4-Hydroperoxy-2-nonenal Is Not Just an Intermediate but a Reactive Molecule That Covalently Modifies Proteins to Generate Unique Intramolecular Oxidation Products

Abstract

␣,␤-Unsaturated aldehydes generated during lipid peroxidation, such as 4-oxoalkenals and 4-hydroxyalkenals, can give rise to protein degeneration in a variety of pathological states. Although the covalent modification of proteins by these end products has been well studied, the reactivity of unstable intermediates possessing a hydroperoxy group, such as 4-hydroperoxy-2-nonenal (HPNE), with protein has received little attention. We have now established a unique protein modification in which the 4-hydroperoxy group of HPNE is involved in the formation of structurally unusual lysine adducts. In addition, we showed that one of the HPNE-specific lysine adducts constitutes the epitope of a monoclonal antibody raised against the HPNEmodified protein. Upon incubation with bovine serum albumin, HPNE preferentially reacted with the lysine residues. By employing N ␣ -benzoylglycyl-lysine, we detected two major products containing one HPNE molecule per peptide. Based on the chemical and spectroscopic evidence, the products were identified to be the N ␣ -benzoylglycyl derivatives of N ⑀ -4-hydroxynonanoic acid-lysine and N ⑀ -4-hydroxy-(2Z)-nonenoyllysine, both of which are suggested to be formed through mechanisms in which the initial HPNE-lysine adducts undergo Baeyer-Villiger-like reactions proceeding through an intramolecular oxidation catalyzed by the hydroperoxy group. On the other hand, using an HPNE-modified protein as the immunogen, we raised a monoclonal antibody against the HPNE-modified protein and identified one of the HPNE-specific lysine adducts, N ⑀ -4-hydroxynonanoic acid-lysine, as an intrinsic epitope of the monoclonal antibody. Furthermore, we demonstrated that the HPNE-specific epitopes were produced not only in the oxidized low density lipoprotein in vitro but also in the atherosclerotic lesions. These results indicated that HPNE is not just an intermediate but also a reactive molecule that could covalently modify proteins in biological systems.

Angiotensin II modification by decomposition products of linoleic acid-derived lipid hydroperoxide

“…6). Our data suggested that oxidative cleavage of the C3-C4 bond in HPNE occurred during incubation to produce MDA, as reported previously [31].…”

“…EDE can also form Michael addition products with His [30]. Although HPNE is a direct precursor of ONE and HNE, it is reported that it can modify Lys to generate adducts of unusual structure through a mechanism involving hydroperoxy group-mediated intramolecular oxidation [31].…”

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

“…However, these signalling processes most probably involve its reaction and adduct formation with signalling proteins, thus altering their activity (usually inhibition), rather than reversible ligand binding [45]. Interestingly, it appears that hydroperoxynonenal is not just a reactive intermediate leading to HNE, but also has biological effects in its own right, as it has been reported to modify proteins [46].…”

Lipid peroxidation generates biologically active phospholipids including oxidatively N-modified phospholipids

“…However, t -butylhydroperoxide is used in synthesis reactions to peroxidize imine bonds, which then lose water to form the amides, so we speculate that similar reaction would occur with lipid peroxides. A similar type of reaction from imine to amide has been proposed during the reaction of 4-hydroperoxy-nonenal with lysine to form N -4-hydroxy-nanenoyl-lysine (Shimozu et al, 2011). Thus the aldehydes formed during fragmentation such as hexanal and 8-oxo-octaenoate-PC could react with PE to initially form an imine bond, and then peroxidation of this bond by LOOH would rationalize the formation of N -hexanoyl-PE and N -C7:1CA-PE, respectively (Fig 22).…”