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.

为了在分子水平上分析内质网应激的传感机制，我们对芽殖酵母内质网应激传感器Ire1的N端管腔区域进行了系统诱变。该结果表明，管腔区域分为五个子区域 I 至 V，并且子区域 II 和 IV 对于应力识别至关重要。正常情况下，内质网伴侣 BiP 与 V 亚区相关，并且 BiP 从 Ire1 解离是激活未折叠蛋白反应 (UPR) 所必需的，从而在 ER 应激下维持 ER 的稳态。然而，BiP 与 Ire1 的解离不足以激活 Ire1，需要另一个步骤才能完全激活。免疫荧光和免疫电镜研究、使用各种 Ire1-luminal 区域突变体的报告基因测定、以及体外 Ire1-luminal 结构域和未折叠蛋白之间直接相互作用的生化分析，这些数据表明 Ire1 的激活是通过两个步骤调节的。第一步，BiP从Ire1解离导致其簇形成，第二步，未折叠蛋白与核心区域的直接相互作用诱导Ire胞质结构域的方向，导致Ire1的完全激活。我们还分析了新型 ER 跨膜蛋白 DNAJB12 的作用，DNAJB12 属于 Hsp40 DnaJ 蛋白家族，位于 ER 膜上。 DNAJB12的过表达会加速ERAD对CFTR（囊性纤维化跨膜电导调节因子）的降解，而DNAJB12的敲除会增加CFTR的形成和成熟。