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

## TL;DR

Researchers developed a stable acetyllysine analog to study protein acetylation effects in cells, overcoming limitations of natural acetylation.

## Contribution

A non-hydrolyzable acetyllysine analog, ketolysine (KeK), is introduced as a functional substitute for acetyllysine in cellular studies.

## Key findings

- Ketolysine (KeK) mimics acetyllysine (AcK) in structural and functional effects when incorporated into ubiquitin.
- KeK remains stable in cells, unlike AcK, which is deacetylated by deacetylases.
- KeK incorporation affects p53-mediated transcription, making it suitable for studying acetylation in vivo.

## 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.

Determining the consequences of acetylation for a protein of interest in cells is a considerable challenge. Using genetic code expansion and p53, this study shows that site-specific incorporation of non-hydrolysable acetyllysine analogues enables functional analysis.