Sinapinic acid can replace ascorbate in the biotin switch assay

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.     

S-nitrosation of proteins during d-galactosamine-induced cell death in human hepatocytes

Nitric oxide (NO) participates in the cell death induced by d-Galactosamine (d-GalN) in hepatocytes, and NO-derived reactive oxygen intermediates are critical contributors to protein modification and hepatocellular injury. It is anticipated that S-nitrosation of proteins will participate in the mechanisms leading to cell death in d-GalN-treated human hepatocytes. In the present study, d-GalN-induced cell death was related to augmented levels of NO production and S-nitrosothiol (SNO) content. The biotin switch assay confirmed that d-GalN increased the levels of S-nitrosated proteins in human hepatocytes. S-nitrosocysteine (CSNO) enhanced protein S-nitrosation and altered cell death parameters that were related to S-nitrosation of the executioner caspase-3. Fifteen S-nitrosated proteins participating in metabolism, antioxidative defense and cellular homeostasis were identified in human hepatocytes treated with CSNO. Among them, seven were also identified in d-GalN-treated hepatocytes. The results here reported underline the importance of the alteration of SNO homeostasis during d-GalN-induced cell death in human hepatocytes.     

Regulation of Protein Function and Signaling by Reversible Cysteine S-Nitrosylation

NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications.     

ESNOQ,Proteomic Quantification of Endogenous S-Nitrosation

S-nitrosation is a post-translational protein modification and is one of the most important mechanisms of NO signaling. Endogenous S-nitrosothiol (SNO) quantification is a challenge for detailed functional studies. Here we developed an ESNOQ (Endogenous SNO Quantification) method which combines the stable isotope labeling by amino acids in cell culture (SILAC) technique with the detergent-free biotin-switch assay and LC-MS/MS. After confirming the accuracy of quantification in this method, we obtained an endogenous S-nitrosation proteome for LPS/IFN-γ induced RAW264.7 cells. 27 S-nitrosated protein targets were confirmed and using our method we were able to obtain quantitative information on the level of S-nitrosation on each modified Cys. With this quantitative information, over 15 more S-nitrosated targets were identified than in previous studies. Based on the quantification results, we found that the S-nitrosation levels of different cysteines varied within one protein, providing direct evidence for differences in the sensitivity of cysteine residues to reactive nitrosative stress and that S-nitrosation is a site-specific modification. Gene ontology clustering shows that S-nitrosation targets in the LPS/IFN-γ induced RAW264.7 cell model were functionally enriched in protein translation and glycolysis, suggesting that S-nitrosation may function by regulating multiple pathways. The ESNOQ method described here thus provides a solution for quantification of multiple endogenous S-nitrosation events, and makes it possible to elucidate the network of relationships between endogenous S-nitrosation targets involved in different cellular processes.     

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

S-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. We used transient kinetic methods to determine a minimal mechanism for spontaneous S-nitrosoglutathione (GSNO)-mediated transnitrosation of human glutathione transferase (GST) P1-1, a major detoxification enzyme and key regulator of cell proliferation. Cys⁴⁷ of GSTP1-1 is S-nitrosated in two steps, with the chemical step limited by a pre-equilibrium between the open and closed conformations of helix α2 at the active site. Cys¹⁰¹, in contrast, is S-nitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. Despite the presence of a GSNO binding site at the active site of GSTP1-1, isothermal titration calorimetry as well as nitrosation experiments using S-nitrosocysteine demonstrate that GSNO binding does not precede S-nitrosation of GSTP1-1. Kinetics experiments using the cellular reductant glutathione show that Cys¹⁰¹-NO is substantially more resistant to denitrosation than Cys⁴⁷-NO, suggesting a potential role for Cys¹⁰¹ in long term nitric oxide storage or transfer. These results constitute the first report of the molecular mechanism of spontaneous protein transnitrosation, providing insight into the post-translational control of GSTP1-1 as well as the process of protein transnitrosation in general.     

S-Nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated (S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation (S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.     

