S-Nitrosoglutathione covalently modifies cysteine residues of human carbonyl reductase 1 and affects its activity

Abstract

Carbonyl reductase 1 (CBR1 or SDR21C1) is a ubiquitously-expressed, cytosolic, monomeric, and NADPH-dependent enzyme. CBR1 participates in apoptosis, carcinogenesis and drug resistance, and has a protective role in oxidative stress, cancer and neurodegeneration. S-Nitrosoglutathione (GSNO) represents the newest addition to its diverse substrate spectrum, which includes a wide range of xenobiotics and endogenous substances. GSNO has also been shown to covalently modify and inhibit CBR1. The aim of the present study was to quantify and characterize the resulting modifications. Of five candidate cysteines for modification by 2 mM GSNO (positions 26, 122, 150, 226, 227), the last four were analyzed using MALDI-TOF/TOF mass spectrometry and then quantified using the Selected Reaction Monitoring Approach on hyphenated HPLC with a triple quadrupole mass spectrometer. The analysis confirmed GSNO concentration-dependent S-glutathionylation of cysteines at positions 122, 150, 226, 227 which was 2–700 times higher compared to wild-type CBR1 (WT-CBR1). Moreover, a disulfide bond between neighboring Cys-226 and Cys-227 was detected. We suggest a role of these two cysteines as a redox-sensitive cysteine pair. The catalytic properties of wild-type and enzyme modified with 2 mM GSNO were also investigated by steady state kinetic experiments with various substrates. GSNO treatment of CBR1 resulted in a 2–5-fold decrease in k_cat with menadione, 4-benzoylpyridine, 2,3-hexanedione, daunorubicin and 1,4-naphthoquinone. In contrast, the same treatment increased k_cat for substrates containing a 1,2-diketo group in a ring structure (1,2-naphthoquinone, 9,10-phenanthrenequinone, isatin). Except for 9,10-phenanthrenequinone, all changes in k_cat were at least in part compensated for by a similar change in K_m, overall yielding no drastic changes in catalytic efficiency. The findings indicate that GSNO-induced covalent modification of cysteine residues affects the kinetic mechanism of CBR1 both in terms of substrate binding and turnover rate, probably by covalent modification of Cys-226 and/or Cys-227.

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.