Rescued Secretion of Misfolded Mutant Proinsulin.

[摘要] In pancreatic beta cells, the insulin precursor proinsulin is folded in the endoplasmic reticulum (ER), forming three critical intramolecular disulfide bonds. After homo-dimerizing, native proinsulin exits the ER en route to secretory vesicles, where it forms hexamers, is endoproteolytically cleaved to mature insulin, and is stored until it is secreted in response to elevated blood glucose. In Mutant Ins-gene induced Diabetes of Youth (MIDY), misfolded mutant proinsulin is retained in the ER and acts in a dominant-negative manner to impair maturation of wild-type (wt) proinsulin, leading to decreased insulin release and eventual ER stress-induced beta cell death. Using cell culture and mouse models, I have investigated two potential mechanisms to improve secretion of misfolded mutant proinsulin. First, I found that intermolecular interactions between proinsulin molecules impact strongly on the fate of those molecules. Misfolded mutant proinsulin molecules dimerize with and impair secretion of co- expressed wt molecules. Interestingly, the opposite is also true; wt proinsulin molecules also stabilize and enhance secretion of mutant molecules. Thus, there is a dynamic bidirectional interaction between dimerization partners, which we hope to exploit pharmacologically to improve clearance of misfolded proteins from the ER and alleviate ER stress-induced cell death.In the second half of my project, I investigated how manipulating the oxidative environment of the ER may impact proinsulin secretion and beta cell health in cells expressing mutant proinsulin. ER Oxidoreductin-1 (Ero1), the best-studied ER oxidant, contributes to oxidative folding of secretory proteins by coupling generation of de novo disulfide bonds with reduction of molecular oxygen. Due to its generation of hydrogen peroxide as a byproduct, Ero1 hyperactivity has been speculated to contribute to cell death in stressed beta cells. Surprisingly, I found the opposite to be true. Overexpression of Ero1 rescued secretion of wt proinsulin in the presence of mutant proinsulin. Furthermore, Ero1 directly rescued a subset of MIDY mutant proinsulins by improving their oxidative folding, resulting in a decrease in mutant proinsulin-induced ER stress response. These findings improve our understanding of proinsulin maturation in beta cells, and may contribute to novel therapeutic approaches in this and other secretory protein conformational diseases.

[发布机构] University of Michigan

[效力级别] Secretory Protein Folding

[关键词] Monogenic Diabetes;Secretory Protein Folding;Biological Chemistry;Molecular;Cellular and Developmental Biology;Physiology;Science;Molecular and Integrative Physiology