Abstract
Arrest defective 1 (ARD1), also known as N(alpha)-acetyltransferase 10 (NAA10) was originally identified as an N-terminal acetyltransferase (NAT) that catalyzes the acetylation of N-termini of newly synthesized peptides. After that, mammalian ARD1/NAA10 expanded its’ role to lysine acetyltransferase (KAT) that post-translationally acetylates internal lysine residues of proteins. ARD1/NAA10 is the only enzyme with both NAT and KAT activities. However, recent studies on the role of human ARD1/NAA10 (hARD1/NAA10) in lysine acetylation are contradictory, as crystal structure and in vitro acetylation assay results revealed the lack of KAT activity. Thus, the role of hARD1/NAA10 in lysine acetylation is still debating. Here, we found a clue that possibly explains these complicated and controversial results on KAT activity of hARD1/NAA10. Recombinant hARD1/NAA10 exhibited KAT activity, which disappeared soon in vitro. Size-exclusion analysis revealed that most recombinant hARD1/NAA10 formed oligomers over time, resulting in the loss of KAT activity. While oligomeric recombinant hARD1/NAA10 lost its ability for lysine acetylation, its monomeric form clearly exhibited lysine acetylation activity in vitro. We also characterized the KAT activity of hARD1/NAA10 that was influenced by several experimental conditions, including concentration of reactants and reaction time. Taken together, our study proves that recombinant hARD1/NAA10 exhibits KAT activity in vitro but only under accurate conditions, including reactant concentrations and reaction duration.
Highlights
Post-translational modifications (PTMs) refer to the chemical alterations in proteins following syntheses to regulate their activities and cellular functions
We described the autoacetylation of rhARD1/N(alpha)-acetyltransferase 10 (NAA10) in vitro [21]
We hypothesized that the lysine acetylation of rhARD1/NAA10 may be unstable and disappear during purification
Summary
Post-translational modifications (PTMs) refer to the chemical alterations in proteins following syntheses to regulate their activities and cellular functions. PTMs characterized with the transfer of an acetyl group from acetyl-coenzyme A to either the N-terminal. Molecules 2020, 25, 588 amino acid or a lysine residue of protein, and is catalyzed with N-terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs), respectively. N-terminal acetylation catalyzed by NATs is one of the most common protein modifications in eukaryotes, affecting about 80% human proteins and 60% yeast proteins [1,2]. N-terminal residues of newly synthesized proteins from ribosomes in an irreversible manner. In contrast to N-terminal acetylation, lysine acetylation catalyzed by KATs is reversibly regulated by lysine deacetyltransferases (KDACs) that remove acetyl groups from lysine residues in proteins [3]
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