Endocrine Abstracts

Proinsulin makes three evolutionarily-conserved disulfide bonds, two of which connect the insulin B and A chains, and one intrachain bond within the A chain. In Type II diabetes mellitus, increasing evidence suggests that insulin-secreting pancreatic β cells show signs of endoplasmic reticulum (ER) stress and an increase in the presence of proinsulin with mispaired disulfide bonds. In addition, heterozygous expression of misfolded mutant proinsulin is known to cause autosomal dominant diabetes, resulting in a loss of β cell mass. ER oxidoreductases are involved in protein chaperone activity as well as redox reactions leading to formation of disulfide bonds of secretory proteins. The ER is a more oxidizing environment than that of the cytosol, at least in part due to the actions of Ero1α and Ero1β which become reduced by ER oxidoreductases. In turn, cargo proteins in the ER lumen transfer reducing equivalents to ER oxidoreductases, which results in formation of disulfide bonds within the secretory cargo. In endocrine pancreatic β cells, the major secretory cargo protein is proinsulin, the precursor in insulin biosynthesis. Thus, proinsulin is a major source for donating reducing equivalents to ER oxidoreductases, yet very little is known about the oxidoreductases that facilitate its proper disulfide bond formation within the ER. To begin to characterize the relationship between ER oxidoreductases and proinsulin within the ER of β cells, I have employed selective RNAi-mediated knock-down of ER oxidoreductases. Using anti-insulin immunoprecipitates from metabolically-labeled cells, I have used Tris-tricine-urea-SDS-PAGE to closely examine disulfide bond formation in endogenous proinsulin. By pinpointing the key ER oxidoreductases involved in forming proinsulin’s three native disulfide bonds, we can potentially manipulate these ER oxidoreductases to enhance proper proinsulin disulfide bond formation. In turn, this may alleviate ER stress, preventing β cell failure and the development of Type II diabetes mellitus.