N-terminal acetylation can stabilize proteins independent of their ubiquitination

• Published in: Scientific reports Vol. 13; no. 1; pp. 5333 - 13
• Main Authors: van de Kooij, Bert, de Vries, Evert, Rooswinkel, Rogier W., Janssen, George M. C., Kok, Frédérique K., van Veelen, Peter A., Borst, Jannie
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 631/337; 631/45; Acetylation; Animals; Humanities and Social Sciences; Humans; Lysine; Lysine - metabolism; Mammalian cells; Mammals - metabolism; multidisciplinary; N-Terminus; Proteasomes; Protein Processing, Post-Translational; Proteins; Proteome - metabolism; Proteomes; Science; Science (multidisciplinary); Ubiquitin; Ubiquitination
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-023-32380-3

The majority of proteins in mammalian cells are modified by covalent attachment of an acetyl-group to the N-terminus (Nt-acetylation). Paradoxically, Nt-acetylation has been suggested to inhibit as well as to promote substrate degradation. Contrasting these findings, proteome-wide stability measurements failed to detect any correlation between Nt-acetylation status and protein stability. Accordingly, by analysis of protein stability datasets, we found that predicted Nt-acetylation positively correlates with protein stability in case of GFP, but this correlation does not hold for the entire proteome. To further resolve this conundrum, we systematically changed the Nt-acetylation and ubiquitination status of model substrates and assessed their stability. For wild-type Bcl-B, which is heavily modified by proteasome-targeting lysine ubiquitination, Nt-acetylation did not correlate with protein stability. For a lysine-less Bcl-B mutant, however, Nt-acetylation correlated with increased protein stability, likely due to prohibition of ubiquitin conjugation to the acetylated N-terminus. In case of GFP, Nt-acetylation correlated with increased protein stability, as predicted, but our data suggest that Nt-acetylation does not affect GFP ubiquitination. Similarly, in case of the naturally lysine-less protein p16, Nt-acetylation correlated with protein stability, regardless of ubiquitination on its N-terminus or on an introduced lysine residue. A direct effect of Nt-acetylation on p16 stability was supported by studies in NatB-deficient cells. Together, our studies argue that Nt-acetylation can stabilize proteins in human cells in a substrate-specific manner, by competition with N-terminal ubiquitination, but also by other mechanisms that are independent of protein ubiquitination status.