遗传 ›› 2013, Vol. 35 ›› Issue (3): 343-351.doi: 10.3724/SP.J.1005.2013.00343

• 研究报告 • 上一篇    下一篇

BiP调控IRE1a启动子转录活性及蛋白表达的效应

周菁华, 韩晓凤, 刘艳娜, 张鹏, 郭风劲   

  1. 重庆医科大学细胞生物学与遗传学教研室, 发育生物学模式动物平台, 重庆 400016
  • 收稿日期:2012-08-27 修回日期:2012-11-26 出版日期:2013-03-20 发布日期:2013-03-25
  • 通讯作者: 郭风劲 E-mail:guo.fengjin@gmail.com
  • 基金资助:

    国家自然科学基金面上项目(编号: 81171697), 重庆市高等学校优秀人才支持计划(编号:渝教人2011-65)和国家人力资源和社会保障部择优项目(编号:渝人社办[2011] 235 号)资助

The effect on BiP regulation of IRE1a promoter transcription activ-ity and protein expression

ZHOU Jing-Hua, HAN Xiao-Feng, LIU Yan-Na, ZHANG Peng, GUO Feng-Jin   

  1. Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing 400016, China
  • Received:2012-08-27 Revised:2012-11-26 Online:2013-03-20 Published:2013-03-25

摘要: 正常条件下, 外分泌或者跨膜的蛋白需要在内质网中完成正确的折叠。在营养缺乏、分化或其他应激状态下, 内质网中的蛋白质会发生错误折叠并不断积聚, 形成内质网应激(Endoplasmic reticulum stress), 引发未折叠蛋白反应(Unfolded protein response)。内质网应激条件下, 内质网腔中的分子伴侣结合免疫球蛋白BiP(Binding immunoglobulin protein)与需肌醇酶IRE1a(Inositol-requiring kinase 1)解离并与未折叠蛋白结合从而帮助蛋白形成正确结构; 同时未折叠蛋白通过与跨膜蛋白IRE1a直接结合活化其内切核糖核酸酶结构域, 活化的IRE1a能够剪切Xbp1的mRNA使其形成有活性的转录因子, 进而起始分子伴侣与未折叠蛋白反应相关蛋白的表达, 使细胞脱离内质网应激状态。文章克隆了IRE1a的启动子, 用荧光素酶报告基因法检测到BiP能够上调IRE1a的启动子活性。通过构建IRE1a启动子一系列截短体的报告基因载体, 确定了IRE1a启动子活性的核心区域; 进一步通过逆转录PCR和免疫印迹在mRNA水平和蛋白水平分别检测BiP对IRE1a启动子的调控作用。结果发现在内质网应激条件下, 分子伴侣BiP通过上调IRE1a启动子转录活性, 增加IRE1a的表达, 进而提高细胞处理内质网应激时未折叠蛋白的能力, 为揭示内质网应激条件下BiP调控IRE1a转录调控机制提供理论依据, 同时为阐明ER stress 各信号分子之间的作用机制奠定实验基础。

关键词: BiP, IRE1a, 内质网应激, 未折叠蛋白反应

Abstract: Usually, secreted or transmembrane proteins complete their three-dimension folding within endoplasmic reticulum (ER). Under the conditions of nutrient depletion, cell differentiation, or other stress statuses, misfolded or unfolded proteins aggregate within ER, and consequently cause ER stress and Unfolded Protein Response (UPR). In response to ER stress, BiP (Binding immunoglobulin protein) dissociates with IRE1a (Inositol-requiring kinase 1) and binds to unfolded proteins as a molecular chaperone in helping maintain their correct structure. Co-related to BiP’s dissociation, IRE1a oglimerizes and activated its endoribonuclease domain by transautophosphorylation. Activated IRE1a then, by cleaving mRNA of Xbp1 and activating its transcription activity, triggers UPR. In this paper, in order to determine effect of BiP on transcription activity of IRE1a, we cloned promoter region of IRE1a into reporter gene analysis vector and found that BiP could upregulate promoter activity of IRE1a. Then, we constructed another 6 truncated promoter reporter vectors of IRE1a and pinpoint the core promoter activity region. Furthermore, both our RT-PCR and Western blot results showed that BiP could upregulate mRNA transcription level and protein expression level of IRE1a. Base on these findings, we can propose that, in order to alleviate ER stress caused by the misfolded or malfolded proteins, BiP could upregulate expression of IRE1a by increase its promoter activity. This study may suggest a novel signal pathway on IRE1a regulation in ER stress.

Key words: BiP, IRE1a, ER stress, unfolded protein response

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