Identification of ubiquitin K33 and K48 as sites of CO2-dependent carbamylation with implications for intracellular CO2 signalling

Abstract

CO2 is a product of aerobic respiration that has been implicated in various signalling pathways in health and disease. However, the exact mechanisms of CO2 signalling are poorly understood. This is due in large part to the lack of technology with which to study protein-CO2 interactions, known as carbamylation, and thus identify CO2 sensors. Recently a method for trapping carbamates, forming a carboxyethyl modification to the target residue, has been developed for downstream analysis of carbamate formation by MS. The aim of this thesis is to use this novel workflow to identify a CO2 sensor. MALDI-TOF MS was performed to identify peptides containing carboxyethyl modification. LC-MS/MS was then performed to identify K33 and K48 as sites of carboxyethyl modification. These residues were demonstrated to be sites of carbamylation by introducing 13C into the workflow. Finally, a cross-linking assay demonstrated CO2-dependent reduction to the rate of ubiquitination. Together these results demonstrate two sites of carbamylation of ubiquitin with consequences to ubiquitination kinetics. Therefore, it is proposed that ubiquitin is a CO2 sensor.