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Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages
Nitric Oxide 12 (2005) 121–126 www.elsevier.com/locate/yniox
Brief Communication
IdentiWcation of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages夽 Chengjiang Gao, Hongtao Guo, Junping Wei, Zhiyong Mi, Philip Y. Wai, Paul C. Kuo¤ Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA Received 2 June 2004; revised 1 November 2004 Available online 25 December 2004
Abstract Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identiWcation of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identiWcation of the “universe” of S-nitrsoylated proteins. In this study using endotoxinstimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identiWed as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins speciWc to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not suYcient. 2004 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Biotin switch; Lipolysaccharide; Proteome
Nitric oxide (NO) is a pleuripotent regulator of multiple cellular functions, in part, as a result of its participation in redox chemistry [1,2]. The formation of S-nitrosothiols exempliWes those pathways of NO oxidation that lead to surrogate NO-like bioactivity and signal transduction with allosteric receptor modiWcation, inhibition of sulfhydryl-enzyme activities, and down-regulation of transcriptional activators. Previous research eVorts to characterize S-nitrosylated proteins have utilized reductionist techniques to focus on single proteins and their associated NO-dependent functions. Recently, JaVrey and Snyder [3] described the “biotin 夽 Supported by NIH AI44629, GM65113 and GM069331 grants to P.C.K. * Corresponding author. Fax: +1 919 684 8716. E-mail address: [email protected] (P.C. Kuo).
1089-8603/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.11.006
switch” technique for isolation of S-nitrosylated proteins. This approach allows investigators to potentially isolate the entire proteome of S-nitrosylated proteins in cell systems. In this study, utilizing RAW264.7 murine macrophages exposed to endotoxin (LPS), we utilize the: (1) “biotin switch” technique for isolation of the family of S-nitrosylated proteins and (2) MALDI-TOF protein sequencing for identiWcation of these proteins. When compared to those proteins S-nitrosylated following exposure of endothelial or mesangial cells to exogenous NO donors, our results indicate distinctly diVerent proteins are modiWed following LPS stimulation of endogenous NO synthesis in RAW264.7 murine macrophages. This suggests that LPS-mediated synthesis of NO can function in an autocrine fashion to alter a wide variety of cellular proteins and their associated functions.
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C. Gao et al. / Nitric Oxide 12 (2005) 121–126
Experimental procedures
Proteins preparation for proteomic analysis
Reagents S-Nitrosoglutathione (GSNO) and GSH were purchased from Sigma. LPS was purchased from R&D Systems. 1400W was puchased from Cayman Biochemicals. Streptavidin–agarose, biotin–HPDP, and goat anti-biotin were obtained from Pierce. Hybond-P membranes and ECL reagents were from Amersham Biosciences.
To prepare cytosolic and membrane-associated proteins, RAW264.7 cells were harvested by scraping, resuspended in 20 volumes of Hen buVer (250 mM Hepes, pH 7.9, 1 mM EDTA, and 0.1 mM neocuproine) containing 0.4% Chaps, and allowed to swell for 15 min at 4 °C. The cells were centrifuged at 2000g for 10 min at 4 °C, and the supernatant was recovered. Protein concentration was determined using the Bio-Rad protein assay system.
Cell culture
Biotin switch
Macrophage RAW264.7 cells were cultured in Dulbecco’s modiWed Eagle’s medium with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 g/ml streptomycin. Macrophages were treated with 100 ng/ml LPS for 12 h to induce inducible nitric oxide synthase (iNOS) expression. In selected instances, the speciWc inhibitor of inducible NO synthase, 1400W (100 M), was added. After incubation for 12 h at 37 °C in 5% CO2, the supernatants and cells were harvested for assays.
The biotin switch procedure was described by JaVrey and Snyder [3]. The cytosolic and membrane-associated proteins (6 mg) from LPS-treated and nontreated macrophages were blocked with 20 mM methyl methanethiosulfonate and 2.5% SDS at 50 °C for 20 min with frequent vortexing. Methyl methanethiosulfonate was removed by precipitation with two volumes of ¡20 °C acetone. After resuspending the proteins in Hen buVer containing 1% SDS, sodium ascorbate solution (1 mM Wnal concentration), and biotin–HPDP (100 M Wnal concentration) were added. The mixtures were incubated for 1.5 h at 25 °C in the dark. Biotinylated nitrosothiols were then acetone-precipitated with two volumes of ¡20 °C acetone. After centrifugation, the pellet was resuspended in 0.1 ml Hen buVer containing 1% SDS/mg of protein in the initial protein sample. Two volumes of neutralization buVer (20 mM Hepes, pH 7.7, 100 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100) were added, and 15 l of streptavidin–agarose/mg of protein used in the initial protein sample were added. The biotinylated proteins were incubated with the resin for 1 h at room temperature. The resin was extensively washed in 10 volumes of neutralization buVer containing 600 mM NaCl. Bound proteins were then eluted in a solution containing 20 mM Hepes, pH 7.7, 100 mM NaCl, 1 mM EDTA, and 100 mM of 2-mercaptoethanol. The samples were then mixed with SDS sample buVer.
