Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules that act through the oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Identifying cysteine oxidation on a wholeproteome scale remains a technical challenge due to the low abundance of oxidised thiols. Redox proteomics techniques therefore use multistep enrichment protocols, but these have inherent limitations and inform only on the enriched proteome. We developed stable isotope cysteine labelling with iodoacetamide (SICyLIA), a simple, unbiased, and robust mass spectrometry-based workflow for thiol oxidation analysis. We applied SICyLIA to diverse cellular models and primary tissues and generated the most in-depth thiol oxidation profiles to date. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys showed oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies. As metabolic proteins were found to be highly sensitive to oxidation, we investigated whether metabolic adaptation to redox stress is regulated through thiol oxidation by combining SICyLIA with metabolomics. We delineated how glycolysis and the pentose phosphate pathway (PPP) are regulated in acute oxidative stress conditions. We generated new biological insight into the mechanism by which GAPDH oxidative inhibition maximises NADPH production in the PPP to help cells adapt to acute oxidative stress, a function previously attributed to PKM2 and TPI. Our results negate a role for PKM2 in this phenomenon: PKM2 is not oxidised and not required in acute oxidative stress. In contrast, we find that PKM2 is oxidised in chronic oxidative stress conditions. Oxidation causes an oligomerisation switch from dimeric to tetrameric PKM2, which depends on a cysteine residue that is conserved in allosterically regulated PK isoforms. Together, this may suggest another adaptation of this PK isoform to confer benefits onto PKM2-overexpressing cancer cells that face high levels of oxidative stress.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:761952 |
| Date | January 2018 |
| Creators | van der Reest, Jiska |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/38983/ |
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