PNNL

Abstract

Levels of improperly processed insulin (i.e., proinsulin) in islets and blood of individuals with stage 1 pre-symptomatic T1D (i.e., prediabetic) are significantly elevated. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and deficiencies or mutations in CPE result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a ß-cell-specific Cpe knockout mouse model. Increases in dibasic-residue-containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion implying paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.