nitrosoproteome: Tools and strategies - Wiley Online Library

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Laura M. López-Sánchez1, Jordi Muntané2, 3, Manuel de la Mata2, 3 and Antonio Rodríguez-Ariza1. 1 Unidad de Investigación, Hospital Universitario Reina ...
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DOI 10.1002/pmic.200800546

Proteomics 2009, 9, 808–818

REVIEW

Unraveling the S-nitrosoproteome: Tools and strategies Laura M. López-Sánchez1, Jordi Muntané2, 3, Manuel de la Mata2, 3 and Antonio Rodríguez-Ariza1 1

Unidad de Investigación, Hospital Universitario Reina Sofía, Córdoba, Spain Liver Research Unit, Hospital Universitario Reina Sofía, Córdoba, Spain 3 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Spain 2

One of the major tasks to be accomplished in the postgenomic era is the characterization of PTMs in proteins. The S-nitrosation of protein thiols is a redox-based PTM that modulating enzymatic activity, subcellular localization, complex formation, and degradation of proteins, largely contributes to the complexity of cellular proteomes. Although the detection of S-nitrosated proteins is problematical due to the lability of S-nitrosothiols, with the improvement of molecular tools an increasing range of proteins has been shown to undergo S-nitrosation. We here review recent proteomic approaches for the systematic assessment of potential targets for protein S-nitrosation. The development of new analytical methods and strategies over the past several years now allows us to investigate the nitrosoproteome on a global scale.

Received: June 30, 2008 Revised: August 28, 2008 Accepted: September 22, 2008

Keywords: Nitric oxide / S-nitrosation / S-nitrosocysteine / S-nitrosoglutahione / S-nitrosylation

1

Introduction

Nitric oxide (NO) is a simple, diatomic molecule possessing unique and captivating chemistry. NO was regarded as an atmospheric pollutant until it was found in 1987 and the following years that is synthesized in vivo, and is responsible for a range of physiological processes such as vasodilation, inhibition of platelet aggregation, neurotransmission, and antimicrobial activity [1–4]. As a result, conditions of NO excess or deficiency are currently believed to be responsible Correspondence: Dr. Antonio Rodríguez-Ariza, Unidad de Investigación, Hospital Universitario Reina Sofía, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain E-mail: [email protected] Fax: 134-957010452 Abbreviations: CMC, carboxymethylcysteine; CSNO, S-nitroso-Lcysteine; GSNO, S-nitrosoglutathione; GSNOR, S-nitrosoglutathione reductase; HASMC, human aortic smooth muscle cell; MMTS, methylmethanethiosulfonate; NO, nitric oxide; SNO, Snitrosothiol

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

to some degree in a variety of biological derangements. Many of NO actions are thought to be mediated by means of stimulation of soluble guanylyl cyclase (sGC) which results in the production of the second messenger cyclic GMP. There is burgeoning evidence, however, that NO can trigger or modulate cell signaling by modifying other proteins. A reaction of NO in biological systems is the formation of nitrosothiols (SNOs), a modification of the sulphur atom on cysteine, also called S-nitrosation or S-nitrosylation. Target thiols include protein or peptide cysteine residues and cysteine itself. Stamler et al. showed that thiols potentiate the action of NO [5] and that SNOs form in vivo [6]. Furthermore, they showed that the SNO pool comprises both low-molecular weight and S-nitrosoprotein species, and postulated that S-nitrosation of proteins represents a PTM that modulates protein function and cell phenotype [7]. Since this pioneering work, numerous studies have provided considerable evidence that the sGC-GMP pathway represents only part of alternative biochemical pathways through which NO can activate or modulate cell signaling. S-nitrosation of proteins is a key reversible modification that regulates enzymatic acwww.proteomics-journal.com

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tivity, subcellular localization, complex formation, and degradation of proteins [8–10]. In addition, the available tools for SNO detection and identification have been considerably improved in recent years, and a mounting range of proteins has been shown to undergo S-nitrosation. Therefore, it is not surprising that an increasing number of proteomic studies has begun to deal with the so-called S-nitrosoproteome, that is, with the systematic identification and characterization of those proteins that undergo S-nitrosation in a particular organism, organ, or cell type. We will here review recent proteomic approaches for the systematic assessment of potential targets for protein S-nitrosation. These strategies include recent developed methods for the identification of the modified cysteines, that will provide researchers with better tools for exploring this PTM and for performing an in depth analysis of the cellular S-nitrosoproteome.

