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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.

SUBMITTER: Wakita C

PROVIDER: S-EPMC2781427 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Stereochemical configuration of 4-hydroxy-2-nonenal-cysteine adducts and their stereoselective formation in a redox-regulated protein.

Wakita Chika C Maeshima Takuya T Yamazaki Atsushi A Shibata Takahiro T Ito Sohei S Akagawa Mitsugu M Ojika Makoto M Yodoi Junji J Uchida Koji K

The Journal of biological chemistry 20090819 42

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- an ...[more]

PMID: 19692331

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Project description:The solution structure of the N1-[1-hydroxy-3-buten-2(R)-yl]-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined in the oligodeoxynucleotide 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3'. This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene (underlined) and was named the ras61 R-N1-BDO-(61,2) adduct. 1H NMR revealed a weak C5 H1' to X6 H8 nuclear Overhauser effects (NOE), followed by an intense X6 H8 to X6 H1' NOE. Simultaneously, the X6 H8 to X6 H3' NOE was weak. The resonances arising from the T16 and T17 imino protons were not observed. 1H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 394 NOE-derived distance restraints and 151 torsion angle restraints yielded a structure in which the modified deoxyinosine was in the syn conformation about the glycosyl bond, with a glycosyl bond angle of 83 degrees , and T17, the complementary nucleotide, was stacked into the helix but not hydrogen bonded with the adducted inosine. The refined structure provides a plausible hypothesis as to why these N1 deoxyinosine adducts strongly code for the incorporation of dCTP during trans lesion DNA replication, irrespective of stereochemistry, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296] and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the syn conformation may facilitate incorporation of dCTP via Hoogsteen type templating with deoxyinosine, generating A to G mutations. However, conformational differences between the R- and the S-N1-BDO-(61,2) adducts, involving the positioning of the butenyl moiety in the major groove of DNA, suggest that adduct stereochemistry plays a secondary role in modulating the biological response to these adducts.

Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disorder with systemic inflammation and may be induced by oxidative stress that affects an inflamed joint. Our objectives were to examine isotypes of autoantibodies against 4-hydroxy-2-nonenal (HNE) modifications in RA and associate them with increased levels of autoantibodies in RA patients.MethodsSerum samples from 155 female patients [60 with RA, 35 with osteoarthritis (OA), and 60 healthy controls (HCs)] were obtained. Four novel differential HNE-modified peptide adducts, complement factor H (CFAH)1211-1230, haptoglobin (HPT)78-108, immunoglobulin (Ig) kappa chain C region (IGKC)2-19, and prothrombin (THRB)328-345, were re-analyzed using tandem mass spectrometric (MS/MS) spectra (ProteomeXchange: PXD004546) from RA patients vs. HCs. Further, we determined serum protein levels of CFAH, HPT, IGKC and THRB, HNE-protein adducts, and autoantibodies against unmodified and HNE-modified peptides. Significant correlations and odds ratios (ORs) were calculated.ResultsLevels of HPT in RA patients were greatly higher than the levels in HCs. Levels of HNE-protein adducts and autoantibodies in RA patients were significantly greater than those of HCs. IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgM anti-IGKC2-19 HNE may be considered as diagnostic biomarkers for RA. Importantly, elevated levels of IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgG anti-THRB328-345 were positively correlated with the disease activity score in 28 joints for C-reactive protein (DAS28-CRP). Further, the ORs of RA development through IgM anti-HPT78-108 HNE (OR 5.235, p < 0.001), IgM anti-IGKC2-19 (OR 12.655, p < 0.001), and IgG anti-THRB328-345 (OR 5.761, p < 0.001) showed an increased risk. Lastly, we incorporated three machine learning models to differentiate RA from HC and OA, and performed feature selection to determine discriminative features. Experimental results showed that our proposed method achieved an area under the receiver operating characteristic curve of 0.92, which demonstrated that our selected autoantibodies combined with machine learning can efficiently detect RA.ConclusionsThis study discovered that some IgG- and IgM-NAAs and anti-HNE M-NAAs may be correlated with inflammation and disease activity in RA. Moreover, our findings suggested that IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgG anti-THRB328-345 may play heavy roles in RA development.

Dataset Information

Stereochemical configuration of 4-hydroxy-2-nonenal-cysteine adducts and their stereoselective formation in a redox-regulated protein.

Publications

Stereochemical configuration of 4-hydroxy-2-nonenal-cysteine adducts and their stereoselective formation in a redox-regulated protein.

Similar Datasets

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