Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification
Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification
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DOI：
10.1111/j.1742-4658.2007.06172.x
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发表时间：
2008-01-01
期刊：
影响因子：
5.4
通讯作者：
Park, Myung Hee
中科院分区：
文献类型：
--
作者：
Cano, Veridiana S. P.;Jeon, Geoung A.;Park, Myung Hee
真核细胞翻译起始因子5A(EIF5A)是唯一一种含有亚硫氨酸[N-epsilon-(4-氨基-2-羟基丁基)赖氨酸]的蛋白质，是其活性所必需的。Hypusine是由脱氧亚硫氨酸合成酶和脱氧亚硫氨酸羟基酶对一种特定的赖氨酸(人eIF5A的Lys50)进行翻译后修饰而形成的。为了研究其活性所需的eIF5A的特征，我们在人eIF5A-1中产生了49个突变，在高度保守的残基上进行了单一氨基酸替换，或者在N端或C端截断，并测试了补充酿酒酵母eIF5A缺失菌株生长的突变蛋白。仅有几个突变型eIF5A(K47D、G49A、K50A、K50D、K50I、K50R、G52A和K55A)在亚硫氨酸修饰位点或其附近发生替换或在N端或C端截断21个氨基酸后，生长促进活性被取消。Lys50替代蛋白的失活明显是由于缺乏脱氧亚精氨酸修饰所致。相反，K47D和G49A是脱氧亚硫氨酸合成酶的有效底物，但不能支持生长，这表明Lys47和Gly49在eIF5A活性中发挥关键作用，可能是它与效应器(S)的相互作用。通过使用携带不稳定eIF5A基因工程的UBHY-R菌株，我们提出了eIF5A在蛋白质合成中主要功能的证据。当选定的eIF5A突变蛋白被测试其在蛋白质合成中的活性时，观察到它们促进蛋白质合成的能力与生长之间的密切相关，进一步支持eIF5A在翻译中的核心作用。
The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.