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PreprintCited 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

Open access
Kienle, Simon Maria, Sigg, Matthias, Schneider, Tobias, Stuber, Katrin, Lehmann, Jan, Jansen, Jasmin, Stengel, Florian, Marx, Andreas, Kovermann, Michael, Scheffner, Martin·2025·Nature Communications
DOI 10.1101/2025.09.16.674391PMC PMC12932645

Preprint — published as 10.1038/s41467-026-69782-6 · Paper page

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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 the AcK surrogate of choice for studying acetylation of a given protein in cells.

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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
openRxiv
Published
2025-09-18
Volume
17
Issue
1
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2025.09.16.674391
PMC
PMC12932645
OpenAlex
W7130857709
Cited by
0
References
1
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, SFB 969 B3, SFB 969 B9, 496470458, 516836828, 471011418)
  • Universität Konstanz
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, HEK293 Cells

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