Oxidative folding of proinsulin in the endoplasmic reticulum (ER) is critical for the proper sorting and secretion of insulin from pancreatic β-cells. Here, by using non-cell-based insulin aggregation assays and mouse insulinoma-derived MIN6 cells, we searched for a candidate molecular chaperone for (pro)insulin when its oxidative folding is compromised. We found that interaction between insulin and calreticulin (CRT), a lectin that acts as an ER-resident chaperone, was enhanced by reductive stress in MIN6 cells. Co-incubation of insulin with recombinant CRT prevented reductant-induced aggregation of insulin. Furthermore, lysosomal degradation of proinsulin, which was facilitated by dithiothreitol-induced reductive stress, depended on CRT in MIN6 cells. Together, our results suggest that CRT may be a protective molecule against (pro)insulin aggregation when oxidative folding is defective, e.g. under reductive stress conditions, in vitro and in cultured cells. Because CRT acts as a molecular chaperone for not only glycosylated proteins but also non-glycosylated polypeptides, we also propose that (pro)insulin is a novel candidate client of the chaperone function of CRT.

中文翻译：

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钙网蛋白可在体外和MIN6细胞中保护胰岛素抵抗还原性应激。

胰岛素原在内质网（ER）中的氧化折叠对于从胰岛β细胞正确分选和分泌胰岛素至关重要。在这里，通过使用基于非细胞的胰岛素聚集测定法和小鼠胰岛素瘤衍生的MIN6细胞，我们在（氧化）氧化折叠受到损害时搜索了（pro）胰岛素的候选分子伴侣。我们发现，胰岛素和钙网蛋白（CRT）之间的相互作用（一种作为ER内分子伴侣的凝集素）通过MIN6细胞中的还原性应激得到增强。将胰岛素与重组CRT共同孵育可防止还原剂诱导的胰岛素聚集。此外，由二硫苏糖醇诱导的还原应激促进的胰岛素原的溶酶体降解取决于MIN6细胞中的CRT。一起，我们的研究结果表明，在体外和培养细胞中，当氧化折叠有缺陷时，例如在还原性应激条件下，CRT可能是针对（原）胰岛素聚集的保护性分子。因为CRT不仅充当糖基化蛋白的分子伴侣，而且还充当非糖基化多肽的分子伴侣，所以我们还提出（原）胰岛素是CRT伴侣功能的新型候选客户。