Mechanism of Translation Elongation Factor 2 Inhibition by Bacterial Toxins

描述（由申请人提供）：白喉链杆菌（白喉毒素）和铜绿假单胞菌（外毒素A）产生的毒素都通过抑制蛋白质合成导致宿主细胞死亡。这两种毒素ADP核糖化一种独特的二苯二胺残基，这种残基是由真核翻译延伸因子2 （eEF2）中特定组氨酸的翻译后修饰产生的。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，二甲氰胺残基位于该蛋白的结构域IV的尖端。低温电镜研究建议将这个尖端定位在核糖体mRNA附近。eEF2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，mRNA移动三个碱基。因此，该位点的adp核糖基化可能会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，人们就知道eEF2是这些毒素的唯一靶点，但其抑制机制仍不清楚。发展中国家仍发生白喉疫情，对再次免疫和继续预防白喉假单胞菌的担忧正在出现，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、遗传和生化研究已经对结构域IV和这种修饰在抑制翻译中的作用提出了假设。我们已经证明，在adp -核糖化eEF2存在的情况下，白喉毒素对eEF2具有显性抗性，这导致了一个新的系统的发展，以探索这些毒素在体内抑制eEF2的机制。我们将验证adp核糖化的eEF2损害核糖体的易位功能，并且可能也影响保真度的假设。我们将利用位于adp -核糖基化位点附近的eEF2结构域IV突变体和adp -核糖基化的eEF2来测试毒素活性在体内和体外的后果。我们将利用酵母遗传系统的优势，进行集中的遗传筛选，结合生化分析，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，利用遗传、生化和分子生物学等新的综合方法来研究白喉及相关毒素的毒性和耐药性机制，可能为设计体内对抗这些毒素的方法提供基础。

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.