Covalent Cross-Linking of Glutathione and Carnosine to Proteins by 4-Oxo-2-nonenal

Abstract

The lipid oxidation product 4-oxo-2-nonenal (ONE) derived from peroxidation of polyunsaturated fatty acids is a highly reactive protein cross-linking reagent. The major family of cross-links reflects conjugate addition of side-chain nucleophiles such as sulfhydryl or imidazole groups to the C=C of ONE to give either a 2- or 3-substituted 4-ketoaldehyde, which then undergoes Paal-Knorr condensation with the primary amine of protein lysine side-chains. If ONE is intercepted in biological fluids by antielectrophiles such as glutathione (GSH) or β-alanylhistidine (carnosine), this would lead to circulating 4-ketoaldehydes that could then bind covalently to the protein Lys residues. This phenomenon was investigated by SDS–PAGE and mass spectrometry (MALDI-TOF and LC-ESI-MS/MS with both tryptic and chymotryptic digestion). Under the reaction conditions of 0.25 mM to 2 mM ONE, 1 mM GSH or carnosine, 0.25 mM bovine β-lactoglobulin (β-LG), 100 mM phosphate buffer (pH 7.4, 10% ethanol), 24 h, 37 °C, virtually every Lys of β-LG was found to be fractionally cross-linked to GSH. Cross-linking of Lys to carnosine was slightly less efficient. Using cytochrome c and RNase A, we showed that ONE becomes more protein-reactive in the presence of GSH, whereas protein modification by 4-hydroxy-2-nonenal is inhibited by GSH. Stable antielectrophile–ONE–protein cross-links may serve as biomarkers of oxidative stress and may represent a novel mechanism of irreversible protein glutathionylation.

Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs

“…We imagine a sequence involving first a Michael addition of glutathione to PL's more electrophilic C2-C3 olefin followed by the formation of a non-covalent complex between the PL-glutathione adduct and a glutathione-binding protein, and finally a Michael addition of a nucleophilic residue of the glutathione-binding protein to the less electrophilic C7-C8 olefin that is accelerated by the formation of the complex. Mechanistically analogous protein glutathionylation under conditions of electrophilic stress has been observed recently for the metabolic byproduct 4-oxo-nonenal (25) and the chemotherapeutic busulfan (26), both of which are bivalent electrophiles. Such a model also provides a chemical rationale for the results of an unbiased quantitative proteomics analysis of proteins binding to PL, which identified numerous glutathione-binding proteins among the highest confidence interactions (5).…”

The sphingosine 1-phosphate breakdown product, (2E)-hexadecenal, forms protein adducts and glutathione conjugates in vitro

“…Another possibility that could be considered was discovered for 4-oxo-2-nonenal (4-ONE) several years ago. This LPO product, once conjugated by GSH via Michael addition, was shown to effectively cross-link GSH to Lys residues of proteins, in the mentioned study shown for the model protein β-lactoglobulin (28). Although this is a plausible mechanism, not least due to the proven Schiff base formation of 2EHD and Lys ( Figure 4D), further clarification is needed.…”

A Comparison of Reversible Versus Irreversible Protein Glutathionylation

“…The polyunsaturated lipid peroxidation product 4-oxo-2-nonenal (ONE) is a highly reactive protein cross-linking agent (Zhu, Gallogly, Mieyal, Anderson, & Sayre, 2009). The compound also adds to GSH, resulting in the formation of a reactive 4-ketoaldehyde that has the potential to covalently add to proteins (Cooper, Pinto, et al, 2011).…”