Assay of NO production NO released from cells in culture was quantiWed by measurement of the NO metabolite, nitrite. Cell culture medium (50 l) was removed from culture dish and centrifuged; the supernatants were mixed with 50 l sulfanilamide (1%) in 0.5 N HCl. After a 5-min incubation at room temperature, an equal volume of 0.02% N(1-naphthyl)ethylenediamine was added. Following incubation for 10 min at room temperature the absorbance of samples at 540 nm was compared with that of an NaNO2 standard on a MAXLINE microplate reader (Molecular Devices, Sunnyvale, CA). Immunoblot analysis Macrophages were washed three times in PBS and incubated with boiling 2£ nonreducing electrophoresis sample buVer for 2 min. Separation was performed by SDS–12% PAGE, and then the products were electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 mA. The membrane was blocked overnight at 4 °C in Tris-buVered saline (TBS) containing 3% BSA. Blocked membranes were incubated with the anti-mouse iNOS MAb (Transduction Laboratories, Lexington, KY), washed three times in TBS–0.1% Tween, and incubated with biotinylated sheep antimouse IgG (Amersham, Arlington Heights, IL) for 1 h. After being washed three additional times, membranes were incubated with a strepavidin–horseradish peroxidase conjugate. After an additional washing, bound antibodies were detected by the ECL detection system (Amersham, Arlington Heights, IL).
Western blot analysis Biotinylated proteins were resolved by SDS–PAGE, transferred to Hybond-P membranes, detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound antibodies were visualized by the ECL chemiluminescence detection system (Amersham Biosciences). Protein sequencing Immediately after aYnity separation by the biotinswitch method, the puriWed proteins mixed with loading buVer were resolved by SDS–PAGE. Gels were then stained using the silver staining method. The 15
C. Gao et al. / Nitric Oxide 12 (2005) 121–126
most prominent protein bands were excised and digested overnight with trypsin. The resulting digest was then injected onto a Microbore high performance liquid chromatography (Beckman 32 K Gold) system, and the fractions were collected. The 10 best fractions were selected for matrix-assisted laser desorption/ionization mass analysis of the intact protein (ABI/Perseptive Voyager DE-Pro); subsequently, these sequenced
123
using the Edman technique (ABI Procise 470). The resulting data were manually interpreted and searched using Sequest against the NCBI nonredundant data base. Statistical analysis Data are presented means § SEM of three experiments. Analysis was performed using Student’s t test; p values less than 0.05 were considered signiWcant.
Results LPS-mediated NO synthesis
Fig. 1. Immunoblot ananlysis of iNOS protein expression in RAW264.7 murine macrophages. RAW264.7 macrophages were treated with 100 ng/ml LPS for 12 h to induce inducible nitric oxide synthase (iNOS) expression. In selected instances, the inhibitor of iNOS, 1400W (100 M), was added. Unstimulated cells served as Controls. Immunoblots were performed as described under Experimental procedures. -Actin served as the loading control. Blot is representative of three experiments.
To conWrm iNOS protein expression in the presence of LPS stimulation, immunoblot analysis was performed (Fig. 1). In the presence of LPS and LPS + 1400W, iNOS protein was expressed in equivalent amounts under both treatment conditions. Nitrite levels were then assayed as a measure of NO synthesis. In Control, LPS + 1400W and 1400W treatment groups, nitrite concentrations were 1.5 § 0.5, 2.5 § 0.6, and 2.2 § 0.6 nmol/mg protein, respectively. These were not signiWcantly diVerent from each other. In contrast, the nitrite level in LPS treated cells was 25.3 § 3.4 nmol/mg protein (p < 0.01 vs Control, LPS + 1400W and 1400W).
Fig. 2. (A) Western blot of biotinylated proteins in Control and LPS-treated RAW264.7 murine macrophages. Proteins were resolved by SDS– PAGE, transferred to Hybond-P membranes, and detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound strepavidin was visualized by the ECL chemiluminescence detection system (Amersham Biosciences). Blot is representative of three experiments. (B) Silver stain of biotinylated proteins in Control and LPS-treated RAW264.7 murine macrophages. Gel is representative of three experiments.
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Western blot analysis To determine the range of biotinylated proteins following biotin switch, the proteins from unstimulated Controls and LPS-treated RAW264.7 cells were examined by immunoblot analysis (Fig. 2A). Biotinylated proteins were resolved by SDS–PAGE, transferred to Hybond-P membranes, detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound antibodies were visualized by the ECL chemiluminescence detection system (Amersham Biosciences). There was a wide range of biotinylated proteins and theoretically, S-nitrosylated proteins, in the LPS cells, but essentially none were present in the unstimulated Controls. These proteins were also resolved using silver staining (Fig. 2B). SpeciWc controls were also performed. Equivalency of protein loading was conWrmed with visualization using Coomassie blue (Fig. 3A). The biotin switch procedure was then performed in the presence and absence of biotin (Fig. 3B). Interestingly, in the presence of biotin, LPS + 1400W cells, the pattern is not diVerent from that noted in Control cells. In Control and LPS cells, omission of biotin results in no endogenously biotinylated proteins that are visibly detectable. Finally, addition of dithiothreitol (DTT, 1 mM) as a reducing agent results in ablation of all signal in the LPS treated cells. Protein sequencing of isolated proteins The 15 “best” fractions that were isolated using the biotin switch technique were sequenced (Table 1). These were consistent among the three experiments performed. The list includes three proteins involved in glycolysis: glyceraldehayde-3-phosphate (GAPDH), pyruvate kinase, and enolase, four structural proteins: -actin, tubulin, plastin-2, and SMC-6, two translational proteins: elongation factor-1 and -2, and two heat shock proteins: hsp84 and hsp86.
