A reference map of the human proinsulin biosynthetic interaction network

Abstract

The beta-cell secretory protein synthetic machinery is dedicated to insulin production, and recent reports suggest that both proinsulin misfolding and accompanying beta-cell oxidative stress could be common features in type 2 diabetes (T2D). Despite the critical role of insulin in organismal homeostasis, the precise network of interactions from early proinsulin synthesis and folding in the ER, to its subsequent trafficking through the secretory pathway, remain poorly defined. In the present study we utilized a human proinsulin-specific monoclonal antibody for affinity purification mass spectrometry, to yield unbiased profiling of the proinsulin interactome in human islets. The data reveal that human proinsulin interacts with a network of ER folding factors (including chaperones: e.g. ERDJ5, ERDJ3, GRP94, and BiP; and oxidoreductases: e.g. QSOX1, DUOX2, and PRDX4) that are remarkably conserved across both genders and 3 ethnicities. Knockdown of one of the most prominent hits, peroxiredoxin-4 (PRDX4) in MIN6 beta-cells, rendered proinsulin more susceptible to misfolding. Additionally, oxidant exposure in human islets enhanced proinsulin:BiP interactions with augmented proinsulin misfolding. Finally, oxidant exposure in human islets also led to sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Interestingly, we observed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of normal islets. Taken together, these data provide a detailed reference map of the human proinsulin interaction network and suggest critical unrecognized areas for study in insulin biosynthesis, beta cell function, and T2D.