Proinsulin Trafficking through the Secretory Pathway.

Abstract

Proinsulin, the insulin precursor protein, is synthesized in the Endoplasmic Reticulum (ER) of pancreatic beta cells, where it folds to the native state, involving the formation of three evolutionarily conserved disulfide bonds. Once folded, proinsulin exits the ER, traverses the secretory pathway to the trans-Golgi Network (TGN) and into budding secretory granules. Proinsulin is then processed by endopeptidases that excise the connecting C-peptide that links the B- and A-chains, leading to the creation of mature, two-chain insulin. Upon secretion to the bloodstream, insulin binds to cell surface insulin receptors on target tissues, resulting in the activation of signaling cascades that promote metabolic homeostasis. This thesis aims to look at two distinct aspects of the proinsulin maturation process. In the first part of my thesis work, I have designed a proinsulin with a shortened linker peptide with the intent to create a bioengineered protein that acts as a single-chain insulin (SCI), i.e., without a requirement for cleavage by endopeptidases. SCIs expressed via gene therapy have been found to be effective in reversing diabetes in rodent models, obviating the need for exogenous insulin injection. However, to date, very little structure-function analysis of SCIs has been performed. In Chapter 2, I have examined the structural features of the linker peptide that would allow for mammalian expression, secretion, and bioactivity of SCIs for development into future diabetes therapeutics. In the second part of my thesis work, I have been attempting to identify the ER oxidoreductase(s) that promote(s) formation of the three disulfide bonds of proinsulin, which heretofore are unknown. In Chapter 3, I present results that point to two key members of the family of PDI-like ER oxidoreductases: Protein Disulfide Isomerase itself, and Endoplasmic Reticulum protein 72 (ERp72), which may both play critical, yet opposing, roles in this process. As the misfolding of proinsulin is implicated in the progression of various forms of diabetes, understanding the key factors that control the balance of proinsulin folding and misfolding (by regulating proinsulin disulfide bond formation) could also provide potential benefit for designing therapies that increase insulin production.