Discussion S-Nitrosylation of thiol groups of cysteine is the prototypical post-translational modiWcation by which NO acts to regulate various cellular functions [4]. It is thought that disruption or dysregulation of S-nitrosothiol signalling leads to impairment of cellular functions. As such, given the ubiquitous presence of NO in physiologic and pathophysiologic settings, identiWcation of the “universe” of Snitrosylated proteins in the proteomic approach is of great interest and potential import. Delineating the functional correlates of S-nitrosylation can begin with identiWcation of the entire population of S-nitrososylated proteins. In fact, these S-nitrosylated proteins have been termed the “nitrosylome” by Martinez-Ruiz and Lamas [5].
Fig. 3. (A) Coomassie blue stain of biotinylated protein in Control and LPS-treated RAW264.7 cells. Gel is representative of three experiments. (B) Western blot of biotinylated proteins in Control and LPStreated RAW264.7 murine macrophages. Proteins were resolved by SDS–PAGE, transferred to Hybond-P membranes, and detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound strepavidin was visualized by the ECL chemiluminescence detection system (Amersham Biosciences). In selected instances, biotin was omitted, 1400W (100 M) was added as a speciWc inhibitor of iNOS activation, or DTT (1 mM) was included as a reducing agent. Blot is representative of three experiments.
Large scale identiWcation of S-nitrosylated proteins has been hampered in the past by lack of techniques which could address the labile nature of this reaction and disruption of the nitrosothiol bond during MALDI. However, over 100 individual proteins have been found to be S-nitrosylated in various systems, typically on the
C. Gao et al. / Nitric Oxide 12 (2005) 121–126
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Table 1 S-Nitrosylated proteins in LPS stimulated RAW264.7 murine macrophages Protein name
Accession No.
Protein MW (kDa)
C.I. (%)
Glyceraldehyde-3-phosphate dehydrogenase -Actin Pyruvate kinase 3 FRAG-6 Enolase-1 Elongation factor-1, chain Elongation factor-2 -Tubulin Plastin-2 GTP binding protein 1 Heat shock protein-hsp84 Heat shock protein-hsp86 CEM-15—ApoB mRNA editing enzyme SMC-6—structure maintenance of chromosomes MKIAA1068—murine homologue of human protein 1068
GI: 6679937 GI: 49868 GI: 31981562 GI: 2707972 GI: 12963491 GI: 72870 GI: 192989 GI: 6678469 GI: 315433113 GI: 6681225 GI: 6680305 GI: 1170384 GI: 26328763 GI: 20071400 GI: 37360216
36 39 57.8 21.3 47.1 50.0 29.9 49.8 70 40.5 83.3 86 95 40.2 12.3
100 100 100 100 100 99.8 100 100 100 100 100 100 100 61 39
basis of in vitro modiWcation or exogenous sources of NO [6]. JaVrey and Snyder [3] described the biotin switch technique, in which a biotin group is substituted at each cysteine thiol modiWed by nitrosylation. These biotinylated proteins can then be isolated by biotin–strepavidin aYnity chromatography or immunoblotting, as we performed in our studies. In previous studies, JaVrey et al. [7] identiWed S-nitrosylated proteins following exposure of murine brain cell lysates to S-nitrosoglutathione (GSNO) with subsequent conWrmation comparing wildtype and nNOS deWcient mice. Among the identiWed proteins were metabolic enzymes such as GAPDH, creatine kinase, hexokinase1, and glycogen phosphorylase; ion channels such as the NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor, the hyperpolarization-activated cation channel isoforms 2 and 3, and the sodium-pumping enzyme Na+/K+ ATPase 1 and 2 subunits; structural proteins such as neuroWlament heavy chain (NF-H), - and -tubulin, and - and -actin; and signalling proteins such as the retinoblastoma gene product (Rb), heat-shock protein 72 (Hsp72), isoforms 1, 2, and 4 of the collapsinresponse-mediator protein (CRMP), and calbindin. Subsequently, using a system of mouse mesangial cells, Kuncewicz et al. [8] identiWed an additional 31 unique Snitrosylated proteins following exposure of their cells to GSNO. Three, uroguanylin, peroxisome proliferatoractivated receptor-, and NADPH-cytochrome p450 reductase, were found to be S-nitrosylated following subsequent exposure of mesangial cells to IL-1. Finally, Martinez-Ruiz and Lamas [5] have also utilized the same technique to identify S-nitrosylated proteins in bovine aortic endothelial cells exposed to GSNO. Nine proteins were isolated. Among these three studies, a great deal of overlap is present, especially with target identiWcation of GAPDH and -actin. Of greater interest, however, is the tremendous degree of disparity amongst these groups in the various target proteins. Using this same biotin switch technique, Foster and Stamler [9] have examined protein
