Carbamylation across the Mammalian Proteome

Abstract

Carbon dioxide (CO2) is a ubiquitous bioactive gas involved in regulating multiple mammalian biochemical pathways. CO2 regulates diverse biological processes through various sensing mechanisms, including the carbamylation post-translational modification (PTM). Carbamylation in mammals has been implicated in the ventilatory response by haemoglobin, cellular conductance by connexin 26 and proteolysis by ubiquitin (Ub). CO2-protein interactions remain an active area of research, with emerging technologies profiling these modifications across the proteome.

This thesis presents a mammalian proteome screen and identifies carbamylation sites across the HEK293 cell line. Proteome coverage was increased 7-fold using a fractionation-based workflow, and a method for carbamate validation was developed. Nine novel carbamylation sites were identified across two distinct screening datasets. Following the identification of carbamylated Ub K48 and Histone H3K79, the biological relevance of carbamylation at these targets was investigated.

Proteolysis targeting chimeras (PROTACs) are an emerging therapeutic technology which relies on the ubiquitin-proteasome system to target specific proteins for degradation. A luminescence assay was used to assess the degradation efficiencies of seven bromodomain containing 4 (BRD4) targeting PROTACs in HEK293 and nine SWI/SNF related, matrix-associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2) targeting PROTACs in NCIH838 when exposed to normoxic and hypercapnic CO2. The data revealed that the PROTACs’ dose-response is independent of CO2 concentration.

Across mass spectrometry (MS) datasets, eleven novel histone carbamylation sites were identified, and carbamylation at H3K79 was selected for further study. H3K79 methylation is a well-characterised histone PTM that results in DNA transcription activation. An in-vitro and in-cellulo assay assessed H3K79 methylation under elevated CO2. The data indicated that H3K79 methylation is enhanced under elevated inorganic carbon and that H3K79 methylation mediated transcriptional change is dependent on the partial pressure of CO2 (PCO2).