Identification and characterization of protein degradation substrates and their receptors

蛋白质降解底物及其受体的鉴定和表征

• 批准号: 5387935
• 负责人: Professor Dr. Claus Schwechheimer
• 金额: --
• 依托单位: Lehrstuhl für Systembiologie der Pflanzen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2002
• 资助国家: 德国
• 起止时间: 2001-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5387935?language=en
• 关键词: Identification; characterization; protein; degradation; substrates

F-box domain containing proteins (FBPs) are the substrate-receptors of E3 ubiquitin ligases and mediate the ubiquitylation of protein substrates prior to their degradation. Analysis of the Arabidopsis genome has revealed that over 500 proteins, thus 2% of the Arabidopsis proteome, contain an F-box domain. However, it is currently not possible to predict the identity of proteins and protein families that are targeted for degradation by FBPs based on sequence similarities. Here, it is proposed to generate random translational fusions between endogenous genes and the luciferase reporter in transgenic Arabidopsis plants that harbor a luciferase bearing gene trap T-DNA construct. In these lines, luciferase activity will be used as an indirect indicator of protein abundance to identify luciferase fusion proteins that are subject to protein degradation upon exposure to specific stimuli. In a complementary approach, degradation substrates that are the targets of a small FBP family will be identified using the yeast two-hybrid system these proteins will then be characterized in planta. The combined approaches are expected to result in the identification of novel protein families that are regulated by protein degradation and of conserved protein domains that confer protein instability.

含F-box结构域蛋白（F-box domain containing proteins，FBPs）是E3泛素连接酶的底物受体，在蛋白底物降解前介导蛋白底物的泛素化。对拟南芥基因组的分析表明，超过500种蛋白质，即拟南芥蛋白质组的2%，含有F-box结构域。然而，目前不可能基于序列相似性来预测被FBPs靶向降解的蛋白质和蛋白质家族的身份。在此，提出在含有荧光素酶携带基因陷阱T-DNA构建体的转基因拟南芥植物中产生内源基因和荧光素酶报告基因之间的随机翻译融合。在这些细胞系中，荧光素酶活性将用作蛋白质丰度的间接指标，以鉴定在暴露于特定刺激物时发生蛋白质降解的荧光素酶融合蛋白。在一个互补的方法中，降解底物是一个小的FBP家族的目标将被确定使用酵母双杂交系统，这些蛋白质，然后将在植物中进行表征。组合的方法，预计将导致在识别新的蛋白质家族，调节蛋白质降解和保守的蛋白质结构域，赋予蛋白质的不稳定性。