S-nitrosylation in rat liver mitochondria. They identify a number of target proteins: sarcosine dehydrogenase, catalase, dihydrolipoamide dehydrogenase, hydroxymethylglutaryl-CoA synthase, glutamate oxaloacetate transaminase, and malate dehydrogenase. In our study, murine RAW264.7 macrophage cells were exposed to endotoxin to induce iNOS expression. Unlike the other studies, there was no component in which cells were exposed to a source of exogenous NO. Like the studies previously mentioned, our results indicate that a large number of proteins are S-nitrosylated in this setting. Again, some proteins common to the other studies were found: GAPDH, -actin, heat shock proteins, and -tubulin. Of interest, a number of proteins unique to this setting were also found: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. Review of the Medline database Wnds no previous publications demonstrating S-nitrosylation of any of these proteins. Enolase and pyruvate kinase are metabolic enzymes required for glycolysis. Although neither has been identiWed as a target for Snitrosylation, both have been found to have NO-dependent functions. With regard to pyruvate kinase, Momken et al. [10] demonstrated that the absence of eNOS in soleus muscle induced a marked decrease in pyruvate kinase (¡26%) activity. Loss of neuronal enolase activity in the presence of NMDA has been found to be ablated by a competitive substrate inhibitor of NOS activity [11]. Elongation factor-1 chain is a large G protein and functions as part of a translational multimolecular complex reported to be a target of CDK1/cyclin B, the universal regulator of M phase [12]. During protein synthesis, EF-1 forms a ternary complex with aminoacylated tRNA and GTP (eEF1A-GTP-aatRNA), and delivers aatRNA to the ribosome after GTP hydrolysis. Protein synthesis elongation factor-2 catalyzes the translocation of the peptidyl-tRNA from the A site to the P site of the ribosome. Most organisms encode a single EF2 protein and its activity is regulated by
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phosphorylation [13]. Of note, is the observation that both EF-1 and EF-2 also function as actin-binding proteins [14]. Plastin-2 or Wmbrin is an actin cross-linking protein [15]. Several multiprotein complexes containing members of the structural maintenance of chromosomes (SMC) family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization; the SMC5/6 complex is required for a coordinated response to DNA damage and normal chromosome integrity [16]. Finally, CEM15, also known as the human protein apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), mediates a newly described form of innate resistance to retroviral infection by catalyzing the deamination of deoxycytidine to deoxyuridine in viral cDNA replication intermediates [17]. FRAG-6 mRNA induction by interferons is IRF-1-independent and it is likely to be activated by the JAK/STAT pathway [18]. In a similar fashion, Zhang and Hogg [19] have recently examined the formation of S-nitrosothiols in RAW cells exposed to LPS. The measured concentration of intracellular S-nitrosothiols was 17.4 § 1.0 pmol/mg protein and was associated with the high molecular weight (>3 kDa) pool. These intracellular nitrosothiols are stable with half-lives of approx. 3 h. The authors postulate that the stable pool of S-nitrosothiols is isolated from the GSH pool and is directly nitrosated. Our results suggest that the biotin switch technique is a viable technique for identiWcation of S-nitrosylated proteins. Certainly, based upon the studies to date, a widely disparate group of setting- and cell-speciWc proteins are modiWed in the presence of NO. The question as to why certain proteins are S-nitrosylated in speciWc settings in speciWc cells is unknown. While the ubiquitous nature of NO argues for some degrees of commonality as with GAPDH and -actin, S-nitrosylation of other proteins such as EF-1 and -2 in the speciWc setting of endotoxin stimulated macrophages suggests a regulatory mechanism for which NO is necessary, but not suYcient. The varying nature of the identiWed target proteins indicates that more is required than the presence of NO. References [1] C. Nathan, Nitric oxide as a secretory product of mammalian cells, FASEB 6 (1992) 1157–1175.
[2] A.B. Wilson, J.M. Harris, R.R.A. Coombs, Interleukin-2 induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors including interleukin-1, Cell. Immunol. 113 (1988) 130–142. [3] S.R. JaVrey, S.H. Snyder, The biotin switch method for the detection of S-nitrosylated proteins, Sci. STKE (2001) PL1. [4] M.W. Foster, T.J. McMahon, J.S. Stamler, S-Nitrosylation in health and disease, Trends Mol. Med. 9 (2003) 160–168. [5] A. Martinez-Ruiz, S. Lamas, Detection and proteomic identiWcation of S-nitrosylated proteins in endothelial cells, Arch. Biochem. Biophys. 423 (2004) 192–199. [6] J.S. Stamler, S. Lamas, F.C. Fang, Nitrosylation. the prototypic redox-based signaling mechanism, Cell 106 (2001) 675–683. [7] S.R. JaVrey, H. Erdjument-Bromage, C.D. Ferris, P. Tempst, S.H. Snyder, Protein S-nitrosylation: a physiological signal for neuronal nitric oxide, Nat. Cell Biol. 3 (2001) 193–197. [8] T. Kuncewicz, E.A. Sheta, I.L. Goldknopf, B.C. Kone, Proteomic analysis of S-nitrosylated proteins in mesangial cells, Mol. Cell Proteomics 2 (2003) 156–163. [9] M.W. Foster, J.S. Stamler, New insights into protein S-nitrosylation. Mitochondria as a model system, J. Biol. Chem. 279 (2004) 25891–25897. [10] I. Momken, D. Fortin, B. Serrurier, X. Bigard, R. Ventura-Clapier, V. Veksler, Endothelial nitric oxide synthase (NOS) deWciency aVects energy metabolism pattern in murine oxidative skeletal muscle, Biochem. J. 368 (2002) 341–347. [11] H. Kollegger, G.J. McBean, K.F. Tipton, Reduction of striatal Nmethyl-D-aspartate toxicity by inhibition of nitric oxide synthase, Biochem. Pharmacol. 