The beta-cell protein synthetic machinery is dedicated to insulin production, and recent reports suggest misfolding of its precursor, proinsulin, is a feature of type 2 diabetes (T2D). Despite the critical role of insulin in organismal homeostasis, the precise network of interactions required for proper proinsulin folding and subsequent trafficking through the Golgi and 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 both genders and 3 ethnicities. Among the most prominent proinsulin interactions was with the ER localized oxidoreductase peroxiredoxin-4 (PRDX4). A functional role for PRDX4 was demonstrated by knockdown studies in MIN6 beta-cells 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 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.


D.T. Tran: None. P. Arvan: None. R.J. Kaufman: None. P. Itkin-Ansari: None.


National Institutes of Health (R24DK110973); JDRF (2-SRA-2015-47-M-R)

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