Departments of Internal Medicine and of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, Houston, Texas 77030
ProteEx, Incorporated, The Woodlands, Texas 77381
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ABSTRACT |
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NO exerts its actions by chemical modification of targets, preferentially interacting with thiol groups, transition metals, and free radicals. S-Nitrosylation of cysteine residues in target proteins is a principal reaction of NO and of several NO-derived species. This redox-based post-translational modification has been implicated in the cGMP-independent control of a broad spectrum of cellular functions in a variety of cell types (5). A growing number of proteins has been found to undergo S-nitrosylation in vivo, including hemoglobin (6), creatine kinase (7), glyceraldehyde-3-phosphate dehydrogenase (8), the N-methyl-D-aspartate receptor (9) and ryanodine receptors (10), and several caspases (11), resulting in altered function. In addition, S-nitrosylation appears to be a mechanism for signal transduction in cells. For example S-nitrosylation of NF-B p50 modifies its function in mediating changes in expression of target genes (12). Gow et al. (13) provided evidence that Ca2+, growth factors, and developmental transitions regulate S-nitrosylation in diverse tissues. The identification of the full complement of S-nitrosylated proteins and the functional consequences of this modification is essential for understanding the mechanisms of actions of NO and the signaling events that arise from its release.
Recently Jaffrey et al. (14) reported a proteomic approach, termed the "biotin-switch" method, to detect proteins that are S-nitrosylated in vitro and in vivo. The method involves the substitution of a biotin group at every Cys sulfur modified by nitrosylation. Biotinylated proteins are then purified by avidin-affinity chromatography or identified by immunoblotting. Using this method in mice, these authors identified 16 proteins S-nitrosylated by NO derived from neuronal NOS (14). In the present study, we identified 31 novel protein targets of S-nitrosylation in NO-treated and cytokine-activated murine mesangial cells using the biotin-switch method combined with two-dimensional (2D) gel electrophoresis and MALDI-TOF MS, and here we discuss their potential relevance to iNOS biology and mesangial cell function.
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EXPERIMENTAL PROCEDURES |
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Cell Culture
Mouse mesangial cells (ATCC CLR-1927) were cultured at 37 °C in complete medium (Dulbeccos modified Eagles medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5% fetal bovine serum). In some instances, as indicated in the text and figure legends, cells were treated for 24 h with IL-1ß (10 ng/ml) to induce iNOS expression.
Sample Preparation for Proteomic Analysis
To prepare extracts of cytosolic and membrane-associated proteins, cells were harvested by scraping, resuspended in 20 volumes of HEN buffer (25 mM HEPES, pH 7.9, 1 mM EDTA, 0.1 mM neocuproine) containing 0.4% CHAPS, and allowed to swell for 15 min at 4 °C. The cells were centrifuged at 2000 x g for 5 min at 4 °C, and the pellet was washed in HEN buffer. The supernatant was recovered, and the particulate fraction was pelleted by centrifugation at 4 °C. The resulting samples were used immediately or stored at -80 °C until used.
In Vitro S-Nitrosylation
The procedure detailed by Jaffrey et al. (14) was followed in detail with a few modifications. The cytosolic and membrane-associated proteins (12 mg) were incubated with the NO donor GSNO (40 µM) or the controls GSH (40 µM) or GSSG (40 µM) in the dark at room temperature for 20 min with constant rotating. To prepare proteins for proteomic identification, 60 mg of protein sample were used. The treated proteins were incubated with 20 mM methyl methanethiosulfonate and 2.5% SDS at 50 °C for 20 min with vortexing. Methyl methanethiosulfonate was removed by precipitation with 2 volumes of -20 °C acetone or by desalting columns. After resuspending the proteins in HEN buffer containing 1% SDS, sodium ascorbate solution (1 mM final concentration) and biotin-HPDP (up to 10 mM final concentration) were added. The mixtures were incubated for 1.5 h at 25 °C in the dark with intermittent vortexing. Biotinylated nitrosothiols were then acetone-precipitated with 2 volumes of -20 °C acetone, or biotin-HPDP was removed by desalting columns. After centrifugation, the pellet was resuspended in 0.1 ml HEN buffer containing 1% SDS/mg of protein in the initial protein sample. Two volumes of Neutralization buffer (20 mM HEPES, pH 7.7, 100 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100) were added, and 15 µl of NeutrAvidin-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 buffer 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 2-mercaptoethanol. The samples were then mixed in SDS sample buffer.