45 (1993) 260–264. [12] C. Cans, B.J. Passer, V. Shalak, V. Nancy-Portebois, V. Crible, N. Amzallag, D. Allanic, R. TuWno, M. Argentini, D. Moras, G. Fiucci, B. Goud, M. Mirande, R. Amson, A. Telerman, Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A, Proc. Natl. Acad. Sci. USA 100 (2003) 13892–13897. [13] T.M. Malave, J.D. Forney, IdentiWcation of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila, Gene 326 (2004) 97–105. [14] M. Bektas, B. Guncer, C. Guven, R. Nurten, E. Bermek, Actin—an inhibitor of eukaryotic elongation factor activities, Biochem. Biophys. Res. Commun. 317 (2004) 1061–1066. [15] K. Ohsawa, Y. Imai, Y. Sasaki, S. Kohsaka, Microglia/macrophage-speciWc protein Iba1 binds to Wmbrin and enhances its actin-bundling activity, J. Neurochem. 88 (2004) 844–856. [16] S.H. Harvey, D.M. Sheedy, A.R. Cuddihy, M.J. O’Connell, Coordination of DNA damage responses via the Smc5/Smc6 complex, Mol. Cell. Biol. 24 (2004) 662–674. [17] A.M. Sheehy, N.C. Gaddis, M.H. Malim, The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif, Nat. Med. 9 (2003) 1404–1407. [18] A.M. Silva, R.L. Bottrel, L.F. Reis, IdentiWcation and partial characterization of FRAG-6, a novel interferon-stimulated gene that is expressed in an IRF-1-independent manner, Cytokine 11 (1999) 813–821. [19] Y. Zhang, N. Hogg, Formation and stability of S-nitrosothiols in RAW 264.7 cells, Am. J. Physiol. Lung Cell Mol. Physiol. 287 (2004) L467–L474.
Brief Communication
IdentiWcation of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages夽 Chengjiang Gao, Hongtao Guo, Junping Wei, Zhiyong Mi, Philip Y. Wai, Paul C. Kuo¤ Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA Received 2 June 2004; revised 1 November 2004 Available online 25 December 2004
Abstract Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identiWcation of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identiWcation of the “universe” of S-nitrsoylated proteins. In this study using endotoxinstimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identiWed as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins speciWc to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not suYcient. 2004 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Biotin switch; Lipolysaccharide; Proteome
Nitric oxide (NO) is a pleuripotent regulator of multiple cellular functions, in part, as a result of its participation in redox chemistry [1,2]. The formation of S-nitrosothiols exempliWes those pathways of NO oxidation that lead to surrogate NO-like bioactivity and signal transduction with allosteric receptor modiWcation, inhibition of sulfhydryl-enzyme activities, and down-regulation of transcriptional activators. Previous research eVorts to characterize S-nitrosylated proteins have utilized reductionist techniques to focus on single proteins and their associated NO-dependent functions. Recently, JaVrey and Snyder [3] described the “biotin 夽 Supported by NIH AI44629, GM65113 and GM069331 grants to P.C.K. * Corresponding author. Fax: +1 919 684 8716. E-mail address: [email protected] (P.C. Kuo).
1089-8603/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.11.006
switch” technique for isolation of S-nitrosylated proteins. This approach allows investigators to potentially isolate the entire proteome of S-nitrosylated proteins in cell systems. In this study, utilizing RAW264.7 murine macrophages exposed to endotoxin (LPS), we utilize the: (1) “biotin switch” technique for isolation of the family of S-nitrosylated proteins and (2) MALDI-TOF protein sequencing for identiWcation of these proteins. When compared to those proteins S-nitrosylated following exposure of endothelial or mesangial cells to exogenous NO donors, our results indicate distinctly diVerent proteins are modiWed following LPS stimulation of endogenous NO synthesis in RAW264.7 murine macrophages. This suggests that LPS-mediated synthesis of NO can function in an autocrine fashion to alter a wide variety of cellular proteins and their associated functions.
122
C. Gao et al. / Nitric Oxide 12 (2005) 121–126
Experimental procedures
Proteins preparation for proteomic analysis
Reagents S-Nitrosoglutathione (GSNO) and GSH were purchased from Sigma. LPS was purchased from R&D Systems. 1400W was puchased from Cayman Biochemicals. Streptavidin–agarose, biotin–HPDP, and goat anti-biotin were obtained from Pierce. Hybond-P membranes and ECL reagents were from Amersham Biosciences.
To prepare cytosolic and membrane-associated proteins, RAW264.7 cells were harvested by scraping, resuspended in 20 volumes of Hen buVer (250 mM Hepes, pH 7.9, 1 mM EDTA, and 0.1 mM neocuproine) containing 0.4% Chaps, and allowed to swell for 15 min at 4 °C. The cells were centrifuged at 2000g for 10 min at 4 °C, and the supernatant was recovered. Protein concentration was determined using the Bio-Rad protein assay system.
