Stereochemical Configuration of 4-Hydroxy-2-nonenal-Cysteine Adducts and Their Stereoselective Formation in a Redox-regulated Protein

• Published in: The Journal of biological chemistry Vol. 284; no. 42; pp. 28810 - 28822
• Main Authors: Wakita, Chika, Maeshima, Takuya, Yamazaki, Atsushi, Shibata, Takahiro, Ito, Sohei, Akagawa, Mitsugu, Ojika, Makoto, Yodoi, Junji, Uchida, Koji
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.10.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Aldehydes - chemistry; Borohydrides - chemistry; Chromatography, High Pressure Liquid; Cysteine - chemistry; HeLa Cells; Humans; Lipid Peroxidation; Magnetic Resonance Spectroscopy - methods; Mass Spectrometry - methods; Models, Chemical; Oxidation-Reduction; Peptides - chemistry; Protein Synthesis, Post-Translational Modification, and Degradation; Proteins - chemistry; Recombinant Proteins - chemistry; Stereoisomerism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.019927

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys73 and, to the lesser extent, the active site Cys32. More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys73, whereas Cys32 exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.