Department of Biochemistry, Keimyung University School of Medicine, Daegu 42601, Korea.
Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
Molecules. 2020 Jan 29;25(3):588. doi: 10.3390/molecules25030588.
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.
ARD1（也称为 NAA10）最初被鉴定为一种 N 端乙酰转移酶（NAT），可催化新合成肽的 N 端乙酰化。此后，哺乳动物 ARD1/NAA10 的作用扩展到赖氨酸乙酰转移酶（KAT），该酶可对蛋白质内部赖氨酸残基进行翻译后乙酰化。ARD1/NAA10 是唯一具有 NAT 和 KAT 活性的酶。然而，最近关于人 ARD1/NAA10（hARD1/NAA10）在赖氨酸乙酰化中作用的研究结果相互矛盾，因为晶体结构和体外乙酰化测定结果表明其缺乏 KAT 活性。因此，hARD1/NAA10 在赖氨酸乙酰化中的作用仍存在争议。在这里，我们发现了一个可能解释 hARD1/NAA10 的 KAT 活性的复杂和有争议的结果的线索。重组 hARD1/NAA10 表现出 KAT 活性，但很快在体外消失。凝胶过滤分析显示，大多数重组 hARD1/NAA10 随时间形成寡聚体，导致 KAT 活性丧失。虽然寡聚重组 hARD1/NAA10 失去了赖氨酸乙酰化的能力，但它的单体形式在体外明显表现出赖氨酸乙酰化活性。我们还研究了 hARD1/NAA10 的 KAT 活性，其受到几种实验条件的影响，包括反应物浓度和反应时间。总之，我们的研究证明重组 hARD1/NAA10 在体外具有 KAT 活性，但仅在反应物浓度和反应持续时间等准确条件下才具有活性。