Cell culture
Biotin switch
Macrophage RAW264.7 cells were cultured in Dulbecco’s modiWed Eagle’s medium with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 g/ml streptomycin. Macrophages were treated with 100 ng/ml LPS for 12 h to induce inducible nitric oxide synthase (iNOS) expression. In selected instances, the speciWc inhibitor of inducible NO synthase, 1400W (100 M), was added. After incubation for 12 h at 37 °C in 5% CO2, the supernatants and cells were harvested for assays.
The biotin switch procedure was described by JaVrey and Snyder [3]. The cytosolic and membrane-associated proteins (6 mg) from LPS-treated and nontreated macrophages were blocked with 20 mM methyl methanethiosulfonate and 2.5% SDS at 50 °C for 20 min with frequent vortexing. Methyl methanethiosulfonate was removed by precipitation with two volumes of ¡20 °C acetone. After resuspending the proteins in Hen buVer containing 1% SDS, sodium ascorbate solution (1 mM Wnal concentration), and biotin–HPDP (100 M Wnal concentration) were added. The mixtures were incubated for 1.5 h at 25 °C in the dark. Biotinylated nitrosothiols were then acetone-precipitated with two volumes of ¡20 °C acetone. After centrifugation, the pellet was resuspended in 0.1 ml Hen buVer containing 1% SDS/mg of protein in the initial protein sample. Two volumes of neutralization buVer (20 mM Hepes, pH 7.7, 100 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100) were added, and 15 l of streptavidin–agarose/mg of protein used in the initial protein sample were added. The biotinylated proteins were incubated with the resin for 1 h at room temperature. The resin was extensively washed in 10 volumes of neutralization buVer containing 600 mM NaCl. Bound proteins were then eluted in a solution containing 20 mM Hepes, pH 7.7, 100 mM NaCl, 1 mM EDTA, and 100 mM of 2-mercaptoethanol. The samples were then mixed with SDS sample buVer.
Assay of NO production NO released from cells in culture was quantiWed by measurement of the NO metabolite, nitrite. Cell culture medium (50 l) was removed from culture dish and centrifuged; the supernatants were mixed with 50 l sulfanilamide (1%) in 0.5 N HCl. After a 5-min incubation at room temperature, an equal volume of 0.02% N(1-naphthyl)ethylenediamine was added. Following incubation for 10 min at room temperature the absorbance of samples at 540 nm was compared with that of an NaNO2 standard on a MAXLINE microplate reader (Molecular Devices, Sunnyvale, CA). Immunoblot analysis Macrophages were washed three times in PBS and incubated with boiling 2£ nonreducing electrophoresis sample buVer for 2 min. Separation was performed by SDS–12% PAGE, and then the products were electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 mA. The membrane was blocked overnight at 4 °C in Tris-buVered saline (TBS) containing 3% BSA. Blocked membranes were incubated with the anti-mouse iNOS MAb (Transduction Laboratories, Lexington, KY), washed three times in TBS–0.1% Tween, and incubated with biotinylated sheep antimouse IgG (Amersham, Arlington Heights, IL) for 1 h. After being washed three additional times, membranes were incubated with a strepavidin–horseradish peroxidase conjugate. After an additional washing, bound antibodies were detected by the ECL detection system (Amersham, Arlington Heights, IL).
Western blot analysis Biotinylated proteins were resolved by SDS–PAGE, transferred to Hybond-P membranes, detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound antibodies were visualized by the ECL chemiluminescence detection system (Amersham Biosciences). Protein sequencing Immediately after aYnity separation by the biotinswitch method, the puriWed proteins mixed with loading buVer were resolved by SDS–PAGE. Gels were then stained using the silver staining method. The 15
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most prominent protein bands were excised and digested overnight with trypsin. The resulting digest was then injected onto a Microbore high performance liquid chromatography (Beckman 32 K Gold) system, and the fractions were collected. The 10 best fractions were selected for matrix-assisted laser desorption/ionization mass analysis of the intact protein (ABI/Perseptive Voyager DE-Pro); subsequently, these sequenced
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using the Edman technique (ABI Procise 470). The resulting data were manually interpreted and searched using Sequest against the NCBI nonredundant data base. Statistical analysis Data are presented means § SEM of three experiments. Analysis was performed using Student’s t test; p values less than 0.05 were considered signiWcant.
Results LPS-mediated NO synthesis
Fig. 1. Immunoblot ananlysis of iNOS protein expression in RAW264.7 murine macrophages. RAW264.7 macrophages were treated with 100 ng/ml LPS for 12 h to induce inducible nitric oxide synthase (iNOS) expression. In selected instances, the inhibitor of iNOS, 1400W (100 M), was added. Unstimulated cells served as Controls. Immunoblots were performed as described under Experimental procedures. -Actin served as the loading control. Blot is representative of three experiments.
To conWrm iNOS protein expression in the presence of LPS stimulation, immunoblot analysis was performed (Fig. 1). In the presence of LPS and LPS + 1400W, iNOS protein was expressed in equivalent amounts under both treatment conditions. Nitrite levels were then assayed as a measure of NO synthesis. In Control, LPS + 1400W and 1400W treatment groups, nitrite concentrations were 1.5 § 0.5, 2.5 § 0.6, and 2.2 § 0.6 nmol/mg protein, respectively. These were not signiWcantly diVerent from each other. In contrast, the nitrite level in LPS treated cells was 25.3 § 3.4 nmol/mg protein (p < 0.01 vs Control, LPS + 1400W and 1400W).
