COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis
COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis
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DOI：
10.1210/me.2015-1012
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发表时间：
2015-08-01
影响因子：
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通讯作者：
Chen, Xuequn
中科院分区：
文献类型：
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作者：
Fang, Jingye;Liu, Ming;Chen, Xuequn
胰腺β细胞在内质网（ER）中具有高度活性的蛋白质合成和输出机制，以适应胰岛素原的大量产生。ER稳态对于β细胞功能至关重要，并且通过蛋白质合成、折叠、输出和降解之间的微妙平衡来维持。引起糖尿病的因素对ER稳态的破坏导致β细胞死亡。在维持β细胞ER稳态的4种成分中，ER输出在胰岛素生物合成中的作用是最不清楚的。为了解决这一知识缺口，本研究调查了胰岛素原ER输出在MIN 6细胞和原代胰岛的分子机制。分泌相关RAS相关蛋白（Sar）1小GT3的两种抑制性突变体，已知特异性阻断外壳蛋白复合物II（COPII）依赖性ER输出，使用重组腺病毒在β细胞中过表达。这种方法的结果，以及小干扰RNA介导的Sar 1敲低，表明缺陷的Sar 1功能阻断胰岛素原ER的输出，并取消其转化为成熟的胰岛素在MIN 6细胞，分离的小鼠和人胰岛。它进一步揭示，使用在体外囊泡形成试验，胰岛素原包装成COPII囊泡在GTP和Sar 1依赖性的方式。通过Sar 1突变体阻断COPII依赖的ER出口强烈诱导ER形态学改变、ER应激反应和β细胞凋亡。这些反应由PKR（双链RNA依赖性激酶）样ER激酶（PERK）/真核翻译起始因子2 α（p-eIF 2 α）和肌醇需要蛋白1（IRE 1）/x盒结合蛋白1（Xbp 1）途径介导，但不通过激活转录因子6（ATF 6）介导。总的来说，研究结果表明，COPII依赖的ER输出在胰岛素生物合成，ER稳态和β细胞存活中起着至关重要的作用。
Pancreatic beta-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for beta-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to beta-cell death. Among the 4 components to maintain ER homeostasis in beta-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar) 1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in beta-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP-and Sar1-dependent manner. Blockage of COPIIdependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and beta-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/ eukaryotic translation initiation factor 2 alpha (p-eIF2 alpha) and inositol- requiring protein 1 (IRE1)/ x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and beta-cell survival.