Development of an In Vitro Proinsulin Folding Assay
Authors: Mackin Robert Brian 
Published: June 2021

Diabetes mellitus (DM) is a chronic metabolic disease with significant long‐term complications. Two forms of the disease, types 1 and 2, are responsible for the majority of cases. Some forms of type 1 DM are caused by mutations in the insulin gene, and some forms of type 2 DM are believed to involve the ER stress response. Both of these particular variants likely involve deficits in proinsulin (PI) folding as part of their disease progression. I therefore set out to develop an assay that could be used to examine PI folding in vitro.

Human PI with an N‐terminal poly‐His affinity tag was expressed in bacteria, inclusion bodies were solubilized and the affinity tag was removed by cyanogen bromide digestion. Following rotary evaporation, PI was sulfonated using sodium sulfite and tetrathionate and the product of this reaction (PI‐SO4) was applied to a Superdex 75 gel filtration column. Subsequent purification was performed using anion‐exchange chromatography. A portion of the sample was reduced using dithiothreitol and the product (PI‐red) was obtained following buffer exchange. Both PI‐SO4 and PI‐red were frozen on dry ice and stored at −20°C. Upon thawing, the PI‐red sample was cloudy (likely due to intermolecular disulfide bond formation), while the PI‐SO4 sample was still transparent.

Human protein disulfide isomerase (PDI), with its N‐terminal signal sequence replaced by a poly‐His affinity tag, was also expressed in bacteria. Soluble proteins were extracted and subjected to IMAC chromatography. The eluted fraction was applied to a Superdex 200 gel filtration column, fractions were pooled and aliquots were frozen in liquid nitrogen and stored at −80°C.

Initial attempts to observe PI folding were performed using the cloudy PI‐red samples in the hope that PDI could rearrange the incorrect disulfide bonds. However, incubation of PI‐red with PDI in either the absence or presence of 1 mM each of reduced and oxidized glutathione (an approximately 200‐fold molar excess of the GSG/GSSG redox pair) did not result in any detectable change in this form of PI. In contrast, results using PI‐SO4 were more interesting. Incubation of PI‐SO4 with PDI in the absence of GSH/GSSG showed no change in PI‐SO4, and incubation of PI‐SO4 with GSH/GSSG alone resulted in the single PI‐SO4 HPLC peak decomposing to a series of smaller peaks with similar retention times. However, incubation of PI‐SO4 (5 μM) with both the GSH/GSSG redox pair (500 μM each) and PDI (1 μM) for 30 min at 30°C and pH 7.4 resulted in a single HPLC peak that eluted with the retention time of properly folded PI.

These results suggest that this assay can be used to quantify the ability of PDI to properly fold both normal and mutant versions of PI. It is hoped that results from future studies can be used to understand the PI folding pathway, shed light on the DM disease process, and evaluate the ability of potential treatments to mitigate improper or inefficient PI folding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.