Fig. 2. (A) Western blot of biotinylated proteins in Control and LPS-treated RAW264.7 murine macrophages. Proteins were resolved by SDS– PAGE, transferred to Hybond-P membranes, and detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound strepavidin was visualized by the ECL chemiluminescence detection system (Amersham Biosciences). Blot is representative of three experiments. (B) Silver stain of biotinylated proteins in Control and LPS-treated RAW264.7 murine macrophages. Gel is representative of three experiments.
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Western blot analysis To determine the range of biotinylated proteins following biotin switch, the proteins from unstimulated Controls and LPS-treated RAW264.7 cells were examined by immunoblot analysis (Fig. 2A). Biotinylated proteins were resolved by SDS–PAGE, transferred to Hybond-P membranes, detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound antibodies were visualized by the ECL chemiluminescence detection system (Amersham Biosciences). There was a wide range of biotinylated proteins and theoretically, S-nitrosylated proteins, in the LPS cells, but essentially none were present in the unstimulated Controls. These proteins were also resolved using silver staining (Fig. 2B). SpeciWc controls were also performed. Equivalency of protein loading was conWrmed with visualization using Coomassie blue (Fig. 3A). The biotin switch procedure was then performed in the presence and absence of biotin (Fig. 3B). Interestingly, in the presence of biotin, LPS + 1400W cells, the pattern is not diVerent from that noted in Control cells. In Control and LPS cells, omission of biotin results in no endogenously biotinylated proteins that are visibly detectable. Finally, addition of dithiothreitol (DTT, 1 mM) as a reducing agent results in ablation of all signal in the LPS treated cells. Protein sequencing of isolated proteins The 15 “best” fractions that were isolated using the biotin switch technique were sequenced (Table 1). These were consistent among the three experiments performed. The list includes three proteins involved in glycolysis: glyceraldehayde-3-phosphate (GAPDH), pyruvate kinase, and enolase, four structural proteins: -actin, tubulin, plastin-2, and SMC-6, two translational proteins: elongation factor-1 and -2, and two heat shock proteins: hsp84 and hsp86.
Discussion S-Nitrosylation of thiol groups of cysteine is the prototypical post-translational modiWcation by which NO acts to regulate various cellular functions [4]. It is thought that disruption or dysregulation of S-nitrosothiol signalling leads to impairment of cellular functions. As such, given the ubiquitous presence of NO in physiologic and pathophysiologic settings, identiWcation of the “universe” of Snitrosylated proteins in the proteomic approach is of great interest and potential import. Delineating the functional correlates of S-nitrosylation can begin with identiWcation of the entire population of S-nitrososylated proteins. In fact, these S-nitrosylated proteins have been termed the “nitrosylome” by Martinez-Ruiz and Lamas [5].
Fig. 3. (A) Coomassie blue stain of biotinylated protein in Control and LPS-treated RAW264.7 cells. Gel is representative of three experiments. (B) Western blot of biotinylated proteins in Control and LPStreated RAW264.7 murine macrophages. Proteins were resolved by SDS–PAGE, transferred to Hybond-P membranes, and detected by using a horseradish peroxidase linked streptavidin according to the manufacturer’s protocol (Amersham). Bound strepavidin was visualized by the ECL chemiluminescence detection system (Amersham Biosciences). In selected instances, biotin was omitted, 1400W (100 M) was added as a speciWc inhibitor of iNOS activation, or DTT (1 mM) was included as a reducing agent. Blot is representative of three experiments.
Large scale identiWcation of S-nitrosylated proteins has been hampered in the past by lack of techniques which could address the labile nature of this reaction and disruption of the nitrosothiol bond during MALDI. However, over 100 individual proteins have been found to be S-nitrosylated in various systems, typically on the
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Table 1 S-Nitrosylated proteins in LPS stimulated RAW264.7 murine macrophages Protein name
Accession No.
