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The targetedproteomics for membrane protein and nuclear protein

膜蛋白和核蛋白的靶向蛋白质组学

基本信息

批准号：
18310142
负责人：
HAMAKUBO Takao
金额：
$ 10.85万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
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

Transcription factors or membrane receptor proteins (e. g., the G protein-coupled receptor [GPCR]) are considered to precisely regulate physiological processes by forming complexes with a number of proteins. As the amounts of these proteins, which are important for cellular responses to stimuli, are very small, and purification is difficult, a dynamic analysis of their functions in vivo has not yet been undertaken. In this study, we have developed a method for the functional analysis of membrane proteins expressed and reconstituted on the baculoviruses (Sakihama T., J Biotechnol, 2008). Furthermore, we have developed a method to analyze the dynamism of protein-protein interactions for the endogenous proteins, by fixing the high affinity monoclonal antibodies obtained using the protein-expressing baculovirus (Saitoh R., et. al., JIM, 2007) to low noise magnetic beads, and performing a mass spectrometric analysis.In other veins, a novel regulation pathway mediated by angiotensin was foun … More d by utilizing monoclonal antibodies of angiotensin type II receptor (AT2) and angiotensin receptor-interacting proteins (ATIPs), prepared using baculovirus. We also found that the WTAP (WT1 associated protein) is involved in the stability of cyclin A2 mRNA thereby regulates the cell cycle. We have reported in the meetings that it is possible to detect endogenous HNF4alpha, a nuclear receptor which is important for liver and pancreas formation, from one 10 cm dish cell sample, and to identify over one hundred associating proteins for HNF4alpha, by establishing a method for affinity purification mass spectrometry using magnetic beads and high-affinity antibodies. The sensitive identification of very small amounts of endogenous proteins makes it possible to analyze the physiological regulation dynamics. The method described above will be a powerful tool for protein modification or protein-protein interaction assay for proteins involved in the pathology of various diseases and pharmacologic actions. Less

转录因子或膜受体蛋白（e.例如，在一个实施例中，G蛋白偶联受体[GPCR]）被认为通过与许多蛋白质形成复合物来精确调节生理过程。由于这些蛋白质对细胞对刺激的反应很重要，它们的量非常小，并且纯化很困难，因此尚未对其在体内的功能进行动态分析。在这项研究中，我们开发了一种用于杆状病毒上表达和重构的膜蛋白的功能分析方法（Sakihama T.，J Biotechnol，2008）。此外，我们已经开发了一种分析内源性蛋白质的蛋白质-蛋白质相互作用的动力学的方法，通过固定使用表达蛋白质的杆状病毒获得的高亲和力单克隆抗体（Saitoh R.，et.例如，JIM，2007）和低噪声磁珠，并进行质谱分析。在其他静脉中，发现了一种由血管紧张素介导的新的调节途径。 ...更多信息 d通过利用利用杆状病毒制备的血管紧张素II型受体（AT 2）和血管紧张素受体相互作用蛋白（ATIP）的单克隆抗体。我们还发现WTAP（WT 1 associated protein，WT 1相关蛋白）参与了cyclin A2 mRNA的稳定性，从而调控细胞周期。我们在会议上报告说，通过建立使用磁珠和高亲和力抗体的亲和纯化质谱法，可以从一个10 cm培养皿细胞样品中检测内源性HNF 4 α，一种对肝脏和胰腺形成很重要的核受体，并鉴定出100多种HNF 4 α相关蛋白。非常少量的内源性蛋白质的灵敏鉴定使得分析生理调节动力学成为可能。上述方法将是用于蛋白质修饰或蛋白质-蛋白质相互作用测定的有力工具，所述蛋白质涉及各种疾病的病理学和药理学作用。少