2

Formation of S-nitrosothols and S-nitrosoproteins

SNOs can be synthesized by the reaction of a thiol with acidified nitrous acid. However, the likely nitrosating species in biological systems is dinitrogen dioxide (N2O3), the formation of which from O2 and NO is kinetically facilitated in hydrophobic environments such as biological membranes [11]. Thiol nitrosation may also occur via the intermediacy of a ferric heme nitrosyl species. Importantly, SNOs can also undergo transnitrosation reactions whereby the nitroso function is transferred from one thiol to another via nucleophilic attack on the nitrogen atom of the SNO [12]. The lowmolecular weight SNO pool exists in equilibrium with the Snitrosoprotein pool, and these transnitrosation reactions have an important role both in SNO cell uptake and transport [13] and in the maintenance of cellular SNO homeostasis [14, 15]. Remarkably, SNOs are capable of releasing NO in the presence of cuprous ion and the photolytic homolysis of the S–NO bond generates both NO and the corresponding thiyl radical. Both reactions are an important issue when working with SNOs. Although the chemical reactions involved in S-nitrosothiol formation are relatively well understood, the mechanisms governing S-nitrosoprotein formation remain obscure. A number of factors, including protein thiol microenvironment, metal ion availability, abundance of ROS, and proximity of NO generating systems or hydrophobic environments, have been considered to play a significant role in the S-nitrosation of protein sulfhydryls [16]. A typical determinant of protein S-nitrosation is the pKa of the sulfhydryl group, that can be anomalously low within the constraint of the tertiary structure of a protein and proximity to charged side-chains, rendering its primary form as thiolate anion. Therefore, the three-dimensional microenvironment of the reactive thiol has been claimed to play an important role in the prediction of enhanced susceptibility to S-nitrosation [8, 17, 18]. Besides, a three-residue degenerate consensus motif © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

for S-nitrosation, (G,S,T,C,Y,N,Q)(K,R,H,D,E)(C)(D,E), has also been suggested after sequence analysis of known Snitrosatable proteins [8, 19]. Other studies have shown that regions of local hydrophobicity within proteins might promote specific S-nitrosation of resident Cys residues [8, 20]. Such hydrophobic compartmentalization may arise from tertiary protein structure and protein–protein interaction. In any case, currently it is not possible to predict if a specific protein will be modified and which of its cysteine residues will be nitrosated. Other factor influencing protein S-nitrosation is the redox status of the immediate environment, that is not only related to subcellular localization of the protein, but also to the proximity to NO and reactive oxygen sources [21, 22]. Colocalization or close proximity of protein targets with NO synthase (NOS) isoforms has been suggested as determinants of specific S-nitrosation [19, 23]. In this sense, the existence of a mitochondrial NOS isoform has been repeatedly reported [24–26] and mitochondria and perimitochondrial compartment have been described as main sites of S-nitrosoprotein localization [22]. In any case, the basis for specific protein S-nitrosation in biological systems is not clear and the availability of proteomic data will undoubtedly clarify the mechanisms governing the selectivity of this PTM.

3

Detection of S-nitrosoproteins: The biotin-switch assay

Detection of S-nitrosated proteins is complicated by a number of factors. As stated above, SNOs are sensitive to light, undergoing homolytic cleavage of the S–NO bond and decomposing to NO and thiyl radical [27]. Furthermore, SNOs are readily reduced by agents such as ascorbate and transition metals, especially Cu(I). Additionally, SNOs and Snitrosoproteins are usually present at low levels in cells and tissues, and sensitive and specific methods for their efficient detection are needed. Phosphorylation studies are highly facilitated by the use of radiolabeled precursors, such as [32P] ATP or [32P]PO4. Unfortunately, there are not available radioactive isotopes of nitrogen or oxygen, the atoms that comprise the S-nitroso adduct, and other sensitive methods are necessitated to specifically detect the SNO moiety. The use of antibodies directed against phosphoamino acids represents other sensitive procedure to detect phosphorylated proteins. However, in the case of S-nitrosoproteins, although both polyclonal and mAb have been raised against the S-nitrosocysteine moiety [28, 29], their usefulness has been mainly demonstrated using immunohistochemical methods. A turning point for S-nitrosation research was the development by Jaffrey et al. [30] of an original approach that permitted the selective tagging of S-nitrosoproteins with biotin. This method is usually termed the “biotin-switch” assay and consists of three steps (Fig. 1). In the first step, free thiols are blocked with the thiol-specific methylating agent methylwww.proteomics-journal.com

