Chemical Approaches for Quantification and Visualization of the Prenylome

有了这个奖项，化学部门的生命过程化学项目正在资助明尼苏达大学的Mark Distefano博士开发量化和成像prenylome的方法。戊烯酰化的总体水平将通过使用含炔或叠氮的底物类似物进行代谢标记，然后使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preylated蛋白（如K-Ras）的定位，含炔或叠氮化物的底物类似物将被代谢加入，然后用荧光探针点击反应，通过共聚焦显微镜观察。我们还将研究基于fret的方法和涉及邻近诱导扩增的策略，以提高可视化的灵敏度和选择性。第一个目标的结果将提高对戊烯酰化水平差异与各种药物治疗和疾病状态之间的关系的理解。第二个目标的结果将有助于阐明如何通过其亚细胞定位的变化来调节烯酰化蛋白的活性。蛋白质前酰化涉及到脂质基团附着在蛋白质上；人类细胞中存在数百种这样的蛋白质。preylated蛋白参与调节细胞行为的许多特征，包括生长和通讯，许多疾病需要这些蛋白的参与。这项建议的重点是开发新的方法来监测preylated蛋白的水平，并跟踪它们在细胞内的位置。如果能够实现这一目标，抗击从癌症到衰老等一系列疾病的新方法可能会出现。该项目还将作为一种工具，通过让他们使用这里开发的方法来研究各种细胞类型和模式生物中的蛋白质戊烯酰化，从而引入一组本科生进行研究。

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.