Mechanism of Translation Elongation Factor 2 Inhibition by Bacterial Toxins

说明(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)产生的毒素均可通过抑制蛋白质合成而导致宿主细胞死亡。这两种毒素都是通过对真核细胞翻译延伸因子2(EEF2)中的特定组氨酸进行翻译后修饰而产生的一种独特的二苯二甲胺残基。最近对同样被这些毒素修饰的酿酒酵母eEF2的结构分析表明，双苯二甲胺残基位于蛋白质IV结构域的末端。这一尖端是由冷冻电子显微镜研究提出的，定位在核糖体中的mRNA附近。EEF2介导了蛋白质合成的易位步骤，新形成的多肽-tRNA从核糖体的A-位移动到P-位，mRNA移动了三个碱基。因此，该位点的ADP-核糖化可能影响该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，eEF2一直是这些毒素的唯一靶点，但其抑制机制仍不清楚。发展中国家仍在经历白喉暴发，对再次免疫和持续预防白喉杆菌的担忧正在浮现，免疫功能低下的患者中也观察到铜绿假单胞菌感染。了解毒性的机制，并潜在地利用这些信息开发药物来防止eEF2的ADP核糖基化的影响，具有重要的生物医学意义。我们最近的结构、遗传和生化研究导致了关于结构域IV和这种修饰在抑制翻译中的作用的假设。在ADP核糖化的eEF2存在下，我们表现出对白喉毒素的显性抗性，这导致了一种新的系统的发展，以探索这些毒素在体内抑制eEF2的机制。我们将检验这一假设，即ADP核糖化的eEF2会损害核糖体的易位功能，可能还会影响保真度。我们将利用位于ADP-核糖化位点附近的第四结构域的eEF2突变体和ADP-核糖化的eEF2来测试体内和体外毒素活性的后果。我们将利用酵母遗传系统的优势进行重点遗传筛选和生化分析，以确定体内抑制ADP-核糖化eEF2翻译所需的核糖体成分。利用酵母，研究白喉和相关毒素的毒性和抗性机制的新的遗传、生化和分子生物学综合方法，可能为设计在体内抵消这些毒素影响的方法提供基础。 与公共卫生相关：白喉棒状杆菌和铜绿假单胞菌分别产生白喉毒素(DT)和外毒素A(ETA)，它们的活性会导致宿主细胞死亡。这些毒素的唯一细胞靶点是必需的真核翻译延长因子2(EEF2)。在发展中国家，即使开展了疫苗接种运动，白喉仍是一个严重的健康问题。在泰国最近的一份报告中，整个20世纪90年代都报告了白喉病例，这与20-39岁的人中有25%的人和10-19岁的人中有14%的人对白喉缺乏免疫力的发现一致。20世纪90年代在俄罗斯及其邻国爆发的疫情，包括1990年至1997年在俄罗斯爆发的11.5万多例病例和3000人死亡，主要发生在成年人身上。白喉的死灰复燃表明人们更好地了解了其毒性机制，预防感染对未接种疫苗的人的影响仍然很重要。感染铜绿假单胞菌是一些患者特别担心的问题，例如癌症、囊性纤维化或免疫功能低下的患者，特别是在出现多药耐药菌株的情况下。令人惊讶的是，eEF2上独特的二苯二甲胺残基的ADP核糖基化抑制翻译从而导致细胞死亡的机制尚不清楚。我们建议利用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，开发策略来降低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.