Establishing a common function for ferlin proteins in membrane fusion using novel genetic code expansion and single molecule techniques.

使用新型遗传密码扩展和单分子技术建立膜融合中 ferlin 蛋白的共同功能。

• 批准号: 2019386
• 负责人: Colin Johnson
• 金额: $ 49.42万
• 依托单位: Oregon State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2019386&HistoricalAwards=false
• 关键词: Establishing; common; function; ferlin; proteins

Membrane trafficking is regarded as a crucial and distinctive characteristic of eukaryotic cells, with both intracellular transport and secretion events playing an essential role in homeostasis and signaling. While much of what is known about membrane trafficking comes from studies in yeast, there are important proteins involved in membrane trafficking in multicellular organisms not seen in yeast. One family of these proteins are the ferlins, an evolutionarily ancient family of trafficking proteins linked to an increasingly diverse list of physiological activities, including fertility, the encoding of sound, muscle development, and repair of damaged cell membranes. This diversity of physiological roles has made establishing a common underlying molecular function for this family challenging. This project will address this gap in our knowledge of the Ferlin family proteins, and in so doing add to our understanding of fertility, hearing, and muscle development. The proposed work will also develop undergraduate laboratory research training opportunities and lab classes focused on membrane biology and provide graduate students with state-of-the-art training in interdisciplinary research.The project tests the idea that ferlins share a common function as calcium sensitive scaffolds for regulated exocytosis and endocytosis. Challenges due to the large size of the proteins, their transmembrane domains, and the high number of endogenous cysteines typical of ferlins have precluded many of the traditional recombinant protein assays typically used to test this hypothetical function. To overcome these challenges a novel single molecule protein-protein interaction assay that allows for probing the contacts of large multivalent membrane proteins has been developed. In addition, genetic code expansion techniques will be exploited to incorporate environmentally sensitive fluorescent unnatural amino acids into the ferlins. These technologies will be exploited to determine the functions that underlie ferlin activity. The proposed studies will define a set of underlying molecular-level functions that unite the ferlins, despite their disparate physiological roles. This information will allow integrating ferlins into the larger picture of membrane trafficking and cell signaling. The development of a novel single molecule fluorescence technique will provide a method for the study of large multi-domain membrane proteins and thus have applications beyond the study of ferlins.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

膜运输被认为是真核细胞的一个重要且独特的特征，细胞内运输和分泌事件在稳态和信号传导中发挥着重要作用。虽然关于膜运输的大部分知识都来自对酵母的研究，但在多细胞生物中，有一些重要的蛋白质参与了膜运输，而在酵母中却没有发现。 这些蛋白质中的一个家族是ferlins，这是一个进化上古老的运输蛋白质家族，与越来越多样化的生理活动有关，包括生育能力、声音编码、肌肉发育和受损细胞膜的修复。这种生理作用的多样性使得为该家族建立共同的潜在分子功能变得具有挑战性。该项目将填补我们对 Ferlin 家族蛋白知识的空白，从而增进我们对生育力、听力和肌肉发育的理解。拟议的工作还将开发本科生实验室研究培训机会和专注于膜生物学的实验室课程，并为研究生提供最先进的跨学科研究培训。该项目测试了ferlins作为钙敏感支架具有共同功能的想法。用于调节胞吐作用和内吞作用。由于蛋白质尺寸大、跨膜结构域以及蕨类植物特有的大量内源半胱氨酸带来的挑战已经排除了许多通常用于测试这种假设功能的传统重组蛋白质测定。为了克服这些挑战，开发了一种新型单分子蛋白质-蛋白质相互作用测定法，可以探测大的多价膜蛋白的接触。 此外，将利用遗传密码扩展技术将环境敏感的荧光非天然氨基酸纳入蕨类植物中。这些技术将被用来确定弗林活性的基础功能。拟议的研究将定义一系列潜在的分子水平功能，尽管它们的生理作用不同，但这些功能将猫科动物团结在一起。这些信息将允许将 ferlins 整合到膜运输和细胞信号传导的更大范围中。新型单分子荧光技术的开发将为研究大型多结构域膜蛋白提供一种方法，从而具有超出ferlins研究范围的应用。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准。