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Figure 1. The biotin switch assay and its modifications. During the biotin switch assay S-nitrosoproteins are selectively tagged with biotin. In the first step, free thiols (–SH) are blocked with the thiol-specific methylating reagent MMTS. During the second step, and under conditions where disulfides (–SSR) are not reduced, S-nitrosothiols (–SNO) are selectively reduced by ascorbate. In the final step these newly formed thiols are reacted with the thiol-specific biotinylating reagent biotin-HPDP. Biotinylated proteins can be immunodetected or, alternatively they can be captured on streptavidin resins for their purification and proteomic identification. Some of the modifications of the assay that have been developed for its use in the proteomic analysis of S-nitrosated proteins are schematized in the figure: (A), “SNOSID” method; (B) “HIS-TAG switch” method; (C) fluorescence-based approaches. See text for details.

methanethiosulfonate (MMTS), while nitrosothiols and disulfide bonds are left untouched. Protein mixtures must be treated with chaotropes or detergents, such as SDS, to ensure that all thiols, including those buried within the protein core, are blocked. The second step involves the selective reduction of protein nitrosothiols to thiols with ascorbate. In the final step, these newly formed thiols are reacted with the sulfhydryl-specific biotinylating reagent N-[6-(biotinamido)hexyl]30 -(20 -pyridyldithio)propionamide (biotin-HPDP). This method offers significant advantages in that biotinylated proteins can be detected on Western blots following incubation with antibiotin antibodies or recognition via streptavidin. Importantly, the possibility to capture biotinylated proteins on streptavidin matrices renders this method amenable to proteomic analysis (Fig. 1). Several issues have been raised regarding the specificity and sensitivity of the biotin switch method. The more worrisome issue is the possible nonspecificity of the SNOs reduction by ascorbate. In this regard, some authors have expressed © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

their concerns that this critical step constitutes a source of pitfalls associated with this method [31–33]. The possible reduction by ascorbate of disulfides or other cysteine oxidation derived modifications such as S-glutathionylation or S-oxidations, including sulfenic, sulfinic, and sulfonic acids, may give false positives in this assay. Importantly, Forrester et al. [34] have recently shown that ascorbate does not directly reduce the S–NO bond, but rather undergoes transnitrosation by SNO to generate O-nitrosoascorbate as intermediate, which rapidly homolyzes to yield the semidehydroascorbate radical and NO. This reaction with ascorbate is unique among cysteine oxidation products, and the authors conclude that it confers specificity to the biotin switch assay [34]. A number of spectrophotometric-, fluorimetric-, or chemiluminescence-based methods can accurately detect the total S-nitrosoprotein content in a variety of biological samples (see ref. [35] for a review). However, these methods do not identify which proteins are modified. To date, the biotinswitch assay is the only method for specific tagging of Swww.proteomics-journal.com

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nitrosoproteins and therefore for the isolation and analysis of the S-nitrosoproteome. As other methods, it poses potential problems and limitations. However, once they are recognized and appropriate controls are performed, a correct interpretation of the data may be achieved. There are several methodological reviews to which the reader is referred for more in-depth procedural details on the biotin-switch assay and its application to different types of samples [36–42].

