Chemical Approaches for Quantification and Visualization of the Prenylome

异戊烯体定量和可视化的化学方法

• 批准号: 1308655
• 负责人: Mark Distefano
• 金额: $ 36万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1308655&HistoricalAwards=false
• 关键词: Chemical; Approaches; Quantification; Visualization; Prenylome

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Mark Distefano from the University of Minnesota to develop methods to quantify and image the prenylome. Overall levels of prenylation will be measured by metabolic labeling with alkyne- or azide-containing substrate analogues followed by click reaction with a fluorescent reagent and subsequent quantification via flow cytometry. To selectively observe the localization of prenylated proteins such as K-Ras, alkyne- or azide-containing substrate analogues will be metabolically incorporated followed by click reaction with fluorescent probes and visualization via confocal microscopy. FRET-based methods and a strategy involving proximity-induced amplification will also be investigated to increase visualization sensitivity and selectivity. Results from the first objective will improve the understanding of how differences in prenylation levels correlate with various drug treatments and disease states. Results from the second objective will help clarify how the activities of prenylated proteins are regulated via changes in their subcellular localization.Protein prenylation involves the attachment of a lipid group to a protein; several hundred of such proteins are present in human cells. Prenylated proteins are involved in regulating many features of cellular behavior including growth and communication and a number of diseases require the participation of such proteins. The focus of this proposal is to develop new methods to monitor the levels of prenylated proteins and track their location within cells. If this can be accomplished, new approaches to combat an array of diseases ranging from cancer to aging may emerge. This project will also be used as a vehicle to introduce a group of undergraduates to research by having them use the methods developed here to investigate protein prenylation in a wide variety of cell types and model organisms.

有了这个奖项，化学部的生命过程化学计划正在资助明尼苏达大学的Mark Distefano博士开发方法来量化和成像prenylome。 异戊烯化的总体水平将通过用含炔或叠氮化物的底物类似物进行代谢标记，然后用荧光试剂进行点击反应，随后通过流式细胞术进行定量来测量。 为了选择性地观察异戊烯化的蛋白质如K-Ras的定位，将代谢掺入含炔或叠氮化物的底物类似物，然后与荧光探针进行点击反应并通过共聚焦显微镜进行可视化。 基于FRET的方法和涉及邻近诱导扩增的策略也将被研究，以增加可视化的灵敏度和选择性。 从第一个目标的结果将提高异戊烯化水平的差异如何与各种药物治疗和疾病状态相关的理解。 第二个目标的结果将有助于阐明异戊二烯化蛋白质的活性是如何通过其亚细胞定位的变化来调节的。蛋白质异戊二烯化涉及脂质基团与蛋白质的连接;数百种这样的蛋白质存在于人类细胞中。 异戊烯化蛋白参与调节细胞行为的许多特征，包括生长和通讯，并且许多疾病需要此类蛋白的参与。 这项提案的重点是开发新的方法来监测异戊二烯化蛋白质的水平，并跟踪它们在细胞内的位置。 如果能够实现这一点，那么可能会出现对抗从癌症到衰老等一系列疾病的新方法。 这个项目也将被用来作为一个工具，介绍一组本科生的研究，让他们使用这里开发的方法来研究蛋白质异戊二烯化在各种各样的细胞类型和模式生物。