Conceived and designed the experiments: FLJ NVB MF DJ. Performed the experiments: FLJ AML EMM SM NSB BOF JHP CEH. Analyzed the data: FLJ DJ. Contributed reagents/materials/analysis tools: JHP CEH NVB. Wrote the paper: DJ. Edited manuscript: MF.
Current address: University of Warwick Medical School, Coventry, United Kingdom
The authors have declared that no competing interests exist.
S-nitrosation – the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins – is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins
The denitrosation of
We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.
The chemistry associated with the production of nitric oxide (NO) in biological systems provides the foundation from which the diverse functions of NO may be interpreted. The nitrosation – i.e. the addition of a nitrosonium (NO+) equivalent – of the sulfhydryl group of cysteine residues in proteins to form S-nitrosothiols (RSNOs) has received increasing attention
The denitrosation or removal of the NO moiety from amino acid residues is essential for protein RSNOs and RNNOs to function as NO storage or signaling intermediates. Although denitrosation pathways have been detailed for RSNOs
The denitrosation of nitrosated Trp residues can be modeled by studying the stability of N-acetyl nitroso Trp (NANT) in solution. In this set of experiments, NANT decomposition was followed spectrophotometrically at 335 nm upon incubation of 100 µM NANT with various concentrations of GSH in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA. As previously shown
The decomposition of NANT (100 µM) was followed spectrophotometrically at 335 nm upon incubation with increasing concentrations of GSH in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA. NANT decay followed apparent first order kinetics and kobs for NANT decomposition was plotted as a function of [GSH]. The values represent the mean ± SEM (n = 4).
In contrast with a previous study
(
Next, we found that superoxide dismutase (SOD) inhibited NANT decomposition by GSH in a fashion similar to that observed in the absence of O2 (
(
Finally, GSH, glutathione disulfide (GSSG), GSNO, nitrite (NO2−), and nitrate (NO3−) formation were analyzed by ion-pairing HPLC (
To evaluate the impact of GSH-induced denitrosation of nitrosated Trp residues in human serum albumin (HSA), we established first that tryptophan and cysteine were the only proteinogenic residues to form nitrosated products in significant amounts upon incubation with NO in oxygenated solution (
(
Amino Acids | Peak Area (mV.sec) |
None (buffer control) | 66.6±12.5 |
Alanine | 127.5±73.6 |
Arginine | 41.9±10.4 |
Asparagine | 38.8±5.5 |
Aspartate | 36.8±1.1 |
Glutamate | 62.6±33.5 |
Glutamine | 32.5±8.7 |
Glycine | 35.4±10.3 |
Histidine | 108.9±53.6 |
Isoleucine | 74.3±31.3 |
Leucine | 35.4±4.0 |
Lysine | 37.7±11.0 |
Methionine | 97.5±26.1 |
Phenylalanine | 87.1±29.9 |
Proline | 36.0±4.5 |
Serine | 34.4±6.6 |
Threonine | 66.3±39.5 |
Tyrosine | 55.5±2.8 |
Valine | 189.0±135.2 |
Acetylated amino acids (1 mM) were incubated for 30 min at 37°C with 20 µM DEA/NO at ambient oxygen concentration in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA. Product formation was determined using a chemiluminescence-based assay as described under
Human serum albumin contains only one tryptophan residue (Trp-214) and one reduced cysteine residue (Cys-34;
The protein secondary structure is shown schematically and the domains are colored-coded as follows: I, blue; II, green; III, yellow; IV, red. Cys-34 and Trp-214 are shown in a space-filling representation and colored by atom types (PDB ID: 1E78;
(
Azide inhibited HSA nitrosation in a concentration-dependent manner such that, in the presence of 1 mM azide, Cys-34 and Trp-214 nitrosation was inhibited by approximately 57% and 71%, respectively (
To evaluate possible denitrosation of HSA, the protein was incubated with DEA/NO for 30 min, incubated with either GSH or ascorbate for an additional 30 min, and then desalted before nitroso species determination. Incubation of nitrosated WT HSA with GSH but not ascorbate eliminated the signal associated with Cys-34 (
HSA (3 mg/ml) was incubated with 20 µM DEA/NO in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA for 30 min at 37°C. Samples were then incubated for an additional 30 min either alone or in the presence of GSH (1 mM) and ascorbate (Asc; 300 µM) in 100 mM phosphate buffer (pH 7.4) containing 100 µM DTPA, after which the samples were desalted. Nitrosation at Cys-34 and Trp-214 was then determined using reductive chemiluminescence. The values represent the mean ± SEM (n = 3).
