Protein lifespan is regulated by co-translational modification by several enzymes, including methionine aminopeptidases and N-alpha-aminoterminal acetyltransferases. The NatB enzymatic complex is an N-terminal acetyltransferase constituted by two subunits, NAA20 and NAA25, whose interaction is necessary to avoid NAA20 catalytic subunit degradation. We found that deletion of the first five amino acids of hNAA20 or fusion of a peptide to its amino terminal end abolishes its interaction with hNAA25. Substitution of the second residue of hNAA20 with amino acids with small, uncharged side-chains allows NatB enzymatic complex formation. However, replacement by residues with large or charged side-chains interferes with its hNAA25 interaction, limiting functional NatB complex formation. Comparison of NAA20 eukaryotic sequences showed that the residue following the initial methionine, an amino acid with a small uncharged side-chain, has been evolutionarily conserved. We have confirmed the relevance of second amino acid characteristics of NAA20 in NatB enzymatic complex formation in Drosophila melanogaster. Moreover, we have evidenced the significance of NAA20 second residue in Saccharomyces cerevisiae using different NAA20 versions to reconstitute NatB formation in a yNAA20-KO yeast strain. The requirement in humans and in fruit flies of an amino acid with a small uncharged side-chain following the initial methionine of NAA20 suggests that methionine aminopeptidase action may be necessary for the NAA20 and NAA25 interaction. We showed that inhibition of MetAP2 expression blocked hNatB enzymatic complex formation by retaining the initial methionine of NAA20. Therefore, NatB-mediated protein N-terminal acetylation is dependent on methionine aminopeptidase, providing a regulatory mechanism for protein N-terminal maturation.

蛋白质的寿命受到几种酶（包括蛋氨酸氨基肽酶和N-α-氨基末端乙酰基转移酶）的共翻译修饰的调节。NatB酶复合物是由两个亚基NAA20和NAA25构成的N末端乙酰基转移酶，它们的相互作用对于避免NAA20催化亚基降解是必不可少的。我们发现，删除hNAA20的前五个氨基酸或将肽融合到其氨基末端消除了其与hNAA25的相互作用。hNAA20的第二个残基被具有小的，不带电荷的侧链的氨基酸取代，可以形成NatB酶复合物。然而，被具有大的或带电的侧链的残基取代会干扰其hNAA25相互作用，从而限制功能性NatB复合物的形成。比较NAA20真核序列，可以发现，最初的蛋氨酸（一种带有少量不带电荷侧链的氨基酸）之后的残基在进化上是保守的。我们已经证实了NAA20的第二个氨基酸特性与大肠杆菌NatB酶复合物的形成有关。果蝇。此外，我们已经证明了使用不同的NAA20版本重构yNAA20-KO酵母菌株中NatB形成的酿酒酵母中NAA20第二残基的重要性。在人类和果蝇中，最初的NAA20甲硫氨酸后需要一条不带电荷的小侧链氨基酸，这表明甲硫氨酸氨肽酶的作用对于NAA20和NAA25的相互作用可能是必需的。我们表明抑制MetAP2表达通过保留NAA20的初始蛋氨酸来阻止hNatB酶复合物的形成。因此，NatB介导的蛋白质N末端乙酰化依赖于蛋氨酸氨基肽酶，为蛋白质N末端成熟提供了调节机制。