Mechanism of Translation Elongation Factor 2 Inhibition by Bacterial Toxins

描述（由申请人提供）：白喉棒状杆菌（白喉毒素）和铜绿假单胞菌（外毒素A）产生的毒素均通过抑制蛋白质合成导致宿主细胞死亡。这两种毒素ADP核糖基化独特的二苯二甲酰胺残基，其通过真核翻译延伸因子2（eEF 2）中的特异性组氨酸的翻译后修饰产生。最近的结构分析eEF 2从酵母酿酒酵母，这也是修改这些毒素，表明的diphthamide残基位于尖端的结构域IV的蛋白质。冷冻电子显微镜研究表明，该尖端位于核糖体中mRNA附近。eEF 2介导蛋白质合成的易位步骤，其中新形成的肽基-tRNA从核糖体的A-位点移动到P-位点，并且mRNA移动三个碱基。因此，该位点的ADP-核糖基化可能影响蛋白质的这一关键功能。令人惊讶的是，尽管自20世纪70年代以来，eEF 2已被认为是这些毒素的唯一靶标，但抑制机制仍然未知。发展中国家仍在经历白喉的爆发，对重新免疫和继续预防C。白喉正在出现，并且在免疫功能低下的患者中观察到铜绿假单胞菌感染。了解毒性的机制，并可能利用这些信息来开发药物，以防止ADP-核糖基化的eEF 2的影响，具有重要的生物医学意义。我们最近的结构，遗传和生物化学的研究已经导致了关于结构域IV和这种修饰在抑制翻译中的作用的假设。我们已经证明了在ADP-核糖基化eEF 2存在下对白喉毒素的显性抗性，这导致了一种新系统的开发，以探索这些毒素在体内抑制eEF 2的机制。我们将测试的假设，ADP-核糖基化的eEF 2妥协易位功能的核糖体，也许也影响保真度。我们将利用eEF 2突变体在结构域IV，附近的ADP-核糖基化的网站，和ADP-核糖基化的eEF 2在体内和体外测试毒素活性的后果。我们将利用酵母遗传系统的优势，进行集中的遗传筛选，再加上生化分析，以确定所需的核糖体成分的翻译抑制ADP-核糖基化的eEF 2在体内。与酵母，新的综合遗传，生物化学和分子生物学的方法来研究白喉和相关毒素的毒性和耐药性的机制可能提供的基础上设计的方法，以抵消这些毒素在体内的影响。 公共卫生相关性： 白喉棒状杆菌和铜绿假单胞菌各自分别产生毒素白喉毒素（DT）和外毒素A（ETA），其活性导致宿主细胞死亡。这些毒素的唯一细胞靶标是必需的真核翻译延伸因子2（eEF 2）。在发展中国家，白喉仍然是一个重大的健康问题，即使有疫苗接种运动。在泰国最近的一份报告中，整个1990年代都报告了白喉病例，这与25%的20-39奥尔兹和14%的10-19奥尔兹缺乏白喉免疫力的调查结果一致。1990年代在俄罗斯和邻国爆发的疫情，包括1990年至1997年俄罗斯的115，000多例病例和3，000例死亡，其中大多数是成年人。白喉的复苏表明，更好地了解其毒性机制和预防感染对未免疫个体的影响仍然很重要。铜绿假单胞菌感染是一个特别关注的一个子集的患者，如那些癌症，囊性纤维化或免疫功能下降，特别是作为多药耐药菌株已经出现。令人惊讶的是，ADP核糖基化eEF 2上独特的二苯二甲酰胺残基抑制翻译并因此导致细胞死亡的机制仍然未知。我们建议利用酿酒酵母中独特的遗传系统来了解这些微生物毒素的毒性作用发生的机制，并从长远来看，制定战略，以减少与这些毒素在细胞中的活性相关的发病率和死亡率。

DESCRIPTION (provided by applicant): Toxins produced by Corynebacterium diphtheriae (diphtheria toxin) and Pseudomonas aeruginosa (exotoxin A) both result in host cell death by the inhibition of protein synthesis. Both of these toxins ADP ribosylate a unique diphthamide residue, produced by the post- translational modification of a specific histidine in the eukaryotic translation Elongation Factor 2 (eEF2). Recent structural analysis of eEF2 from the yeast Saccharomyces cerevisiae, which is also modified by these toxins, demonstrates the diphthamide residue lies on the tip of domain IV of the protein. This tip is proposed by cryo-electron microscopic studies to be positioned near the mRNA in the ribosome. eEF2 mediates the translocation step of protein synthesis, where the newly formed peptidyl-tRNA is moved from the A- to the P-site of the ribosome and the mRNA is moved by three bases. As such, ADP-ribosylation of this site likely affects this key function of the protein. Surprisingly, even though eEF2 has been known to be the sole target for these toxins since the 1970s, the mechanism of inhibition remains unknown. Developing countries still experience outbreaks of Diphtheria, concerns regarding re-immunization and continued protection against C. diphtheriae are emerging, and P. aeruginosa infections are observed in immunocompromised patients. Understanding the mechanism of toxicity, and potentially utilizing this information to develop drugs to prevent the effects of ADP-ribosylation of eEF2, has important biomedical implications. Our recent structural, genetic and biochemical studies have led to hypotheses on the role of domain IV and this modification in inhibiting translation. We have demonstrated dominant resistance to diphtheria toxin in the presence of ADP-ribosylated eEF2, which has led to the development of a novel system to explore the mechanism of inhibition of eEF2 by these toxins in vivo. We will test the hypothesis that ADP-ribosylated eEF2 compromises translocation function at the ribosome, and perhaps also affects fidelity. We will utilize eEF2 mutants in domain IV, near the site of ADP-ribosylation, and ADP-ribosylated eEF2 to test the consequences of toxin activity in vivo and in vitro. We will utilize the advantages of the yeast genetic system to perform focused genetic screens coupled with biochemical analyses to determine the ribosome components that are required for the inhibition of translation by ADP-ribosylated eEF2 in vivo. With yeast, novel integrated genetic, biochemical and molecular biological approaches to the study of the mechanism of toxicity and resistance to diphtheria and related toxins may provide the basis for the design of approaches to counteract the effects of these toxins in vivo. PUBLIC HEALTH RELEVANCE: Corynebacterium diphtheriae and Pseudomonas aeruginosa each produce toxins, diphtheria toxin (DT) and exotoxin A (ETA) respectively, whose activity results in host cell death. The only cellular target of these toxins is the essential eukaryotic translation Elongation Factor 2 (eEF2). In the developing world Diphtheria remains a significant health concern, even with vaccination campaigns. In a recent report from Thailand, diphtheria cases were reported throughout the 1990s, consistent with the findings that 25% of 20-39 year olds and 14% of 10-19 year olds lacked immunity to diphtheria. In Russia and neighboring country outbreaks in the 1990s, including more than 115,000 cases and 3,000 deaths from 1990 to 1997 in Russia, were mostly among adults. The resurgence of Diphtheria indicates a greater understanding of its mechanism of toxicity and prevention of the effects of infection on unimmunized individuals remains important. Infections with P. aeruginosa are a particular concern for a subset of patients such as those with cancer, cystic fibrosis or reduced immune function, in particular as multidrug-resistant strains have emerged. Surprisingly, the mechanism by which ADP ribosylation of a unique diphthamide residue on eEF2 inhibits translation and thus results in cell death remains unknown. We propose to utilize a unique genetic system in the yeast Saccharomyces cerevisiae to understanding the mechanism by which the toxic effects of these microbial toxins occurs, and in the long term to develop strategies to reduce the morbidity and mortality associated with the activity of these toxins in the cell.