PDF(Pubmed)
Abstract：
Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we\'ve challenged β-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.
摘要：
突变胰岛素原折叠的原发性缺陷可导致内质网（ER）中显性阴性胰岛素原积累，顺行胰岛素原贩运受损，扰动的ER稳态，胰岛素生产减少，和β细胞功能障碍。相反，如果ER到高尔基体运输的原发性损害（也扰乱ER稳态）驱动非突变胰岛素原的错误折叠-这可能表明双向进入常见的病理表型（胰岛素原错误折叠，扰动的ER稳态，和胰岛素原的内质网输出不足），最终导致胰岛素储存减少和糖尿病。这里,我们已经用损害ER到高尔基运输的条件挑战β细胞，并设计了一种准确的方法，使用新型非还原SDS-PAGE/免疫印迹方案评估胰岛素原的不同折叠/错误折叠形式的相对丰度。我们证实了在引入糖尿病性INS突变时大量的胰岛素原错误折叠，或在db/db小鼠的胰岛中。而高尔基体/后高尔基体区室中胰岛素原运输的阻断导致适当折叠的胰岛素原（带有天然二硫键）的细胞内积累，ER向高尔基体运输的损害(无论这种损害是通过遗传还是药理学手段实现的)导致天然胰岛素原减少,同时存在更多的错误折叠胰岛素原.值得注意的是,可逆的ER到高尔基运输缺陷（如用brefeldinA或细胞能量消耗处理）在逆转时迅速恢复ER折叠环境，导致预先存在的错误折叠的胰岛素原消失，同时保留带有天然二硫键的胰岛素原。因此，ER到高尔基体运输的适当稳态平衡与更有利的胰岛素原折叠(以及运输)结果有关。
