Analyses on the novel functions of the COP9 signalosome, a master regulator responding to environm ental stimuli

响应环境刺激的主调节器 COP9 信号体的新功能分析

基本信息

批准号：
18570041
负责人：
TSUGE Tomohiko
金额：
$ 2.63万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

We have previously reported that COP9 signalosome (CSN) regulates signal transduction by mediating proteolysis in the nuclei. We have also identified novel CSN1 function that does not involve proteolysis in its regulation. Here, we focused on identifying proteins that directly bind to the functional portion of the CSN1 protein, using the Arabidopsis system to investigate the novel regulation of CSN1. The findings are summarized below.1. The putative CSN1-binding protein, SAP130 (spliceosome associated protein 130) was identified in a systematic pull-down using the functional domain of CSN1 as bait. In Arabidopsis, SAP130 was coded by two closely mapped genes, and its mRNA was detected in all of the tested organs. Plants harboring T-DNA insertions in the promoter or coding region of these genes did not display detectable changes. In attempt to analyze the loss-of-function and gain-of-function of these genes, we constructed transgenic plants that knock-down and over-express the two genes … More , by over-expression and by RNAi methods, together with an inducible system. Further analyses are in progress.2. SAP130 was shown to directly bind CSN1 in both mammalian and plant systems. This binding was mediated through the N-terminal half of CSN1. Plants expressing mutations in this binding-interface have been created to reveal the endogenous nature of this binding. Furthermore we revealed that, SAP130 is homologous to DDB1, directly binds cullin subunit of Cullin-RING ubiquitin E3 ligases, and possess polyubiquitinating activity.3. Since SAP130 has been identified in multiple complexes such as SF3b, STAGA, and TFTC, we tagged CSN1 and SAP130 with various fluorescence tags to investigate the localization patterns in detail, in combination with external stimuli. The proteins were dominantly detected in the nucleus and further analyses is in progress. These results have been reported through international and domestic conferences and through high-profiled international journal publications. Less

我们之前报道过 COP9 信号体 (CSN) 通过介导细胞核中的蛋白水解来调节信号转导。我们还鉴定了不涉及蛋白水解的新功能。在此，我们重点鉴定直接与功能部分结合的蛋白质。拟南芥系统研究了CSN1蛋白的调控机制，结果总结如下： 1．使用 CSN1 的功能域作为诱饵，在系统下拉中鉴定出 SAP130（剪接体相关蛋白 130）。在拟南芥中，SAP130 由两个紧密定位的基因编码，并且在所有含有 T 的植物器官中都检测到了它的 mRNA。 -这些基因的启动子或编码区中的DNA插入没有显示出可检测到的变化。为了分析这些基因的功能丧失和功能获得，我们构建了能够敲低和获得功能的转基因植物。通过过度表达和 RNAi 方法以及诱导系统，SAP130 被证明可以在哺乳动物和植物系统中直接结合 CSN1。 SAP130 与 DDB1 是同源的，可以直接结合。 Cullin-RING泛素E3连接酶的cullin亚基，并具有多泛素化活性。3.由于SAP130已在SF3b、STAGA和TFTC等多种复合物中被鉴定，因此我们用各种荧光标签标记CSN1和SAP130以详细研究其定位模式。，结合外部刺激，这些蛋白质主要在细胞核中被检测到，并且进一步的分析正在进行中，这些结果已通过国际和国内会议报告。以及通过备受瞩目的国际期刊出版物。