S-Nitrosoglutathione covalently modifies cysteine residues of human carbonyl reductase 1 and affects its activity
Introduction
Human CBR1 (carbonyl reductase 1, EC 1.1.1.184), or according to the new nomenclature SDR21C1 [1], is a ubiquitously-expressed, monomeric, cytosolic enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily [2]. CBR1 catalyzes the NADPH-dependent reduction of a variety of structurally diverse substrates, mostly carbonyls. The best known xenobiotic substrates include quinones (such as the vitamin K precursor menadione and 9,10-phenanthrenequinone, both often used as model substrates), anthracyclines, ketoaldehydes, aromatic aldehydes, and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), the carcinogenic nitrosamine of tobacco smoke [3], [4]. CBR1 also reduces a range of endogenous substances including prostaglandins, steroids and other aliphatic aldehydes and ketones along with the endogenous indole isatin and S-nitrosothiol GSNO (S-nitrosoglutathione) [5], [6]. Moreover, CBR1 plays a protective role in oxidative stress, tumor metastasis, neurodegeneration and apoptosis [7], [8]. The underlying molecular mechanisms are poorly understood. One study has shown that CBR1 inactivates the lipid aldehyde 4-oxo-2-nonenal [9], a lipid peroxidation product formed during oxidative stress. Hence CBR1 might protect from oxidative stress by eliminating reactive oxygen species [10].
Before the discovery of GSNO as CBR1 substrate in 2008 [6], CBR1 had only been known to convert carbonyl groups to alcohols. GSNO reduction thus represents a new mechanism, where an NO bond is reduced. The kinetic parameters of CBR1 and GSNO are comparable to those of model CBR1 substrates as isatin or menadione [6]. CBR1 seems to be specific for GSNO, because S-nitrosocysteine is not reduced by CBR1. Moreover, CBR1-dependent GSNO reduction has been described in A549 lung adenocarcinoma cell lysates. This indicates that CBR1 also acts as GSNO reductase in vivo.
GSNO is a key endogenous S-nitrosothiol, which serves as a reservoir and donor of NO in organisms [11], [12], [13]. In humans, GSNO is physiologically present up to micromolar levels [11], plays a role in apoptosis [14], has a neuroprotective role [15], inhibits platelet activation [16], and has a strong bronchodilatation effect in asthma [17]. Until 2008, only GSNOR (GSNO-reductase, also termed alcohol dehydrogenase 3) was known to reduce GSNO, resulting in no NO release but in NO signaling termination [18].
The first indication that CBR1 can contain a glutathione binding site had been suggested already by Wermuth in 1981. He had found that the glutathione adduct of prostaglandin PGA1 is reduced by CBR1 while free PGA is not [2]. Another fact supporting this hypothesis was that CBR1 from human placenta was inhibited by oxidized GSH adducts [20]. Later, the same research group suggested that a cysteine residue could play an important role in glutathione binding and enzyme activity [21]. In 2008, the X-ray crystal structure of the GSH-binding site was found in close structural proximity to the active site of CBR1 [7].
Recent studies have revealed that GSNO causes covalent modification of CBR1, which results in loss of enzyme activity at a concentration around 100 μM GSNO [19]. The fact that treatment with dithiothreitol (DTT) restored the enzyme activity, while incubation with ascorbic acid did not, indicated that S-glutathionylation was the mechanism responsible for the enzyme inhibition. Further indirect evidence pointed towards Cys-227 as the glutathionylated residue and, in agreement with a previous study that has provided strong support for Cys-227 as the reactive residue [20], Cys-227 was hypothesized to be subject to glutathionylation [19].
In the present study, we sought to identify the cysteine residues in CBR1 that are modified by GSNO and to characterize and quantify the modifications in a mass spectrometry approach. CBR1 contains 5 cysteine residues in its sequence (positions 26, 122, 150, 226, 227) (Fig. 1), of which mutation of two (Cys-226, Cys-227) is known to affect activities for substrates like menadione, 4-benzoylpyridine and daunorubicin [20].
To detect these modifications, we used MALDI-TOF/TOF mass spectrometry followed by a selected reaction monitoring (SRM) approach on hyphenated liquid chromatography with triple quadrupole mass spectrometer. Next, we investigated the influence of these modifications upon other substrates of CBR1 by spectrophotometric and HPLC kinetic studies.
Section snippets
Cloning, protein overexpression and GSNO preparation
Cloning, overexpression and purification of CBR1 were done as described in [19] with the exceptions that PCR primers, including restriction sites, were synthesized by Generi Biotech (Hradec Kralove, Czech Republic), and the primer sequences were as follows: forward primer 5′-GGA TTC CAT ATG TCG TCC GGC ATC CA-3′, reverse primer 5′-CGC CTC GAG TCA CCA CTG TTC AAC TC-3′. Next NdeI and XhoI restriction enzymes and commercially available pET-28b(+) vector (Novagen, Darmstadt, Germany) were used.
