title: Stereochemical configuration of 4-hydroxy-2-nonenal-cysteine adducts and their stereoselective formation in a redox-regulated protein.
abstract: 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.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Chika
authors.initials: C
authors.lastname: Wakita
authors.forename: Takuya
authors.initials: T
authors.lastname: Maeshima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamazaki
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shibata
authors.forename: Sohei
authors.initials: S
authors.lastname: Ito
authors.forename: Mitsugu
authors.initials: M
authors.lastname: Akagawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Ojika
authors.forename: Junji
authors.initials: J
authors.lastname: Yodoi
authors.forename: Koji
authors.initials: K
authors.lastname: Uchida
mesh_terms: 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
chemical_list: D000447:Aldehydes; D001894:Borohydrides; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C009282:sodium cyanoborohydride; C027576:4-hydroxy-2-nonenal; D003545:Cysteine
references.citation: Curr Top Cell Regul. 2000;36:151-80
references.pmid: 10842751
references.citation: Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9745-9
references.pmid: 12119401
references.citation: Biochemistry. 1994 Oct 18;33(41):12487-94
references.pmid: 7918471
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Aug;60(Pt 8):1355-63
references.pmid: 15272157
references.citation: Free Radic Biol Med. 2000 Jun 15;28(12):1685-96
references.pmid: 10946210
references.citation: Am J Pathol. 2007 Nov;171(5):1670-81
references.pmid: 17982132
references.citation: J Biol Chem. 1993 Mar 25;268(9):6388-93
references.pmid: 8454610
references.citation: Eur J Biochem. 2000 Oct;267(20):6102-9
references.pmid: 11012661
references.citation: EMBO J. 1989 Mar;8(3):757-64
references.pmid: 2785919
references.citation: J Biol Chem. 1993 Feb 15;268(5):3342-7
references.pmid: 8429010
references.citation: Annu Rev Biochem. 1985;54:237-71
references.pmid: 3896121
references.citation: Free Radic Biol Med. 1999 Sep;27(5-6):504-14
references.pmid: 10490269
references.citation: Prog Lipid Res. 2003 Jul;42(4):318-43
references.pmid: 12689622
references.citation: Annu Rev Immunol. 1997;15:351-69
references.pmid: 9143692
references.citation: J Clin Invest. 2003 Mar;111(5):583-93
references.pmid: 12618510
references.citation: Trends Biochem Sci. 1990 Apr;15(4):129-35
references.pmid: 2187293
references.citation: Clin Cancer Res. 2005 Jan 15;11(2 Pt 1):529-36
references.pmid: 15701837
references.citation: Proc Natl Acad Sci U S A. 1992 May 15;89(10):4544-8
references.pmid: 1584790
references.citation: J Am Chem Soc. 2006 Feb 15;128(6):1879-85
references.pmid: 16464088
references.citation: J Biol Chem. 2008 Jun 13;283(24):16702-10
references.pmid: 18424441
references.citation: Chem Res Toxicol. 1997 Aug;10(8):895-905
references.pmid: 9282839
references.citation: J Biol Chem. 2003 Aug 29;278(35):33408-15
references.pmid: 12816947
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Structure. 1996 May 15;4(5):613-20
references.pmid: 8736558
references.citation: Structure. 1995 Mar 15;3(3):289-97
references.pmid: 7788295
references.citation: Biochem Pharmacol. 1998 Apr 1;55(7):987-94
references.pmid: 9605422
references.citation: Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5611-5
references.pmid: 1608970
references.citation: Free Radic Biol Med. 1991;11(1):81-128
references.pmid: 1937131
references.citation: Immunol Today. 1992 Oct;13(10):405-11
references.pmid: 1418377
references.citation: Biochem Biophys Res Commun. 1978 Nov 14;85(1):257-63
references.pmid: 743278
references.citation: J Biol Chem. 2003 Feb 14;278(7):5044-51
references.pmid: 12473681
references.citation: J Biol Chem. 2001 Jun 15;276(24):20831-8
references.pmid: 11259420
references.citation: Structure. 1996 Jun 15;4(6):735-51
references.pmid: 8805557