Structural studies of stress-responsive sensor protein kinases

应激反应传感器蛋白激酶的结构研究

基本信息

批准号：
15370046
负责人：
HAKOSHIMA Toshio
金额：
$ 9.86万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15370046/
关键词：
stress protein separation purification IRE TRAF IRF

项目摘要

The accumulation of unfolded proteins in the lumen of the endoplasmic reticulum induces an unfolded protein response. The response alleviates stress by upregulating protein folding and degradation pathways in the endoplasmic reticulum and inhibiting protein synthesis. This response is mediated by a novel membrane-integrate protein serine/threonine kinase, IRE. IRE consists of a sensor domain in the lumen, a transmembrane helix region, and a cytoplasmic domain. The cytoplasmic domain has a linker at the juxtamembrane region, which is followed by protein kinase domain and nuclease domain. Interestingly, the nuclease activity was regulated by the kinase that is activated by the oligomerization of the protein through the sensor domain in the lumen. This nuclease domain resembles to that of RNase L, which is known to be a component of the interferon response pathway, and, again, the organization of the kinase-nuclease in one polypeptide chain is also common in these proteins. In this study, we tried to establish the protein preparation of IREs, human IREα,IREβ and yeast Ire1p, for the three-dimensional structural studies. We produced several kinds of constructs for protein expression in both E.coli cells and insect cells and tried to purify the expressed proteins. We found high protein expression and relatively efficient purification procedure of a protein using a construct of the cytoplasmic domain from Ire1p. We also found that constructs of the sensor domain of Ire1p provide proteins that can be efficiently purified. These advances in protein preparation enable us to prepare protein crystals for X-ray diffraction studies or to measure NMR spectra for structural analyses and determination.

内质网腔中展开的蛋白质的积累会诱导展开的蛋白质反应。该反应通过上调内质网中的蛋白质折叠和降解途径来减轻压力，并抑制蛋白质合成。这种反应是由一种新型的膜融合蛋白丝氨酸/苏氨酸激酶IRE介导的。 IRE由管腔，跨膜螺旋区和细胞质结构域中的传感器结构域组成。细胞质结构域在近去膜区域具有接头，其后是蛋白激酶结构域和核酸酶结构域。有趣的是，核酸酶活性受激酶调节，该激酶通过蛋白质通过管腔中的传感器结构域的寡聚而激活。该核酸酶结构域类似于RNase L的结构，该结构域已知是干扰素反应途径的组成部分，并且在一个多肽链中的激酶核酸酶的组织在这些蛋白中也很常见。在这项研究中，我们试图为三维结构研究建立IRE，人IREα，IREβ和酵母IRE1P的蛋白质制备。我们在大肠杆菌细胞和昆虫细胞中生产了几种用于蛋白质表达的构建体，并试图纯化表达的蛋白质。我们发现使用IRE1P的细胞质结构域的构建体发现了蛋白质的高蛋白表达和相对有效的纯化程序。我们还发现，IRE1P传感器结构域的构建体提供可以有效纯化的蛋白质。这些蛋白质制备方面的进展使我们能够为X射线衍射研究制备蛋白质晶体或测量NMR光谱以进行结构分析和测定。