Mass Spectrometry‐based Proteomics in Deciphering Hormonal Regulation of Ubiquitin Linkages

Abstract

Hormones such as insulin and glucagon regulate metabolic organ liver. Ubiquitin (Ub) is a 76‐amino acid protein that can be attached on a lysine of the substrate protein. Ub posttranslational modification (PTM) is unique in its diversity as many possible mono‐ and multiple Ub molecules can be attached to protein substrates leading to homotypic and mixed linkages. The type of linkages added determine the functional outcomes such as protein degradation or localization changes. Hormones regulate protein PTM and metabolic disorders such as diabetes involve dysregulation of hormonal signaling. We hypothesized that hormones regulate Ub linkage profile and dysfunction of this regulation is a part of diabetes pathophysiology. We studied the effect of insulin and glucagon on protein ubiquitination in HepG2 cells. We utilized Tandem Ubiquitin Binding Entities (TUBE) and immunoprecipitation strategies for linkage‐specific ubiquitin protein enrichment and further analyzed them by western blotting and proteomics. Our preliminary data indicates differential regulation by hormones of the protein Ub linkage profile. Significant leads obtained from proteomic studies will be further characterized. In addition, we aim to study the ubiquitin linkage profiles in tissues obtained from rodent diabetes models. Our study has potential to find key targets involved in hormonal action and their dysfunction in diabetes.