The regulation of protein synthesis by oxygen

氧对蛋白质合成的调节

• 批准号: RGPIN-2015-04807
• 负责人: Uniacke, Jim
• 金额: $ 2.84万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667380
• 关键词: regulation; protein; synthesis; oxygen

Living organisms are characterized by their ability to respond to stimuli and adapt. My research program focuses on these characteristics with regard to the protein synthesis machinery. Also known as translation, protein synthesis is a fundamental biological process in the flow of genetic information from messenger ribonucleic acid (mRNA) into protein. Stressful environmental changes lead to a refocusing of translation efforts away from the maintenance of basic cell functions and toward the production of stress response proteins. This occurs by repressing the primary translation initiation machinery, which accesses mRNAs through their cap structure located at the 5' end and accounts for >95% of translation (cap-dependent translation). My research program addresses a major question in cell biology: how do cells translate their mRNAs into proteins during periods of stress when cap-dependent translation is repressed? My focus is on hypoxia (low oxygen) in human cells because the acquisition of adequate oxygen is crucial to produce enough ATP for their proper functioning, especially in cells that constitute multicellular animals. It was believed that hypoxic cells switch from canonical cap-dependent translation (mediated by the cap-binding protein eIF4E) to cap-independent processes to translate select mRNAs during stress. I recently demonstrated (Uniacke et al. (2012) Nature) that a non-canonical cap-dependent translation pathway is required for human cells to adapt to hypoxia. This pathway utilizes the cap-binding protein homolog eIF4E2, but many unanswered questions remain. The projects outlined in this proposal aim to demonstrate that hypoxia creates a specialized cap-dependent translation response through the recruitment of specialized translation factors (project 1) and mRNAs via common regulatory sequences (project 2). These will be achieved through co-immunoprecipitation, mass spectrometry, polysome profiling, RNA sequencing, site-directed mutagenesis, and luciferase reporter assays. We will also investigate which cap-dependent translation machineries are dominant in a range of oxygen conditions that include ambient air, physiological, and hypoxia (project 3). This will be achieved by monitoring the activity of the on and off switches of these machineries through western blot and mRNA translation assays. My long-term goals are to highlight the versatility of cap-dependent translation in response to a range of cellular stresses, and to demonstrate that these specialized machineries have prominent roles when cells are cultured in physiological oxygen conditions. The identification of hypoxic eIF4E2 was covered by media such as the CBC and National Post. My research program will continue to have impacts on the natural sciences by influencing mammalian cell culture practices to include oxygen as a variable, and update current models of translational control.

活生物体的特征是它们对刺激和适应的反应能力。我的研究计划着重于蛋白质合成机制的这些特征。蛋白质合成也称为翻译，是从信使核糖核酸（mRNA）流向蛋白质的基本生物学过程。压力的环境变化导致翻译工作重新集中于基本细胞功能的维持和压力反应蛋白的产生。这是通过抑制主要翻译起始机械来发生的，该机制通过其位于5'端的帽结构访问mRNA，占翻译的> 95％（依赖CAP依赖性翻译）。我的研究计划解决了细胞生物学的一个主要问题：在压抑帽依赖性翻译时，细胞如何将其mRNA转化为蛋白质？我的重点是人类细胞中缺氧（低氧），因为获得足够的氧气对于产生足够的ATP至关重要，以使其适当的功能，尤其是在构成多细胞动物的细胞中。人们认为，缺氧细胞从规范帽依赖性翻译（由帽结合蛋白EIF4E介导）转换为盖无依赖性过程，以在应力期间翻译精选的mRNA。我最近证明（Uniacke等人（2012）性质），人类细胞适应缺氧需要非依赖帽依赖性翻译途径。该途径利用了结合蛋白质同源性EIF4E2，但仍然存在许多未解决的问题。该提案中概述的项目旨在证明缺氧通过募集专门的翻译因子（项目1）和MRNA通过共同的调节序列（项目2）创造了专门的CAP依赖性翻译响应。这些将通过共免疫沉淀，质谱法，多质谱分析，RNA测序，定点诱变和荧光素酶报告基因测定法。我们还将研究哪些依赖帽的翻译机器在包括环境空气，生理和缺氧在内的一系列氧气条件下占主导地位（项目3）。这将通过通过Western印迹和mRNA翻译测定法监测这些机器的开关开关的活动和关闭开关的活性来实现。我的长期目标是强调响应一系列细胞应力的帽依赖性翻译的多功能性，并证明当细胞在生理氧气条件下培养细胞时，这些专业的机械具有显着的作用。 CBC和National Post等媒体涵盖了低氧EIF4E2的识别。我的研究计划将通过影响哺乳动物细胞培养实践将氧气作为变量，并更新当前的转化控制模型，从而继续对自然科学产生影响。