Detection and modification of pyruvate kinase isoforms by myeloperoxidase-derived oxidants HOCl and HOSCN

Abstract

Pyruvate kinase M2 is a glycolytic rate-limiting enzyme that has been shown to induce apoptosis through a caspase-independent pathway in response to oxidative damage by H2O2. Recent studies examining the potential redox regulation of PKM2 showed this form of programmed cell death was linked to the oxidation of a single cysteine amino acid Cys358. Myeloperoxidase (MPO), a leukocyte-derived heme enzyme, and the oxidants it generates, play a key role in killing invading pathogens at sites of inflammation, but can also generate host tissue damage, including within the inflamed artery wall in the context of cardiovascular disease and atherosclerosis. Two oxidants produced by this enzyme, hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), react with several biomolecules including amino acids such as Cys, Met, cystine, His, Lys, Trp and Tyr in the case of HOCl, while HOSCN is considered to be a relatively specific oxidant of cysteine residues. In the current study we have examined whether and how oxidation of PKM1 and PKM2 are induced by myeloperoxidase-derived oxidants formed at sites of inflammation. These studies indicate that HOCl and HOSCN rapidly inhibit the catalytic activity of pyruvate kinase in a dose-dependent manner for isolated PKM1 and PKM2 (1.25-80 μM HOSCN and 2-16 μM HOCl, P < 0.01) and for HCAEC (150-400 μM HOSCN and HOCl, P < 0.01) and HCASMC cellular systems (25-400 μM HOSCN and 100-400 μM HOCl, P < 0.01), with this loss of activity associated with oxidation of Cys residues as determined by Thio-Glo assay and the formation of reducible protein aggregates. Peptide mass mapping experiments provided confirmation of Cys oxidation through the derivatization of sulfenic acid / sulfenyl thiocyanate residues with dimedone, where the amino acids Cys 49,152, 165, 326, 358 and 474 were found to contain the 138 Da dimedone adduct. This research also presents the first comprehensive evidence identifying the presence of PKM2 in human coronary endothelial and smooth muscle cells, mammary artery clinical specimens and carotid or abdominal atherosclerotic lesions using a combination of activity measurements, western blot and mass spectrometry techniques.