METHOD DEVELOPMENT FOR MEASURING RELATIVE CHANGES IN PROTEIN LEVEL

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们正在开发代谢标记方法，以测量蛋白质水平的相对变化。这项技术的初步应用将是测量线虫daf-2单突变体和daf-2；daf-16双突变体之间蛋白质水平的变化。在线虫体内，胰岛素/胰岛素样生长因子途径调节新陈代谢、生长和寿命。研究发现，胰岛素/胰岛素样生长因子样受体daf-2的微弱功能丧失突变会延长寿命。DAF-2最终作用于抑制该途径下游的DAF-16转录因子。在没有DAF-2抑制的情况下，DAF-16调节脂肪和糖原储存的增加。Daf-2突变体的寿命延长被认为是卡路里限制的结果，需要野生型DAF-16。因此，长寿的单个daf-2突变体和正常寿命的daf-16；daf-2双突变体的蛋白质水平之间的差异应该揭示了受DAF-16转录调控的蛋白质，因此可能参与衰老过程。胰岛素/胰岛素样生长因子信号通路在高等真核生物中高度保守，因此这一方法可能对研究其他生物中的胰岛素信号和糖代谢有价值。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are developing metabolic labeling methodologies for measuring relative changes in protein level. The initial application of this technology will be in the measurement of changes in protein level between the daf-2 single mutant and the daf-2; daf-16 double mutant of C. elegans. In C. elegans the insulin/IGF-like pathway regulates metabolism, growth and longevity. Weak loss-of-function mutations in daf-2, the insulin/IGF-like receptor, have been found to have an increased lifespan. DAF-2 ultimately acts to inhibit the DAF-16 transcription factor downstream in the pathway. Without DAF-2 inhibition, DAF-16 regulates the increase of fat and glycogen storage. The increased lifespan phenotype of the daf-2 mutant is thought to be a result of caloric restriction and requires wild-type DAF-16. Hence, differences between the level of proteins in the long-lived single daf-2 mutant and the normal-lived daf-16; daf-2 double mutant should reveal proteins that are transcriptionally regulated by DAF-16 and therefore may be involved in the process of aging. The insulin/IGF-like signaling pathway is highly conserved between higher eukaryotes and therefore this approach will likely be valuable for studying insulin signaling and sugar metabolism in other organisms.