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

Kienle, S. M.; Sigg, M.; Schneider, T.; Stuber, K.; Lehmann, J.; Jansen, J.; Stengel, F.; Marx, A.; Scheffner, M.

2025-09-18 biochemistry
10.1101/2025.09.16.674391 bioRxiv
Show 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.

Published in Nature Communications (predicted rank #1) · training set

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