The role of thioredoxin in the regulation of cellular processes by S-nitrosylation

Background

S-nitrosylation (or S-nitrosation) by Nitric Oxide (NO), i.e., the covalent attachment of a NO group to a cysteine thiol and formation of S-nitrosothiols (R-S-N=O or RSNO), has emerged as an important feature of NO biology and pathobiology. Many NO-related biological functions have been directly associated with the S-nitrosothiols and a considerable number of S-nitrosylated proteins have been identified which can positively or negatively regulate various cellular processes including signaling and metabolic pathways.

Scope of the review

Taking account of the recent progress in the field of research, this review focuses on the regulation of cellular processes by S-nitrosylation and Trx-mediated cellular homeostasis of S-nitrosothiols.

Major conclusions

Thioredoxin (Trx) system in mammalian cells utilizes thiol and selenol groups to maintain a reducing intracellular environment to combat oxidative/nitrosative stress. Reduced glutathione (GSH) and Trx system perform the major role in denitrosylation of S-nitrosylated proteins. However, under certain conditions, oxidized form of mammalian Trx can be S-nitrosylated and then it can trans-S-nitrosylate target proteins, such as caspase 3.

General significance

Investigations on the role of thioredoxin system in relation to biologically relevant RSNOs, their functions, and the mechanisms of S-denitrosylation facilitate the development of drugs and therapies. This article is part of a Special Issue entitled Regulation of Cellular Processes.     

REACH coarse-grained biomolecular simulation: transferability between different protein structural classes

Coarse graining of protein interactions provides a means of simulating large biological systems. The REACH (Realistic Extension Algorithm via Covariance Hessian) coarse-graining method, in which the force constants of a residue-scale elastic network model are calculated from the variance-covariance matrix obtained from atomistic molecular dynamics (MD) simulation, involves direct mapping between scales without the need for iterative optimization. Here, the transferability of the REACH force field is examined between protein molecules of different structural classes. As test cases, myoglobin (all α), plastocyanin (all β), and dihydrofolate reductase (α/β) are taken. The force constants derived are found to be closely similar in all three proteins. An MD version of REACH is presented, and low-temperature coarse-grained (CG) REACH MD simulations of the three proteins are compared with atomistic MD results. The mean-square fluctuations of the atomistic MD are well reproduced by the CGMD. Model functions for the CG interactions, derived by averaging over the three proteins, are also shown to produce fluctuations in good agreement with the atomistic MD. The results indicate that, similarly to the use of atomistic force fields, it is now possible to use a single, generic REACH force field for all protein studies, without having first to derive parameters from atomistic MD simulation for each individual system studied. The REACH method is thus likely to be a reliable way of determining spatiotemporal motion of a variety of proteins without the need for expensive computation of long atomistic MD simulations.     

Nitric oxide and dihydrolipoic acid modulate the activity of caspase 3 in HepG2 cells

Herein, we report that dihydrolipoic acid and lipoic acid (LA) plus lipoamide dehydrogenase and NADH denitrosate S-nitrosocaspase 3 (CASP-SNO). In HepG2 cells, S-nitroso-l-cysteine ethyl ester (SNCEE) impeded the activity of caspase 3 (CASP-SH), while a subsequent incubation of the cells in SNCEE-free medium resulted in endogenous denitrosation and reactivation of CASP-SH. The latter process was inhibited in thioredoxin reductase-deficient HepG2 cells, in which, however, LA markedly reactivated CASP-SH. The data obtained are discussed with focus on low molecular mass dithiols that mimic the activity of thioredoxin in reactions of protein S-denitrosation.     

Distant Neighbor Base Sequence Context Effects in Human Nucleotide Excision Repair of a Benzo[a]pyrene-derived DNA Lesion

The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N²-dG (G?) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G?-C-T-A-C-C-3′ (CG?C-I), and 5′-C-A-C3-A4-C5-G?-C-A-C-A-C-3′ (CG?C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(± 0.2)-fold greater in the case of the CG?C-II than the CG?C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG?C-II duplex is more bent than the CG?C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG?C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson-Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG?C-II than in CG?C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG?C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N²-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.     