4

Improving tools and strategies

A number of significant modifications and enhancements of the biotin switch assay have been reported. Jaffrey et al. [23] amended their original method by substituting a radioactive tag in place of the biotin tag used in biotin-HPDP to facilitate nitrosopeptide mapping. The authors applied this technique to Dexras 1 and found that a single residue is preferentially S-nitrosated in response to NO donors. In addition, because the degree of incorporation of 35S can be measured by scintillation counting, this method is also useful in experiments in which quantitation of S-nitrosylation is desired. A related approach was used by Lu et al. [43] in which free thiols are blocked with MMTS and S-nitrosated cysteines are reacted with iodoacetic acid (12C/13C, 50% ratio) to form carboxymethylcysteine (CMC) whereas disulfides, including the free cysteines blocked with MMTS, are converted to carboxyamidomethylcysteines during 2-D gel sample preparation. After tryptic digestion the S-nitrosopeptides are identified by LC-MS/MS using the two resulting paired CMC immonium ions. The authors were able to identify a single S-nitrosation site in immunoprecipitated PKBa/Akt1 using this approach. Site-specific identification of S-nitrosation has been also accomplished by direct measurements of the 29 Da or the 30 Da mass changes due to the addition of the NO group or its neutral loss from S-nitrosated peptides, respectively [44, 45]. However, the results from these studies have been controversial and it has been suggested that the NO-related mass changes detected from parent ions may be artifacts. Interestingly, it has been recently reported that S-nitrosopeptides undergo an unusual fragmentation pattern under CID that may aid in future MS-based attempts to define the S-nitrosoproteome [46]. The above-mentioned techniques were all performed on purified proteins or on isolated peptides and could not be easily coupled to a proteomic approach aimed to identify both S-nitrosoproteins and modification sites. However, other recently developed extensions of the original biotin switch approach may help in identifying S-nitrosation sites in a complex mixture of proteins. One of these methods is the SNOSID (SNO Site IDentification) method, and introduces a tryptic digestion step before avidin capture [47, 48]. The added step permits the selective isolation of peptides that previously contained SNO-Cys residues, rather than intact SNO proteins, for their later sequencing by LC-MS/MS (Fig. 1). However, as already stated by its developers, the © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

811 SNOSID method poses several limitations, one being that the identification of a protein depends on MS/MS-based sequence information inferred from a single peptide ion. Other limitation is the relatively large amount (1 g) of starting tissue/cells required. Interestingly, it has been recently reported that when a detergent-free biotin-switch method is used, in which urea rather than SDS is used during thiol blockage and biotinylation, the LC-MS/MS performance is significantly improved and the amount of sample for analysis is significantly reduced [49]. In other study [29] the elution of biotin-tagged peptides is performed with formic acid rather than under reducing conditions. In this manner, the peptides retain the Cys-HPDP-biotin adduct and a positive identification of the putative S-nitrosation site is obtained after LC-MS/MS analysis. Other variation of the biotin-switch method that has been developed for the unambiguous recognition of the modified Cysteine is the HIS-TAG switch method [50]. In this case, biotin-HPDP is substituted by a conjugate of iodoacetamide and a His-tag containing peptide (Fig. 1). This novel alkylating agent irreversibly binds to Cys residues, ensuring that proteins are tagged through all purification steps. In addition, the sequence of the His-tag has been designed so that tryptic digestion cleaves part of the alkylating reagent and label of previously S-nitrosated peptides can be detected as a final mass shift of 271.12 Da and by the production of a reporter ion (MH1 = 912.4 Da) which is also detectable in MS spectra. Using the HIS-TAG switch method, its developers were able to identify 28 S-nitrosated peptides belonging to 19 different proteins in rat brain lysates incubated with S-nitrosoglutathione (GSNO) [50]. These approaches will undoubtedly shed light on site specificity of S-nitrosation and will provide fundamental information for further studies on how this PTM affects the function of each protein. However, other recently developed methodologies will also be useful to address other outstanding questions. For example, it is not known whether there are proteins that are preferential targets for S-nitrosation. Similarly, there is paucity of information regarding the amount of a particular protein that is S-nitrosated under different physiologically relevant conditions. Kettenhofen et al. [51] have suggested a modified 2-D gel-based biotin-switch approach to detect thiols that are selectively S-nitrosated. A general thiol reduction is performed, including endogenous disulfides, by replacing ascorbate with DTT. In this way, although there is a lack of specificity, using appropriate untreated controls this method permits the detection of thiols that are clearly modified after exposure of whole cells to low S-nitroso-L-cysteine (CSNO) levels. The authors also showed the enhancement of this method by using difference gel electrophoresis (DIGE) methodology [51]. After reducing with DTT or ascorbate, thiols from control and SNO-treated samples are labeled with monofunctional maleimide-conjugated cyanine dyes (Cy3 and Cy5) and equal amounts of each sample are mixed together and run on the same 2-D gel (Fig. 1). In this manner, those spots in the gel with a difference in the fluorescent intensity of the two dyes will indicate www.proteomics-journal.com

