Disulfide Mispairing During Proinsulin Folding in the Endoplasmic Reticulum

Abstract

Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene–induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of β-cells. Interestingly, formation of these two “interchain” disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes.

Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes

“…Using a cellular system validated to compare mutant and WT proinsulin ubiquitination and degradation in the presence of HRD1 (Allen et al, 2004), we confirm that CE increase HRD1-mediated ubiquitination of C 96 Y proinsulin, which is associated with a selective C 96 Y proinsulin destabilization. Thus, in the presence of HRD1, CE promote the preferential degradation of misfolded C 96 Y over WT proinsulin, potentially decreasing the dominant-negative effect of C 96 Y proinsulin that prevents WT proinsulin from exiting the ER, and being secreted as insulin (Haataja et al, 2016). We show that CE treatment prevents SEL1L protein degradation, and this is critical to stabilize HRD1 protein.…”

Biosynthesis, structure, and folding of the insulin precursor protein

“…The ER maintains a highly oxidizing intraluminal environment, which is considered crucial for proper proinsulin folding . The protein disulphide isomerase (PDI) family of ER‐resident proteins are strongly implicated in the oxidative folding of secretory proteins including proinsulin .…”

“…Although it was initially thought that misfolded proinsulin (as occurs in Akita mice) cannot promote formation of aberrant intermolecular disulphide bonds, this conclusion was inconsistent with the demonstration of an increase in disulphide‐bonded proinsulin aggregates in Akita islets It is now thought that aberrant intermolecular disulphide‐bonded proinsulin is not only a notable feature in Akita mouse islets, but also occurs in WT mouse islets, underscoring that WT proinsulin already has a propensity to misfold within the ER of pancreatic β‐cells …”

New Insights into the Physiological Role of Endoplasmic Reticulum-Associated Degradation

“…Most of the mutations affect the folding and maturation of nascent proteins that transverse the secretory pathway. In the gain-of-toxic-function mutations, a dominant negative behavior in the ER is commonly seen as a result of mispairing of disulfide bonds that may engage both mutant and wildtype oligomerization partners (84). The Sel1L-Hrd1 ERAD complex participate in the turnover of mutant proinsulin in the ER (85), but the extent to which it is involved in the clearance of other disease-associated mutants requires further studies.…”