Lapteva Yulia S, Vologzhannikova Alisa A, Sokolov Andrey S, Ismailov Ramis G, Uversky Vladimir N, Permyakov Sergei E
Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center "Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences", Pushchino, Moscow Region, 142290, Russia.
Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, USA.
Appl Biochem Biotechnol. 2021 May;193(5):1365-1378. doi: 10.1007/s12010-020-03324-8. Epub 2020 May 11.
Most eukaryotic proteins are N-terminally acetylated (Nt-acetylated) by specific N-terminal acetyltransferases (NATs). Although this co-/post-translational protein modification may affect different aspects of protein functioning, it is typically neglected in studies of bacterially expressed eukaryotic proteins, lacking this modification. To overcome this limitation of bacterial expression, we have probed the efficiency of recombinant Escherichia coli NATs (RimI, RimJ, and RimL) with regard to in vitro Nt-acetylation of several parvalbumins (PAs) expressed in E. coli. PA is a calcium-binding protein of vertebrates, which is sensitive to Nt-acetylation. Our analyses revealed that only metal-free PAs were prone to Nt-acetylation (up to 100%), whereas Ca binding abolished this modification, thereby indicating that Ca-induced structural stabilization of PAs impedes their Nt-acetylation. RimJ and RimL were active towards all PAs with N-terminal serine. Their activity towards PAs beginning with alanine was PA-specific, suggesting the importance of the subsequent residues. RimI showed the least activity regardless of the PA studied. Overall, NATs from E. coli are suited for post-translational Nt-acetylation of bacterially expressed eukaryotic proteins with decreased structural stability.
大多数真核生物蛋白质在N端被特定的N端乙酰转移酶（NATs）乙酰化（Nt-乙酰化）。尽管这种共翻译/翻译后蛋白质修饰可能会影响蛋白质功能的不同方面，但在缺乏这种修饰的细菌表达真核生物蛋白质的研究中，它通常被忽视。为了克服细菌表达的这一局限性，我们研究了重组大肠杆菌NATs（RimI、RimJ和RimL）对几种在大肠杆菌中表达的小白蛋白（PAs）进行体外Nt-乙酰化的效率。PA是脊椎动物的一种钙结合蛋白，对Nt-乙酰化敏感。我们的分析表明，只有无金属的PAs易于Nt-乙酰化（高达100%），而钙结合则消除了这种修饰，从而表明钙诱导的PA结构稳定阻碍了它们的Nt-乙酰化。RimJ和RimL对所有N端为丝氨酸的PAs都有活性。它们对以丙氨酸开头的PAs的活性具有PA特异性，这表明后续残基的重要性。无论研究哪种PA，RimI的活性都最低。总体而言，大肠杆菌的NATs适用于对结构稳定性降低的细菌表达真核生物蛋白质进行翻译后Nt-乙酰化。