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thiols that have been selectively modified by the SNO treatment. In addition, the direct comparison of the fluorescence intensity from both dyes provides the relative level of thiol modification. Other exciting new DIGE-based approach has been recently applied to the detection of S-nitrosoproteins [52]. The DyLight Fluor DIGE proteomic approach uses the newly developed DyLight maleimide sulfhydryl reactive fluors (Pierce) to replace the biotin-HPDP in the biotinswitch method. After labeling control and test samples with different DyLight fluors, they are pooled and run on the same 2-D gel. DyLight labeling not only allows each of the individual samples to be visualized independently by selecting appropriate excitation and emission wavelenghts, but also causes in every single spot an acidic shift and a minor upward shift, since each DyLight fluor molecule contains 3– 4 negative charges and a approximate 1 kDa mass. Protein spots with a shifted DyLight pattern can be directly picked from the gel or after poststaining with SYPRO Ruby if more sample is needed for MS identification. By using this method a relative S-nitrosation level for each protein may be obtained from the direct comparison of fluorescent intensity from each DyLight fluor in a single spot. Interestingly enough, other fluorescence-based modification of the biotin-switch method has been very recently reported [53]. In this method, the biotin-HPDP label is replaced by the fluorophore AMCA (7-amino-4-methyl coumarin-3-acetic-acid)-HPDP, and the previously S-nitrosated proteins can be directly visualized on gels after nonreducing SDS-PAGE and UV exposure. Other method for the in situ detection of S-nitrosated proteins is based on the UV-photolysis of protein SNO bonds in gels coated with the NO-sensitive fluorescent probe diaminofluorescein (DAF) [54]. Compared with this DAF-based method, the AMCA switch method combined with the LCMS/MS assay of detected protein bands may give information on modification sites by the shift mass on peptide ions due to AMCA-HPDP adducts in MS/MS spectra. However, this method was only applied to purified proteins and its potential application to the analysis of S-nitrosation in more complex protein mixtures remains to be investigated.

5

The S-nitrosoproteome

About 100 proteins in which the S-nitrosation of protein cysteines had been reported were listed in a 2001 meeting review [10], and with the subsequent proteomic studies for the specific identification of S-nitrosated proteins (summarized in Table 1) this number is increasingly growing. The reported S-nitrosoproteins are involved in diverse cell functions including transcription factors, ion channels, membrane receptors, structural proteins, and enzymes [8]. However, the proteomic studies have been influenced by the relatively low sensitivity of the biotin-switch assay and, since S-nitrosoprotein content in vivo is usually too low, most of them have relied on exogenous treatments to increase the intracellular SNO pool. For that reason, some authors prefer © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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the term S-nitrosatable proteome” to S-nitrosoproteome. Table 1 summarizes the different treatments in vivo and in vitro as well as the methodology used in different studies to address the S-nitrosoproteome in diverse organisms, organs, and cells. Several proteomic analyses aimed to S-nitrosoprotein identification in organs such as brain [30, 47], kidney [55], liver [56], and heart [52]. Other studies identified Snitrosoproteins in different types of cells such as endothelial [22, 57, 58], mesangial [59], vascular smooth muscle [29], macrophages [31], colon epithelial [60], HeLa [49], hepatocytes [61, 62], and spermatozoa [63]. Different groups have also reported the proteomic identification of S-nitrosoproteins in plant leaf extracts [64–66] and cell suspensions [64], and in bacteria [67]. The mechanism of S-nitrosation in vivo is unknown and it is not clear which cell treatment will resemble more closely physiological S-nitrosation. Therefore, different methods of manipulating S-nitrosoprotein levels have been used in different experimental models (Table 1). Some approaches have used exogenous or endogenous methods of increasing NO. Exogenous methods of increasing NO are based on the addition of compounds that donate NO, whereas endogenous methods have basically relied on manipulation of NOS, the enzyme that synthetizes NO. For example, Jaffrey et al. [30] demonstrated that a group of endogenously S-nitrosated proteins in mice brain lysates lost their modification in animals lacking neuronal NOS. Other study has reported the Snitrosation of proteins in murine macrophages with augmented endogenous NO synthesis after endotoxin stimulation [68]. However, as shown in Table 1, the majority of studies have used treatments with chemical donors of NO. An NO donor is a compound that releases NO under physiological conditions. The various groups of NO donors include organic nitrates, inorganic nitrites, inorganic nitroso compounds, and S-nitrosothiols. It should be noted that different NO donors have different potencies, half-lives, activation thresholds, and mechanism of NO release, some compounds decomposing chemically, while others need enzyme catalysis. The mostly used NO donors in the proteomic identification of S-nitrosation targets were NONOates (diazeniumdiolates) and S-nitrosothiols. NONOates are stable as solids but decompose in solution releasing NO. S-nitrosothiols commonly used to generate NO in solution include GSNO and CSNO and these compounds may also act as transnitrosating agents rather than NO releasers. Importantly, these physiologically relevant SNOs elicit cellular responses that are not reproduced by NO itself and transnitrosation via low molecular weight SNOs may constitute a physiological mechanism of S-nitrosoprotein formation [13, 15]. However, it is also important to define S-nitrosation targets in cells exposed to increased levels of extracellular NO release. The majority of studies did not evaluate different classes of NO donors in the same experimental model and, to date, there are scarce data that allow to compare S-nitrosation targets under different conditions of NO production. Jaffrey et al. found and identical subset of proteins when mice brain www.proteomics-journal.com

