Structural studies of stress-responsive sensor protein kinases

未折叠蛋白在内质网管腔内的积累诱导了未折叠蛋白反应。这种反应通过上调内质网中的蛋白质折叠和降解途径以及抑制蛋白质合成来缓解应激。这种反应是由一种新的膜整合蛋白丝氨酸/苏氨酸激酶IRE介导的。IRE由腔内的传感器结构域、跨膜螺旋结构域和细胞质结构域组成。细胞质结构域在近膜区有一个连接体，其后是蛋白激酶结构域和核酸酶结构域。有趣的是，核酸酶的活性是由激酶调节的，激酶通过管腔中的传感器结构域被蛋白质的寡聚化激活。这个核酸酶结构域类似于RNase L的结构域，已知RNase L是干扰素反应途径的一个组成部分，同样，激酶-核酸酶在一个多肽链中的组织在这些蛋白质中也很常见。在本研究中，我们试图建立IREs、人IREα、IREβ和酵母Ire1p的蛋白制备，进行三维结构研究。我们制备了几种在大肠杆菌细胞和昆虫细胞中表达蛋白的构建体，并试图纯化表达蛋白。利用Ire1p细胞质结构域的构建物，我们发现了高表达和相对高效的蛋白纯化过程。我们还发现Ire1p的传感器结构域的构建提供了可以有效纯化的蛋白质。蛋白质制备的这些进步使我们能够制备用于x射线衍射研究的蛋白质晶体或测量用于结构分析和测定的核磁共振光谱。

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.