The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.
The amidation step of diphthamide biosynthesis in yeast requires DPH6, a gene identified through mining the DPH1-DPH5 interaction network.
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DOI：
10.1371/journal.pgen.1003334
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Schaffrath R
中科院分区：
文献类型：
--
作者：
Uthman S;Bär C;Scheidt V;Liu S;ten Have S;Giorgini F;Stark MJ;Schaffrath R
白喉酰胺是真核翻译延伸因子2（eEF 2）中高度修饰的组氨酸残基，是白喉毒素（DT）不可逆ADP核糖基化的靶点。在酿酒酵母（Saccharomyces cerevisiae）中，二苯二甲酰胺生物合成的初始步骤得到了很好的表征，并且需要DPH 1-DPH 5基因。然而，最后的途径步骤-中间体白喉酰胺化为白喉酰胺-是不明确的。在这里，我们挖掘DPH 1-DPH 5的遗传相互作用景观，以确定一个候选基因的难以捉摸的酰胺酶（YLR 143 w/DPH 6），并确认参与第二个基因（YBR 246 w/DPH 7）在酰胺化步骤。与dph 1-dph 5一样，dph 6和dph 7突变体保持eEF 2形式，逃避DT和sordarin（一种依赖于二苯二甲酰胺的抗真菌剂）的抑制。此外，质谱显示dph 6和dph 7突变体特异性地积累白喉修饰的eEF 2，证明未能完成最终的酰胺化步骤。与白喉酰胺化中预期的ATP需求一致，Dph 6含有必需的腺嘌呤核苷酸水解酶结构域并与eEF 2结合。因此，Dph 6是难以捉摸的酰胺酶的候选者，而Dph 7显然将白喉合酶（Dph 5）与白喉酰胺化偶联。后一个结论是基于我们的观察，dph 7突变体显示Dph 5和eEF 2之间的相互作用急剧上调，表明它们的关联被Dph 7控制。在生理上，最佳的翻译准确性和细胞生长需要完成二苯二甲酰胺的合成，如dph突变体之间的共同特征所示，包括增加核糖体-1移码和改变对翻译抑制剂的反应。通过鉴定Dph 6和Dph 7作为联苯二甲酰胺途径的酰胺化步骤所需的组分，我们的工作为详细了解联苯二甲酰胺形成的机理铺平了道路。Diphthamide是一种不寻常的修饰氨基酸，仅存在于单一蛋白质eEF 2中，是细胞合成新蛋白质所必需的。该名称指的是其通过白喉毒素（由病原体白喉棒杆菌产生的疾病诱导剂）灭活eEF 2的靶向功能。为什么细胞需要eEF 2含有联苯酰胺尚不清楚，尽管小鼠无法使其无法完成胚胎发育。细胞通过使用三步生物合成途径修饰eEF 2中的特定组氨酸残基来产生二苯二甲酰胺，其中前两步是明确定义的。然而，参与最终酰胺化步骤的酶是未知的。在这里，我们整合基因组和分子的方法，以确定一个候选人的难以捉摸的酰胺酶（Dph 6），并确认参与的第二个蛋白质（Dph 7）在酰胺化步骤，表明未能合成diphthamide影响蛋白质合成的准确性。然而，与Dph 6相反，Dph 7可能是调节性的。我们的数据强烈表明，它促进解离eEF 2从白喉合酶（Dph 5），进行第二步的二苯二甲酰胺合成，和Dph 5有一个新的作用，作为eEF 2抑制剂时，二苯二甲酰胺合成不完全。
Diphthamide is a highly modified histidine residue in eukaryal translation elongation factor 2 (eEF2) that is the target for irreversible ADP ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step—amidation of the intermediate diphthine to diphthamide—is ill-defined. Here we mine the genetic interaction landscapes of DPH1-DPH5 to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants maintain eEF2 forms that evade inhibition by DT and sordarin, a diphthamide-dependent antifungal. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Consistent with an expected requirement for ATP in diphthine amidation, Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2. Dph6 is therefore a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal −1 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, our work paves the way for a detailed mechanistic understanding of diphthamide formation. Diphthamide is an unusual modified amino acid found uniquely in a single protein, eEF2, which is required for cells to synthesize new proteins. The name refers to its target function for eEF2 inactivation by diphtheria toxin, the disease-inducing agent produced by the pathogen Corynebacterium diphtheriae. Why cells require eEF2 to contain diphthamide is unclear, although mice unable to make it fail to complete embryogenesis. Cells generate diphthamide by modifying a specific histidine residue in eEF2 using a three-step biosynthetic pathway, the first two steps of which are well defined. However, the enzyme(s) involved in the final amidation step are unknown. Here we integrate genomic and molecular approaches to identify a candidate for the elusive amidase (Dph6) and confirm involvement of a second protein (Dph7) in the amidation step, showing that failure to synthesize diphthamide affects the accuracy of protein synthesis. In contrast to Dph6, however, Dph7 may be regulatory. Our data strongly suggest that it promotes dissociation of eEF2 from diphthine synthase (Dph5), which carries out the second step of diphthamide synthesis, and that Dph5 has a novel role as an eEF2 inhibitor when diphthamide synthesis is incomplete.
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影响因子：
56.9
作者：
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作者：
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通讯作者：
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