posted on 2021-08-12, 15:37authored byPanyue Chen, Tiago Jose Paschoal Sobreira, Mark C. Hall, Tony R. Hazbun
Protein
α-N-methylation is an underexplored post-translational
modification involving the covalent addition of methyl groups to the
free α-amino group at protein N-termini. To systematically explore
the extent of α-N-terminal methylation in yeast and humans,
we reanalyzed publicly accessible proteomic datasets to identify N-terminal
peptides contributing to the α-N-terminal methylome. This repurposing
approach found evidence of α-N-methylation of established and
novel protein substrates with canonical N-terminal motifs of established
α-N-terminal methyltransferases, including human NTMT1/2 and
yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but
have unclear and context-dependent roles. Moreover, α-N-methylation
of noncanonical sequences was surprisingly prevalent, suggesting unappreciated
and cryptic methylation events. Analysis of the amino acid frequencies
of α-N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif.
We delineated the distribution of the two types of prevalent N-terminal
modifications, acetylation and methylation, on amino acids at the
first position. We tested three potentially methylated proteins and
confirmed the α-N-terminal methylation of Hsp31 by additional
proteomic analysis and immunoblotting. The other two proteins, Vma1
and Ssa3, were found to be predominantly acetylated, indicating that
proteomic searching for α-N-terminal methylation requires careful
consideration of mass spectra. This study demonstrates the feasibility
of reprocessing proteomic data for global α-N-terminal methylome
investigations.