. L., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837 (1998). · DOI

Metadata

Authors
Kienle, S. M., Sigg, M., Schneider, T., Stuber, K., Lehmann, J., Jansen, J., Stengel, F., Marx, A., Kovermann, M., & Scheffner, M.
Affiliations
University of Konstanz (DE)
Corresponding author
  • Kienle, Simon Maria
  • Scheffner, Martin
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2026-02-21
Volume
17
Issue
1
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-026-69782-6
PMC
PMC12932645
OpenAlex
W7130857709
Cited by
0
References
6
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • KOPS (University of Konstanz) (submittedVersion)
Funders
Deutsche Forschungsgemeinschaft (SFB 969 B3, 516836828, 496470458, TRR353/1 – 471011418, SFB969 B3, SFB969 B9)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
Keywords
lysine acetylation, site-specific incorporation, genetic code expansion, ubiquitin, p53, protein deacetylation, post-translational modification
MeSH terms
Acetylation, Animals, Humans, Lysine, Protein Processing, Post-Translational, Tumor Suppressor Protein p53, Ubiquitin

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