Misfolded Proinsulin Presenting an Unpaired Thiol is Recognized and Targeted for ERAD by Specific Lumenal Components in the ER

Abstract

Objective The accumulation of misfolded proinsulin within the ER of pancreatic β-cells is toxic to the cell, leading to ER stress and cell death. Little is known about how misfolded proteins without glycosylation sites (i.e. proinsulin) are targeted for degradation. Mutant proinsulin, C96Y, (lacks a disulfide bond and has a reactive thiol) has been shown to accumulate and aggregate within the ER, leading to augmented cell apoptosis. We examined whether it is possible for an unpaired thiol on C96Y to act as an endoplasmic reticulum associated degradation (ERAD) recognition signal. Methods: Wild type and mutant (C96Y) proinsulins, labeled with a fluorescent (BODIPY) or radioactive probe (35S-Met), were made using an in vitro transcription/translation system and targeted to the ER lumen of canine pancreatic microsomes. ER microsomes containing proinsulin and lumenal proteins were resuspended in the cystolic medium containing rabbit reticulocyte lysate, which possesses all the required cystolic components for retrotranslocation ERAD. Rates of retrotranslocation and degradation were observed. Results: Our studies, utilizing 35S-Met, show that the ERAD of proinsulin increases when C96Y is present. The rate of retrotranslocation of BODIPY labeled C96Y proinsulin was found to be greater than that of wild type proinsulin. In retrotranslocation studies utilizing reconstituted microsomes, the rate of retrotranslocation was found to be linearly related to lumenal protein concentration. Conclusion Misfolded proinsulin presenting an unpaired thiol is recognized and targeted for ERAD by specific lumenal components in the ER. Funded by: Western University of Health Sciences New Faculty Startup Fund