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Proteomics 2009, 9, 808–818 Table 1. Summary of proteomic studies on S-nitrosation

Sample

Treatmenta)

Arabidopsis cell suspensions Arabidopsis leaf extracts Arabidopsis leaf extracts K. pinnata leaf extracts Bovine endothelial cells Human aortic endothelial cells HASMC

250 mM GSNO, in vitro

Human colon adenocarcinoma cells Human epithelial cells (HeLa) Human hepatocytes

Human hepatocytes Human umbilical vein endothelial cells Human spermatozoa Mouse brain lysates Mouse heart membrane fractions Mouse mesangial cells Mouse macrophages Mouse macrophages Mycobacterium tuberculosis Rat cerebellum lysates Rat cerebellum lysates Rat liver

Methodologyc)

Method of identification

Reference

63

BS

LC-MS/MS

[64]

NO gas, in vivo Hypersenssitive response 250 mM GSNO, in vitro 1 mM CSNO, in vivo 2 mM DEA NONOate, in vivo 100 mM CSNO, in vivo 2 mM PAPANO, in vivo 500 mM Deoxycholate, in vivo 100 mM GSNO, in vitro

52 16 19 9 5

BS 1 2-D BS 1 1-D BS 1 1-D BS 1 2-D

PMF PMF PMF 1 LC-MS/MS PMF

[65] [66] [38] [22]

16 4 18

BS#

LC-MS/MS

[29]

BS 1 2-D

LC-MS/MS

[60]

40

BS#

LC-MS/MS

[49]

5 mM CSNO, in vivo 20 mM D-Galactosamine, in vivo 5 mM GSNO, in vitro 5 mM CSNO, in vivo 30 mM Glucose, in vivo

15 7

BS 1 1-D

PMF 1 LC-MS/MS

[61]

25 20 (4) 2

BS

LC-MS/MS

[62]

BS 1 1-D

PMF

[58]

240

BS 1 1-D

PMF

[63]

15

BS 1 1-D

PMF

[30]

9 17

BS#

PMF

[52]

34 3 15 29

BS 1 2-D BS 1 1-D BS 1 1-D BS 1 1-D

PMF PMF Edman sequencing PMF 1 MS/MS

[59] [31] [68] [67]

56 (4) 19 28

BS# HTS 1 1-D BS 1 2-D

LC-MS/MS PMF 1 LC-MS/MS PMF

[47] [50] [56]

100 mM GSNO, 100 mM CSNO, in vivo 40 mM GSNO, 40 mM DEA NONOate, in vitro Ischemic preconditioning GSNO preconditioning (100 mM), in vivo 40 mM GSNO, in vitro 250 mM GSNO, in vivo Endotoxin induction of INOS 30 mM NaNO2 (pH 5.5), in vivo 2–10 mM GSNO, in vitro 10 mM GSNO, in vitro Alcohol feeding

Number of S-nitrosoproteins identifiedb)

a) In vitro, treatment done in cell lysates. In vivo, treatment done in cells or tissues. b) The numbers in parentheses indicate those S-nitrosoproteins identified in control or untreated samples. c) BS, biotin-switch method; HTS, His-tag switch method; 1-D, 1-D SDS-PAGE; 2-D, 2-D SDS-PAGE; #, identification of Cys S-nitrosation sites.

