Stereochemical configuration of 4-hydroxy-2-nonenal-cysteine adducts and their stereoselective formation in a redox-regulated protein.
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 Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) 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.
Wakita
Chika
C
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Maeshima
Takuya
T
Yamazaki
Atsushi
A
Shibata
Takahiro
T
Ito
Sohei
S
Akagawa
Mitsugu
M
Ojika
Makoto
M
Yodoi
Junji
J
Uchida
Koji
K
D000447:Aldehydes / Q000737:chemistry*; D001894:Borohydrides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D015227:Lipid Peroxidation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013237:Stereoisomerism
D000447:Aldehydes; D001894:Borohydrides; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C009282:sodium cyanoborohydride; C027576:4-hydroxy-2-nonenal; D003545:Cysteine
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