Mir, S. A. | Search bioRxiv

bioRxiv2019

Role of Proinsulin Self-Association in Mutant INS gene-induced Diabetes of Youth

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin in the endoplasmic reticulum (ER) drive the molecular pathogenesis of Mutant-INS-gene induced Diabetes of Youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, proinsulin-WT dimerizes in the ER. Here, we suggest that the normal proinsulin-proinsulin contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that proinsulin Tyr-B16, which is a key residue in normal proinsulin dimerization, helps confer dominant-negative behavior of MIDY mutant proinsulin-C(A7)Y. Substitutions of Tyr-B16 with ether Ala, Asp, or Pro in proinsulin-C(A7)Y each decrease the abnormal interactions between the MIDY mutant and proinsulin-WT, rescuing proinsulin-WT export, limiting ER stress, and increasing insulin production in {beta}-cells and human islets. This study reveals the first evidence indicating that noncovalent proinsulin-proinsulin contact initiates dominant-negative behavior of misfolded proinsulin, pointing to a novel therapeutic target to enhance bystander proinsulin export for increased insulin production.

cell biology↗

bioRxiv2019

A reference map of the human proinsulin biosynthetic interaction network

The beta-cell protein synthetic machinery is dedicated to the production of insulin, which plays a critical role in organismal homeostasis. Insulin synthesis requires the proper folding and trafficking of its precursor, proinsulin, yet the precise network of proinsulin protein interactions in the secretory pathway remains poorly defined. In the present study we conducted unbiased profiling of the proinsulin interactome in human islets, utilizing a human proinsulin-specific monoclonal antibody for affinity purification and mass spectrometry. Stringent analysis identified a central node of interactions between human proinsulin and sequential secretory pathway proteins that is remarkably conserved across 3 ethnicities and both genders. Among the most prominent proinsulin interactions was with ER-localized peroxiredoxin-4 (PRDX4). A functional role for PRDX4 in beta-cells was demonstrated by gene silencing that rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress. Conversely, exogenous PRDX4 improved proinsulin folding. Notably, oxidative stress and even high glucose treatment alone induced proinsulin misfolding in human islets and MIN6 cells, and this was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. This finding prompted PRDX4 analysis in a panel of human islet samples that revealed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of healthy individuals. Taken together, these data highlight the importance of elucidating the complete proinsulin interactome in human islets in order to understand critical steps controlling insulin biosynthesis, beta cell function, and T2D.

biochemistry↗