%0 Journal Article
%T Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis.
%A Arunagiri A
%A Alam M
%A Haataja L
%A Draz H
%A Alasad B
%A Samy P
%A Sadique N
%A Tong Y
%A Cai Y
%A Shakeri H
%A Fantuzzi F
%A Ibrahim H
%A Jang I
%A Sidarala V
%A Soleimanpour SA
%A Satin LS
%A Otonkoski T
%A Cnop M
%A Itkin-Ansari P
%A Kaufman RJ
%A Liu M
%A Arvan P
%J Protein Sci
%V 33
%N 4
%D 2024 Apr
%M 38511500
%F 6.993
%R 10.1002/pro.4949
%X 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.