N-terminal acetylation can stabilize proteins independent of their ubiquitination

Abstract

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.

Tumor Pigmentation Does Not Affect Light-Activated Belzupacap Sarotalocan Treatment but Influences Macrophage Polarization in a Murine Melanoma Model

“…Cells were cultured in a T75 flask and when the confluence reached 75%, the proteins were isolated and analyzed by mass spectrometry, as described. 31 Then, the raw data were analyzed by proteome discoverer version 2.2 (Thermo Electron). The presence of tyrosinase and other melanocyte differentiation antigens (microphthalmia-associated transcription factor [MITF], tyrosinase-related protein 1 [TYRP-1], tyrosinase-related protein 2 [TYRP-2], glycoprotein 100 [gp-100], and Melan-A) was determined by proteomics analysis.…”