Study of the inclusion of the (R)- and (S)-camphor enantiomers in α-cyclodextrin by X-ray crystallography and molecular dynamics

The inclusion of (R)- and (S)-camphor compounds in α-cyclodextrin has been studied by X-ray crystallography. The crystal structures of the complexes reveal that one guest molecule is accommodated inside the cavity formed by a head-to-head cyclodextrin dimer. In the crystal lattice, the dimers form layers which are successively shifted by half a dimer. In both (R)- and (S)-cases, the camphor molecule exhibits disorder and occupies three major sites with orientations that can be described as either ‘polar’ or ‘equatorial’. Molecular dynamics simulations performed for the observed complexes indicate that although the carbonyl oxygen of both (R)- and (S)-camphor switches between different hydrogen bonding partners, it maintains the observed mode of ‘polar’ or ‘equatorial’ alignment.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.     

Protein arginine methyltransferase 7 has a novel homodimer-like structure formed by tandem repeats

Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to nitrogen atoms on arginine residues. Here, we describe the crystal structure of Caenorhabditis elegans PRMT7 in complex with its reaction product S-adenosyl-l-homocysteine. The structural data indicated that PRMT7 harbors two tandem repeated PRMT core domains that form a novel homodimer-like structure. S-adenosyl-l-homocysteine bound to the N-terminal catalytic site only; the C-terminal catalytic site is occupied by a loop that inhibits cofactor binding. Mutagenesis demonstrated that only the N-terminal catalytic site of PRMT7 is responsible for cofactor binding.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

Identification of protein nitrosothiols using phosphine-mediated selective reduction

Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.     

Molecular dynamics simulations of thioredoxin with S-glutathiolated cysteine-73

Thioredoxin-1 (Trx) becomes inactive when cysteine-73 forms a mixed disulfide with glutathione. This reversible S-glutathiolation may serve as a regulatory mechanism. However, molecular basis for the glutathiolation-induced inhibition has not been established due to the lack of its structure. Molecular dynamics (MD) simulations were performed with Gromacs to obtain structural information on glutathiolated Trx. Glutathiolation did not cause a large change in overall shape of Trx although small local changes in the secondary structures were evident. The glutathione moiety was much more flexible than the peptide and spanned a large conformational space. It remained very close to the active site for a large part of the simulation time. Therefore inhibition of Trx by glutathiolation appears to be due to steric hindrance imposed by the covalently attached glutathione.     

Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties

Background

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.

Methods

The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR).

Results

Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.

Conclusions and general significance

Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.     

A Novel Family of S-Nitrosothiols: Chemical Synthesis and Biological Actions

S-Nitrosothiols are a class of chemical compounds that decompose to release nitric oxide and show promise in the treatment of a variety of cardiovascular diseases. Some of these are present in vivo and others have been synthesized in vitro. However, those discovered or synthesized to date have very little tissue selectivity or specificity. We synthesized a number of novel S-nitrosated dipeptides of high purity and examined their effects on vasorelaxation using rat mesenteric arteries and on inhibition of platelet aggregation using platelets from healthyhuman subjects. For comparison, we also tested the effects of S-nitroso- -glutathione (GSNO, an S-nitrosothiol present in vivo) and S-nitroso-N-acetyl- -β,β-dimethylcysteine (SNAP(D), the -isomer of SNAP, a commonly used S-nitrosothiol previously synthesized in vitro) in these biological systems. Satisfactory elemental analyses were obtained for all compounds synthesized (less than ± 0.3%), and all accurate mass measurements were within 1–5 ppm of the expected mass. The novel S-nitrosated dipeptides all elicited vasorelaxation with significantly higher potency, of the order of one log molar unit, than either GSNO or SNAP(D). However, all compounds inhibited U46619-induced platelet aggregation with similar potency to GSNO and SNAP(D). These findings indicate a degree of tissue selectivity which may prove to be of therapeutic usefulness.