Protein MW (kDa)
C.I. (%)
Glyceraldehyde-3-phosphate dehydrogenase -Actin Pyruvate kinase 3 FRAG-6 Enolase-1 Elongation factor-1, chain Elongation factor-2 -Tubulin Plastin-2 GTP binding protein 1 Heat shock protein-hsp84 Heat shock protein-hsp86 CEM-15—ApoB mRNA editing enzyme SMC-6—structure maintenance of chromosomes MKIAA1068—murine homologue of human protein 1068
GI: 6679937 GI: 49868 GI: 31981562 GI: 2707972 GI: 12963491 GI: 72870 GI: 192989 GI: 6678469 GI: 315433113 GI: 6681225 GI: 6680305 GI: 1170384 GI: 26328763 GI: 20071400 GI: 37360216
36 39 57.8 21.3 47.1 50.0 29.9 49.8 70 40.5 83.3 86 95 40.2 12.3
100 100 100 100 100 99.8 100 100 100 100 100 100 100 61 39
basis of in vitro modiWcation or exogenous sources of NO [6]. JaVrey and Snyder [3] described the biotin switch technique, in which a biotin group is substituted at each cysteine thiol modiWed by nitrosylation. These biotinylated proteins can then be isolated by biotin–strepavidin aYnity chromatography or immunoblotting, as we performed in our studies. In previous studies, JaVrey et al. [7] identiWed S-nitrosylated proteins following exposure of murine brain cell lysates to S-nitrosoglutathione (GSNO) with subsequent conWrmation comparing wildtype and nNOS deWcient mice. Among the identiWed proteins were metabolic enzymes such as GAPDH, creatine kinase, hexokinase1, and glycogen phosphorylase; ion channels such as the NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) glutamate receptor, the hyperpolarization-activated cation channel isoforms 2 and 3, and the sodium-pumping enzyme Na+/K+ ATPase 1 and 2 subunits; structural proteins such as neuroWlament heavy chain (NF-H), - and -tubulin, and - and -actin; and signalling proteins such as the retinoblastoma gene product (Rb), heat-shock protein 72 (Hsp72), isoforms 1, 2, and 4 of the collapsinresponse-mediator protein (CRMP), and calbindin. Subsequently, using a system of mouse mesangial cells, Kuncewicz et al. [8] identiWed an additional 31 unique Snitrosylated proteins following exposure of their cells to GSNO. Three, uroguanylin, peroxisome proliferatoractivated receptor-, and NADPH-cytochrome p450 reductase, were found to be S-nitrosylated following subsequent exposure of mesangial cells to IL-1. Finally, Martinez-Ruiz and Lamas [5] have also utilized the same technique to identify S-nitrosylated proteins in bovine aortic endothelial cells exposed to GSNO. Nine proteins were isolated. Among these three studies, a great deal of overlap is present, especially with target identiWcation of GAPDH and -actin. Of greater interest, however, is the tremendous degree of disparity amongst these groups in the various target proteins. Using this same biotin switch technique, Foster and Stamler [9] have examined protein
S-nitrosylation in rat liver mitochondria. They identify a number of target proteins: sarcosine dehydrogenase, catalase, dihydrolipoamide dehydrogenase, hydroxymethylglutaryl-CoA synthase, glutamate oxaloacetate transaminase, and malate dehydrogenase. In our study, murine RAW264.7 macrophage cells were exposed to endotoxin to induce iNOS expression. Unlike the other studies, there was no component in which cells were exposed to a source of exogenous NO. Like the studies previously mentioned, our results indicate that a large number of proteins are S-nitrosylated in this setting. Again, some proteins common to the other studies were found: GAPDH, -actin, heat shock proteins, and -tubulin. Of interest, a number of proteins unique to this setting were also found: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. Review of the Medline database Wnds no previous publications demonstrating S-nitrosylation of any of these proteins. Enolase and pyruvate kinase are metabolic enzymes required for glycolysis. Although neither has been identiWed as a target for Snitrosylation, both have been found to have NO-dependent functions. With regard to pyruvate kinase, Momken et al. [10] demonstrated that the absence of eNOS in soleus muscle induced a marked decrease in pyruvate kinase (¡26%) activity. Loss of neuronal enolase activity in the presence of NMDA has been found to be ablated by a competitive substrate inhibitor of NOS activity [11]. Elongation factor-1 chain is a large G protein and functions as part of a translational multimolecular complex reported to be a target of CDK1/cyclin B, the universal regulator of M phase [12]. During protein synthesis, EF-1 forms a ternary complex with aminoacylated tRNA and GTP (eEF1A-GTP-aatRNA), and delivers aatRNA to the ribosome after GTP hydrolysis. Protein synthesis elongation factor-2 catalyzes the translocation of the peptidyl-tRNA from the A site to the P site of the ribosome. Most organisms encode a single EF2 protein and its activity is regulated by
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phosphorylation [13]. Of note, is the observation that both EF-1 and EF-2 also function as actin-binding proteins [14]. Plastin-2 or Wmbrin is an actin cross-linking protein [15]. Several multiprotein complexes containing members of the structural maintenance of chromosomes (SMC) family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization; the SMC5/6 complex is required for a coordinated response to DNA damage and normal chromosome integrity [16]. Finally, CEM15, also known as the human protein apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), mediates a newly described form of innate resistance to retroviral infection by catalyzing the deamination of deoxycytidine to deoxyuridine in viral cDNA replication intermediates [17]. FRAG-6 mRNA induction by interferons is IRF-1-independent and it is likely to be activated by the JAK/STAT pathway [18]. In a similar fashion, Zhang and Hogg [19] have recently examined the formation of S-nitrosothiols in RAW cells exposed to LPS. The measured concentration of intracellular S-nitrosothiols was 17.4 § 1.0 pmol/mg protein and was associated with the high molecular weight (>3 kDa) pool. These intracellular nitrosothiols are stable with half-lives of approx. 3 h. The authors postulate that the stable pool of S-nitrosothiols is isolated from the GSH pool and is directly nitrosated. Our results suggest that the biotin switch technique is a viable technique for identiWcation of S-nitrosylated proteins. Certainly, based upon the studies to date, a widely disparate group of setting- and cell-speciWc proteins are modiWed in the presence of NO. The question as to why certain proteins are S-nitrosylated in speciWc settings in speciWc cells is unknown. While the ubiquitous nature of NO argues for some degrees of commonality as with GAPDH and -actin, S-nitrosylation of other proteins such as EF-1 and -2 in the speciWc setting of endotoxin stimulated macrophages suggests a regulatory mechanism for which NO is necessary, but not suYcient. The varying nature of the identiWed target proteins indicates that more is required than the presence of NO. References [1] C. Nathan, Nitric oxide as a secretory product of mammalian cells, FASEB 6 (1992) 1157–1175.
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