Inhibition of protein synthesis by didemnin B:: How EF-1α mediates inhibition of translocation
Inhibition of protein synthesis by didemnin B:: How EF-1α mediates inhibition of translocation
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DOI：
10.1021/bi992202h
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发表时间：
2000-04-18
期刊：
影响因子：
2.9
通讯作者：
Toogood, PL
中科院分区：
文献类型：
--
作者：
Ahuja, D;Vera, MD;Toogood, PL
抗肿瘤环状脱脂肽Db(Didemnin B)抑制细胞内和体外蛋白质合成。DB抑制细胞内蛋白质合成的阶段尚不清楚，尽管脱氢二氢异黄素B在多肽伸长阶段阻止了翻译。DB在体外对蛋白质合成的抑制也发生在伸长阶段，以前的研究表明，在蛋白质合成的部分反应模型中，DB阻止了EF-2依赖的易位。这种对易位的抑制显示了对EF-1α的绝对要求；然而，对EF-1α的依赖之前还没有解释。结果表明，DB在溶液中只与EF-1α/GTP弱结合，但与核糖体结合。EF-1α络合物的解离常数K-d=4mU·Fhus，DB对蛋白质合成的抑制似乎涉及到与EF-1α和核糖体的相互作用，这三种成分都是必需的。利用白喉毒素介导的ADP核糖化来检测EF-2，证明了DB阻断EF-2与转位前核糖体EF-1α复合体的结合，从而防止核糖体转位，基于DB抑制蛋白质合成的这个模型和夫西地酸的作用机制，证据支持了关于EF-1α功能的Grasmuk模型，在该模型中，该延长因子在多肽延伸过程中不会完全离开核糖体。
The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1 alpha; however, the dependence upon EF-1 alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1 alpha/GTP in solution, but binds to ribosome.EF-1 alpha complexes with a dissociation constant K-d = 4 mu M.'Fhus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1 alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome EF-1 alpha complexes, thus preventing ribosomal translocation, Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1 alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.