Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells

Costa Sousa, Bebiana, Ahmed, Tanzim, Dann, William L., Ashman, Jed, Guy, Alexandre, Durand, Thierry, Pitt, Andrew R and Spickett, Corinne M (2019). Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells. Free Radical Biology and Medicine, 144 , pp. 223-233.

Abstract

Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residues in vitro and in vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observed in vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity.

Publication DOI: https://doi.org/10.1016/j.freeradbiomed.2019.05.028
Divisions: Life & Health Sciences > Pharmacy
Life & Health Sciences
Life & Health Sciences > Cellular and Molecular Biomedicine
Life & Health Sciences > Biosciences
Life & Health Sciences > Chronic and Communicable Conditions
Additional Information: © 2019 The Authors. Published by Elsevier Inc. CC BY-NC-ND. Funding: European Union’s Horizon 2020 research and innovation programme under the Marie Sklowdowska-Curie grant agreement number 675132.
Uncontrolled Keywords: 4-Hydroxy-2-hexenal,Acrolein,Lipoxidation,Malondialdehyde,Mass spectrometry,Warburg effect,Biochemistry,Physiology (medical)
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
Published Online Date: 2019-11-20
Authors: Costa Sousa, Bebiana
Ahmed, Tanzim
Dann, William L.
Ashman, Jed
Guy, Alexandre
Durand, Thierry
Pitt, Andrew R ( 0000-0003-3619-6503)
Spickett, Corinne M ( 0000-0003-4054-9279)

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