lysates were incubated with GSNO or with DEA-NONOate [30], but in the study by Greco et al. [29] only half of the Snitrosoproteins identified in human aortic smooth muscle cells (HASMC) exposed to PAPANO were also identified when cells were exposed to CSNO. Regardless of the higher efficiency of S-nitrosation of HASMC proteins by CSNO (3 nmol of protein SNO per mg of protein, 16 identified Snitrosoproteins) compared to that of the NO donor (0.4 nmol of protein SNO per mg of protein, four identified S-nitrosoproteins), the results of this latter study suggest the potential selectivity of S-nitrosation of cellular proteins under different © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

conditions of nitrosative stress. Also in this regard, S-nitrosation of mitochondrial membrane proteins has been shown to occur upon exposure to low molecular weight SNOs, but not to free NO released from DETA-NONOate [69]. It is noteworthy that the different set of S-nitrosated proteins reported in these proteomic studies, although some overlapping exists. For instance, roughly half of the S-nitrosoproteins identified in the medicinal plant Kalanchoe pinnata [66] had been previously identified as S-nitrosation targets not only in Arabidopsis thaliana but also in animals. Also in this regard, many of the proteins identified as targets for www.proteomics-journal.com

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S-nitrosation in human hepatocytes exposed to CSNO [62] were also detected as S-nitrosoproteins in the liver of alcohol fed rats [56], suggesting that exposure of human hepatocytes to this low-molecular weight SNO constitutes a suitable model of diseased liver, and also pointing to the relevance of a particular S-nitrosoproteome pertaining to the pathophysiology of liver disease. Data from the nitrosoproteomic studies summarized in Table 1 should be used for the further investigation of the S-nitrosation status of each identified Snitrosoprotein. These targeted studies, that may involve NO release from immunoprecipitated proteins or immunodetection after capture on strepatividin in the biotin-switch assay, should be performed in the same or similar experimental models but under appropriate physiological or pathophysiological conditions, prior to suggest a biological significance for the modification of the observed S-nitrosoprotein. In the complex cellular environment, the amount of Snitrosation and the stability of this PTM highly depend on multiple cell-specific processes such as NO production, glutathione metabolism, antioxidant status, and nitrosothiol homeostasis. For example, after exposing cells to SNOs, only three S-nitrosoproteins were identified in murine macrophages [31], a cell type with a high-output NO pathway that may demand an improved maintenance of SNO homeostasis. However, more than 200 S-nitrosoproteins were identified in SNO-exposed human spermatozoa [63], that are highly specialized cells with a limited defense against oxidative and nitrosative stress [70]. Several enzymatic mechanisms that are dependent on cell type play important roles in SNO homeostasis. Thus, different NOS isoforms have been associated with the production of cellular SNOs [8, 14, 30, 71–73], although the mechanisms of this NOS-associated SNO formation are not clearly understood [74]. Additionally, GSNO reductase (GSNOR) has been identified as a highly conserved metabolizing enzyme involved in SNO homeostasis [14, 15, 73, 75]. This enzyme is formally an alcohol dehydrogenase, but shows much greater activity toward GSNO than toward any other substrate [76]. Mice lacking GSNOR have higher cellular quantity of both GSNO and protein SNO, and exhibit higher tissue damage and mortality following NOS induction after endotoxic or bacterial challenge [14, 15]. These studies provide convincing evidence that S-nitrosoproteins and GSNO are in dynamic equilibrium, and that transnitrosation via GSNO is an integral mechanism of S-nitrosoprotein formation. Other enzymes might also influence SNO and S-nitrosoprotein levels. In this regard, the thioredoxin/thioredoxin reductase system has been described to cleave GSNO [77] and S-nitrosoproteins [78]. Furthermore, a recent study suggests that thioredoxin system may constitute a specific enzymatic mechanism of regulating basal and stimulus-induced protein denitrosation in distinct cellular compartments [79]. Other enzyme catalyzing denitrosation is copper-zinc superoxide dismutase (SOD1) and mutations in SOD1 gene have been shown to be responsible for an aberrant decrease in SNOs in © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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cell and animal models of amyotrophic lateral sclerosis [80, 81]. The alteration or manipulation of the levels of these enzymatic systems that are involved in SNO homeostasis may constitute a more rational approach to investigate Snitrosation targets in specific cell types. For example, stabilization of modified proteins through RNA silencing techniques targeted to these enzymatic systems, rather than treating cells with high nonphysiological SNOs concentrations, could represent a convenient experimental design in future nitrosoproteomic studies.