S-nitrosocysteine and other low molecular weight RSNOs have been used as alternates to NO donors in order to nitrosate cellular proteins
Cell lysates obtained from murine fibroblasts were incubated alone (Control), with 10 µM nitroso-
The denitrosation of NANT occurs through nucleophilic attack on the protonated form of the nitrosamine, and the reversibility of the reaction is suppressed upon addition of a trap for the NO species liberated
Human serum albumin is unique among other mammalian orthologs as it contains only one reduced cysteine (Cys-34) and one tryptophan residue (Trp-214)
The location of Cys-34 and Trp-214 in distinct regions of HSA also offered a testable paradigm related to the stability of the nitroso species associated with HSA. In short, although nitrosated Trp residues are susceptible to denitrosation by GSH, the solubility of GSH (and other low molecular weight thiols) would preclude access to Trp-214 buried within one of the hydrophobic pockets of HSA. Conversely, Cys-34 is accessible to GSH (as evidenced by its thiolation
In summary, several conclusions may be drawn from our results:
The denitrosation of N-nitrosated Trp derivatives in the presence of excess GSH occurs through homolytic cleavage of the N-NO bond driven by the oxidation of GSH and formation of superoxide and peroxynitrite (
As illustrated with HSA, the primary factor limiting RNNO denitrosation by low molecular weight antioxidants such as GSH is the steric accessibility of the nitrosated Trp; as a result, only appropriately located residues within proteins should be available for denitrosation. This issue was previously conceptualized with regard to Trp nitrosation and should also be considered as far as protein RNNO denitrosation is concerned
Techniques for the determination of RNNOs such as chemiluminescence-based assays require preincubation periods that may lead to thiol induced degradation of RNNOs
The denitrosation of nitrosated Trp residues by GSH is slow and should allow for the accumulation of significant amounts of RNNOs
Efficient transnitrosation from N-nitrosated Trp derivatives to GSH (or other low molecular thiols) does not occur unless molecular oxygen is absent or SOD present. Thus, RSNO formation
DEA-NONOate (DEA/NO) was obtained from Cayman Chemicals (Ann Arbor, MI). All N-acetylated L-amino acids were purchased from Sigma except for leucine, histidine, and valine which were purchased from Fisher Scientific (Hampton, NH). The peptides AFKAFAVAR and AFKAWAVAR were purchased from GenScript Corp. (Piscataway, NJ). The anti-biotin antibody was from Bethyl Laboratories (Montgomery, TX) and the anti-rabbit HRP antibody from Amersham Pharmacia (Piscataway, NJ). The HRP detection kit was from Pierce (Rockford, IL). The Lipidex-1000 columns were from Packard Instruments. All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO).
Specific mutations were introduced into the HSA-coding region in a plasmid vector containing the entire HSA coding region as previously described
The decomposition of NANT (100 µM) at 37°C was followed by monitoring the absorbance change at 335 nm (ε = 6100 M−1.cm−1
In a typical experiment, a one mL reaction volume containing various concentrations of NANT and GSH was incubated in 100 mM phosphate buffer (pH 7.4). After 30 min incubation at 37°C, the samples were prepared for analysis by high performance liquid chromatography or chemiluminescence detection as described below. All experiments were performed in the presence of the metal chelator DTPA (100 µM) and in the absence of light to limit the artefactual decomposition of NANT and the reaction products.
NANT was directly quantified by reversed-phase HPLC. Samples were injected onto a 250×4.6 mm 5-µm octadecyl silane C18 Prevail column isocratically running at a flow rate of 1 ml/min with distilled water containing trifluoro acetic acid (TFA; 0.1%) and acetonitrile. The acetonitrile concentration was increased from 20% to 27% in a linear gradient from 13 min to 38 min after injection, with the reactions products detected at 335 nm.
The products obtained from the denitrosation of NANT by GSH were also studied by ion-pairing HPLC as previously described
Nitric oxide formation was measured using a Clark-type NO electrode (Iso-NO with 2 mm shielded sensor; WPI, Sarasota, FL). Changes in current output were recorded and NO release was quantified by comparison with a standard curve constructed by addition of increasing concentrations of NaNO2 under reducing conditions (KI/H2SO4).
In a typical experiment, 800 µl of sample was transferred to a glass tube containing 100 µl of 100 mM NEM. The samples was kept on ice and in the dark for 15 min before addition of 100 µl of 100 mM sulfanilamide in 1 M HCl and incubation for another 15 min on ice to scavenge nitrite. Paired samples were also incubated with or without HgCl2 (final concentration 4.9 mM) for an additional 15 min to evaluate for the presence of RSNOs. The concentration of nitroso species in the samples was evaluated by quantification of the amount of NO liberated after injection of the sample into a purge vessel containing 4.5 ml of glacial acetic acid and 500 µl of an aqueous mixture comprised of 450 mM potassium iodide and 100 mM iodine
Cell lysates were prepared from NIH 3T3 cells
For groups of three or more, the data were analyzed by one-way analysis of variance, and when a significant difference was suggested, the Tukey test was used as a post-hoc test. Comparisons restricted to two groups were analyzed using the Student's t-test. A probability value of less than 0.05 was considered to represent a statistically significant difference.