Regulation of elF4E activity during oxidant stress

描述(由申请人提供)： 本申请包括为培养临床医生科学家而设计的为期5年的培训计划。首席研究员是达特茅斯医学院儿科的助理教授，对氧化剂在支气管肺发育不良发病机制中的作用感兴趣。该项目将提高候选人在分子生物学和细胞信号方面的知识，并培养作为一名独立研究人员取得成功所必需的技术技能。药理学和毒理学系副教授亚伦·巴乔夫斯基博士将担任导师。他被公认为肺和血管系统中氧化剂介导的细胞信号传递方面的权威。巴乔斯基博士培养了许多研究生和几名博士后研究员。肯塔基大学免疫学和微生物学副教授斯蒂芬·G·齐默博士将获得蛋白质合成机制方面的其他专业知识。齐默博士是翻译调控和细胞生长研究领域的领军人物。最后，由药理学和生理学领域备受尊敬的研究人员组成的咨询委员会将提供进一步的指导和职业方向。这项建议的目的是确定eIF4E活性在氧化剂暴露后调节细胞生长和蛋白质合成中的作用。初步证据表明，氧化剂减缓了肺上皮细胞的生长，并改变了关键翻译调节蛋白的磷酸化。具体目标包括：1)确定氧化剂增加eIF4E活性；2)确定增加的eIF4E活性保护细胞免受氧化剂介导的生长停滞和细胞死亡；3)确定eIF4E的激活是通过CaZ+/CaMK介导的。实验设计包括建立H202对A549细胞的生长停滞，通过分析磷酸化状态、表达、切割和结合来分析eIF4E的活性；通过细胞转化和定点突变来改变eIF4E的活性；以及确定与eIF4E活性变化相结合的激酶和磷酸酶的活性。这些研究将产生关于翻译和细胞生长相互依赖的新信息，并为药物干预减少氧化损伤提供新的基础。

DESCRIPTION (provided by applicant): This application encompasses a 5-year training program designed for the development of a clinician scientist. The principal investigator is an Assistant Professor in the Department of Pediatrics at the Dartmouth Medical School with an interest in the role of oxidants in the pathogenesis of bronchopulmonary dysplasia. This program will advance the candidate's knowledge of molecular biology and cell signaling and develop technical skills necessary for success as an independent researcher. Dr. Aaron Barchowsky, an Associate Professor in the Department of Pharmacology and Toxicology, will serve as the mentor. He is well recognized as an authority in oxidant-mediated cell signaling in the lung and vasculature. Dr. Barchowsky has trained numerous graduate level students and several post-doctoral fellows. Additional expertise in protein synthetic mechanisms will be garnered from Dr. Stephen G. Zimmer, Associate Professor of Immunology and Microbiology at the University of Kentucky. Dr. Zimmer is well known as a leader in the investigation of translational regulation and cell growth. Lastly, an advisory committee composed of highly regarded researchers in pharmacology and physiology will provide further guidance and career direction. The goal of this proposal is to establish the role of eIF4E activity in the regulation of cell growth and protein synthesis following oxidant exposure. Preliminary evidence indicates that oxidants slow pulmonary epithelial cell growth and alter the phosphorylation of key translational regulatory proteins. The specific aims entail: 1) determining that oxidants increase eIF4E activity; 2) establishing that increased eIF4E activity protects cells from oxidant-mediated growth arrest and cell death; and 3) determining that activation of eIF4E is mediated through CaZ+/CaMK. Experimental design includes establishing growth arrest in A549 cells with H202, analyzing eIF4E activity through analysis of phosphorylation state, expression, cleavage, and binding; altering eIF4E activity by cellular transformation and site-directed mutagenesis; and determining kinase and phosphatase activities integral to the changes in eIF4E activity. These studies will generate new information concerning the inter-dependence of translation and cell growth and form a novel basis for pharmacological intervention to minimize oxidative injury.