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Nitrosoproteomics in the study of cell signaling by nitric oxide

NO signaling through S-nitrosation is one area of burgeoning interest in cell signaling studies. However, several inherent aspects of S-nitrosation are hampering the progress of proteomic approaches on this subject. Relevant cell signaling proteins that are known to be regulated by S-nitrosation, such as NF-k B subunits [82, 83], caspases [21, 71, 84], protein tyrosine phosphatases [85], or JNK [86] and ASK1 [87] kinases, are nevertheless almost absent from the lists of Snitrosoproteins identified in the proteomic studies summarized in Table 1. Most of these proteins have been identified in targeted studies involving in vitro S-nitrosation of purified candidate proteins or by analysis of NO release from immunoprecipitated proteins. These methods may detect proteins that are S-nitrosated at low levels, but require preselection of candidate proteins. It is obvious that more refined and sensitive detection technologies have to be devised in order to study S-nitrosation in the context of NO-signaling by using proteomic approaches. However, some exciting new strategies are under development. In this respect, a recently developed ELISA assay for detection of specific S-nitrosated intracellular proteins has been described [88]. This method is based on immobilization of the desired protein in plate wells coated with a specific capture antibody and then the NO released from S–NO groups after incubation with HgCl2 is assayed. The previous specific biotin-tagging of S-nitrosoproteins followed by detection with HRP-labeled streptavidin may increase sensitivity. This approach has only been tested for overexpressed intracellular proteins [88] and further improvement is needed for the analysis of S-nitrosation of proteins with normal or even low expression levels. It is reasonable to expect that the development and refinement of this or other antibody-based techniques will make specific Snitrosation protein chips available in the future. The improvement of these new available analytical tools and the use of new strategies, such as the stabilization of modified proteins through RNA silencing techniques targeted to the enzymatic systems that participate in SNO homeostasis, may help to identify the “deep nitrosoproteome,” those low abundant S-nitrosated proteins that participate in NO-mediated signal transduction. www.proteomics-journal.com

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The selectivity of NO-signaling via S-nitrosation is other important subject that nitrosoproteomic analysis may deal with. The S-nitrosation of specific Cys residues in proteins entails a selective response of proteins to changes in cellular NO levels. It has been suggested that some protein Cys residues may have evolved as “NO sensors” and that these molecular adaptations made Snitrosation a preeminent form of NO signaling [89]. The development of new analytical methods and strategies over the past several years permits now the study of the nitrosoproteome on a global scale and many studies have begun to provide proteomic data that are essential to reveal the molecular principles governing the selective Snitrosation of protein thiols and its functional consequences. Thus, in an interesting nitrosoproteomic study [29], the proteomic data provided direct evidence supporting two S-nitrosation motifs that may govern the selectivity of this modification. Sequence alignment of Snitrosated peptides revealed a high occurrence of acidic (D, E) residues at positions -3 and -4, and basic (K,R,H) residues at position 2, relative to modified cysteine. In addition, sequence analysis showed that S-nitrosocysteines in some of the S-nitrosoproteins were located in discrete motifs of increased hydrophobicity.

7

Concluding remarks

Among the reversible thiol-based protein modifications, S-nitrosation of Cys residues is of particular interest because it may enable the regulation of protein function by NO as well as contributing to nitrosative stress. Although the purification of S-nitrosoproteins is still a challenging methodological problem, proteomic technologies have begun to contribute to the analysis of the cellular nitrosoproteome in diverse organisms. In this review we have detailed recent proteomic approaches that are currently used for the systematic assessment of potential targets for protein S-nitrosation. However, more physiologically relevant methods of altering cellular S-nitrosoprotein content must be explored. In this sense, the effects of manipulating endogenous production of NO or altering levels of the enzymatic systems involved in SNO homeostasis must be further investigated in the context of nitrosoproteomic screening. Compartmentalization of NO production in relation to selective protein S-nitrosation should also be addressed using proteomic approaches, helping to illuminate some of the biological functions of NO in cell organelles such as nuclei or mitochondria. Progress in and continuous development of new highly specific and sensitive methodologies to identify S-nitrosation sites in proteins will expand our current knowledge of the biological functions of this remarkable PTM. Undoubtedly, unanticipated new protein characters will enter the stage of NO biology. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

This work was supported by a grant to A. R.-A. from the Programa de Promoción de la Investigación en Salud del Ministerio de Sanidad y Consumo (07/0159). Ciberehd is funded by the Instituto de Salud Carlos III. The authors have declared no conflict of interest.

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