ER Stress-sensing system and Analyses of its signal transduction mechanism.

ER压力传感系统及其信号转导机制分析。

基本信息

批准号：
14037240
负责人：
KOHNO Kenji
金额：
$ 89.15万
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 on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2006
项目状态：
已结题

项目摘要

In order to analyze the sensing mechanism of endoplasmic reticulum (ER)-stress at the molecular level, we have done systematic mutagenesis of N-terminal luminal region of ER stress sensor Ire1 in budding yeast. This result shows that the luminal region is divided into five subregions I to V and that subresions II and IV are essential for stress recognition. ER chaperone BiP associates subregion V under normal condition and the dissociation of BiP from Ire1 is required for the activation of unfolded protein response (UPR), which maintains the homeostasis of the ER under ER stress. However, dissociation of BiP from Ire1 is not sufficient for the activation of Ire1, another step is needed for full activation. Immunofluorescence and immunoelectron microscopic studies, reporter assay using various Ire1-luminal region mutants, and biochemical analysis of direct interaction between Ire1-luminal domain and unfolded proteins in vitro, these data suggested that activation of Ire1 is regulated by two steps. In the first step, BiP dissociation from Ire1 leads to its cluster formation, and in the second step, direct interaction of unfolded proteins with the core region induces the orientation of cytosolic domain of Ire, resulting in the full activation of Ire1.We also analyzed the role of novel ER transmembrane protein, DNAJB12, which belongs to Hsp40 DnaJ protein family and locates in the ER membrane. Overexpression of DNAJB12 accelerates the degradation of CFTR (Cystic fibrosis transmembrane conductance regulator) by ERAD, while the knock down of DNAJB12 increases the formation and maturation of CFTR.

为了分析分子水平上内质网（ER）压力的感应机制，我们在萌芽的酵母中进行了ER应力传感器IRE1的N端腔内腔区域的系统诱变。该结果表明，腔区域分为五个子区域I至V，该子分子II和IV对于压力识别至关重要。 ER伴侣BIP联合子区域V在正常状态下，BIP与IRE1的解离是激活展开的蛋白质反应（UPR）所必需的，该反应维持ER的稳态在ER应力下。但是，BIP与IRE1的解离不足以激活IRE1，需要另一个步骤才能充分激活。免疫荧光和免疫电子显微镜研究，使用各种IRE1-亮部分突变体的记者测定，以及在体外对IRE1-亮内结构域和展开的蛋白质之间直接相互作用的生化分析，这些数据表明，IRE1的激活通过两步来调节IRE1的激活。在第一步中，BIP与IRE1的解离会导致其簇的形成，在第二步中，展开的蛋白质与核心区域的直接相互作用会诱导IRE的细胞质体域的取向，从而完全激活IRE1。我们还分析了新型Er transmbrane prote and ssp 4的作用。膜。 DNAJB12的过表达加速了ERAD的CFTR（囊性纤维化跨膜电导调节剂）的降解，而DNAJB12的敲低会增加CFTR的形成和成熟。