Department of Biochemistry, Wakayama Medical University, Wakayama, 641-8509, Japan.
Department of Biochemistry, Wakayama Medical University, Wakayama, 641-8509, Japan.
Biochimie. 2020 Apr-May;171-172:1-11. doi: 10.1016/j.biochi.2020.01.011. Epub 2020 Jan 29.
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.
胰岛素原在粗面内质网（ER）中的氧化折叠对于胰岛素从胰腺β细胞的正确分拣和分泌至关重要。在这里，我们通过使用非细胞基础的胰岛素聚集测定法和小鼠胰岛素瘤衍生的 MIN6 细胞，在（pro）胰岛素的氧化折叠受到损害时，寻找胰岛素的候选分子伴侣。我们发现，MIN6 细胞中，还原应激增强了胰岛素与钙网蛋白（CRT）之间的相互作用，CRT 是一种作为内质网驻留伴侣的凝集素。胰岛素与重组 CRT 共孵育可防止还原剂诱导的胰岛素聚集。此外，在 MIN6 细胞中，二硫苏糖醇诱导的还原应激促进了胰岛素原的溶酶体降解，这一过程依赖于 CRT。总之，我们的研究结果表明，CRT 可能是一种保护性分子，可防止（pro）胰岛素聚集，特别是在氧化折叠缺陷的情况下，例如在体外和培养细胞中还原应激条件下。因为 CRT 不仅作为糖基化蛋白的分子伴侣，而且还作为非糖基化多肽的分子伴侣，我们还提出（pro）胰岛素是 CRT 伴侣功能的新型候选客户。