MALDI-TOF/TOF analysis of CBR1 protein
Tryptic digestion of CBR1 produces 4 cysteine-containing peptides, 3 carrying one cysteinyl residue each (positions 26, 122, and 150) and 1 peptide with two cysteines (position 226 and 227) (Fig. 2).
To verify the earlier hypothesis regarding modification of cysteine residues 226 and 227 in CBR1 by S-glutathionylation upon incubation with GSNO [19], we performed MALDI-TOF/TOF MS and MS/MS analyses of tryptic peptides resulting from CBR1 protein treated with GSNO. In silico digestion of CBR1
Conclusion
Human CBR1 is a well characterized enzyme with broad substrate specificity and is known to play a role in many pathophysiological conditions [41]. Inspired by earlier studies [19], [20], we analyzed 4 out of 5 cysteines of CBR1 and evaluated their modification upon incubation with GSNO. Using MALDI TOF/TOF and LC-SRM analyses, we confirmed the formation of GSH-mixed disulfides on all those cysteines analyzed, as well as the formation of a disulfide bond between the adjacent cysteines 226 and
Conflicts of interest
There are no competing interests.
Acknowledgements
Our thanks to Dr. Hans-Jörg Martin for interesting discussions and his help in any situation.
This project was supported by the Grant Agency of Charles University (Grant No. 347211/C/2011), by Charles University Project SVV 265 004, by the Institutional program of the University Hospital Hradec Králové, by Postdoctoral fellowship project No. CZ.1.07/2.3.00/30.0012. The publication is co-financed by the European Social Fund and the state budget of the Czech Republic, TEAB, project no.
References (44)
- et al.
The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative
Chem. Biol. Interact.
(2009) Purification and properties of an NADPH-dependent carbonyl reductase from human brain. Relationship to prostaglandin 9-ketoreductase and xenobiotic ketone reductase
J. Biol. Chem.
(1981)- et al.
Characterization of enzymes participating in carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human placenta
Chem. Biol. Interact.
(2001) - et al.
Expression and NNK reducing activities of carbonyl reductase and 11beta-hydroxysteroid dehydrogenase type 1 in human lung
Chem. Biol. Interact.
(2001) - et al.
Carbonyl reductase
Chem. Biol. Interact.
(2000) - et al.
Human carbonyl reductase 1 is an S-nitrosoglutathione reductase
J. Biol. Chem.
(2008) - et al.
The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration
Curr. Biol.
(2004) - et al.
The dual role of S-nitrosoglutathione (GSNO) during thymocyte apoptosis
Cell. Signal.
(1996) - et al.
Nitrosothiol formation catalyzed by ceruloplasmin. Implication for cytoprotective mechanism in vivo
J. Biol. Chem.
(1999) - et al.
Genetic variation in S-nitrosoglutathione reductase (GSNOR) and childhood asthma
J. Allergy Clin. Immunol.
(2007)
Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3
Chem. Biol. Interact.
Some observations concerning the S-nitroso and S-phenylsulphonyl derivatives of L-cysteine and glutathione
Tetrahedron Lett.
S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide
J. Biol. Chem.
S-glutathionylation in protein redox regulation
Free Radic. Biol. Med.
Expression, crystallization and preliminary crystallographic analysis of human carbonyl reductase
J. Mol. Biol.
Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis
Chem. Biol. Interact.
Induction of a human carbonyl reductase gene located on chromosome 21
Biochim. Biophys. Acta
The emerging roles of protein glutathionylation in chloroplasts
Plant Sci.
Glutathione metabolism and its selective modification
J. Biol. Chem.
The changing faces of glutathione, a cellular protagonist
Biochem. Pharmacol.
Carbonyl reductase: a novel metastasis-modulating function
Cancer Res.
Human carbonyl reductase catalyzes reduction of 4-oxonon-2-enal
Biochemistry
Cited by (9)
S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme
2017, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :It was shown that papain, creatine phosphokinase, and glyceraldehyde-3-phosphate dehydrogenase were both S-nitrosylated and S-glutathionylated by GSNO, whereas alcohol dehydrogenase, bovine serum albumin, and actin were nearly only S-nitrosylated [32]. In the case of carbonyl reductase 1, GSNO resulted in S-glutathionylation of cysteine residues, and also in the formation of the intramolecular disulfide bond between neighboring cysteine residues [33]. Consequently, modification by GSNO may result in various products depending on conditions and the nature of the modified protein.
Molecular dynamics simulation of carbonyl reductase 1 clarifies the structural switch in drug metabolism
2020, Journal of Taibah University for ScienceSubstructural dynamics of the phase-I drug metabolizing enzyme, carbonyl reductase 1, in response to various substrate and inhibitor configurations
2019, Tropical Journal of Pharmaceutical ResearchProtein S-Nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-Nitrosothiol-Based Signaling
2019, Antioxidants and Redox SignalingCBR1 rs9024 genotype status impacts the bioactivation of loxoprofen in human liver
2018, Biopharmaceutics and Drug Disposition
- 1
Present address: Institute for Clinical Radiology, Ludwig Maximilians University, 80336 Munich, Germany.