Two-dimensional Gel Electrophoresis and Gel Image Analysis
Immediately after affinity separation by the biotin-switch method, the purified proteins were suspended in a buffer containing 8 M urea, 2 M thiourea, 1% Triton X-100, 1% dithiothreitol, and 1% ampholytes pH 310 (15). An aliquot of 100 µg of protein isolated by the biotin-switch method was loaded onto an 11-cm isoelectric focusing strip, pH 47. Focusing was conducted on isoelectric focusing cells at 250 V for 20 min followed by a linear increase to 8000 V for 2 h. The focusing was terminated at 20,000 V-h. Strips were then equilibrated in 375 mM Tris buffer, pH 8.8, containing 6 M urea, 20% glycerol, and 2% SDS (16). Fresh dithiothreitol was added to the buffer at a concentration of 30 mg/ml. Fifteen minutes later, fresh buffer was added containing 40 mg/ml iodoacetamide. Strips were equilibrated for an additional 15 min and then loaded onto the second dimension using Criterion gradient gels (Bio-Rad) with an acrylamide gradient of 1020%. Gels were then stained using SyproRuby fluorescent dye (17). The results of digital fluorescent image analysis of gel images from samples treated with NO donors were compared with their respective control samples using PDQuest software (Bio-Rad). Analysis included spot detection and comparison of protein patterns using internal protein standards as landmarks.
Tryptic Digestion, MALDI-TOF MS, and Peptide Mass Fingerprinting
For protein spot cutting and MS identification, gels were loaded with 200 µg of the proteins isolated by the biotin-switch method. Following software analysis, unique spots were excised from the gel using the ProteomeWorks robotic spot cutter (Bio-Rad). In-gel spots were robotically digested on a MultiPROBE II (Packard Instrument Co.) as follows. Gel spots were washed twice in 100 mM NH4HCO3 buffer followed by soaking in 100% acetonitrile for 5 min, aspiration of the acetonitrile, and drying of the gels for 30 min. Rehydration of the gels using 20 µg/ml trypsin (Promega, Madison, WI) suspended in 25 mM NH4HCO3 buffer was followed by incubation at 37 °C for 1420 h. The digested peptides were extracted twice using a solution of 50% acetonitrile and 5% trifluoroacetic acid for 40 min. Peptide extracts were desalted and concentrated using Zip-tips (Millipore, Bedford, MA) and robotically placed on MALDI chips using SymBiot I (Applied Biosystems, Foster City, CA). Mass spectral analyses were conducted on a MALDI-TOF Voyager DE PRO mass spectrometer (Applied Biosystems). Data searches were performed using the National Center for Biotechnology Information (NCBI) protein database with a minimum matching peptides setting of 4, a mass tolerance setting of 50200 ppm, and a single trypsin miss cut setting.
Western Analysis
Proteins isolated by the biotin-switch method were also resolved by SDS-PAGE, electroblotted to Hybond-P membranes, and immunoblotted with primary antibodies as indicated in the text and figure legends. Bound antibody was visualized by the ECL chemiluminescence detection system (Amersham Biosciences) using peroxidase-conjugated sheep anti-mouse or goat anti-rabbit IgG as appropriate.
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RESULTS |
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Under basal conditions in the absence of NO donors, relatively few peptide spots were evident on 2D gels of the biotin-switch protein isolates (Fig. 1A). Similarly only a few peptide spots were evident in the negative control samples first treated with GSH and then subjected to the biotin-switch assay (data not shown). In contrast 790 protein spots were evident in the samples treated with GSNO (Fig. 1B). The protein pattern of the 2D gel in Fig. 1B reveals that most of these proteins were detected in the acidic region of the pH gradient. A similar pattern and number of protein spots were identified in the GSSG-treated samples (data not shown) in keeping with the biochemistry of the biotin-switch method and published reports of S-glutathiolation of target cysteine residues in proteins that are also S-nitrosylated (1821). To identify the S-nitrosylated proteins of interest in the NO donor-treated samples, additional 2D gels were produced with the same cellular extracts obtained by the biotin-switch method and stained as described under "Experimental Procedures." Forty-four protein spots of interest were then excised from the gels, digested with trypsin, and subsequently analyzed by MALDI-TOF mass spectrometry. The resulting spectra were used to identify the tryptic peptide mass fingerprints (Fig. 1B and Table I). Of these 44 spots excised from the gels, 35 S-nitrosylated proteins, 34 of which were known proteins, were unambiguously identified (Table I). The proteins identified were members of specific functional families including receptors and membrane proteins, signaling proteins, cytoskeletal and cell matrix proteins, and cytoplasmic proteins (Table I). Three of these, glyceraldehyde-3-phosphate dehydrogenase, caspase-6, and dimethylarginine dimethylaminohydrolase, have been previously reported to be S-nitrosylated in other systems (8, 22, 23).
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DISCUSSION |
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In the course of glomerular injury, mesangial cells are exposed to endogenously produced NO and to NO generated from local phagocytes (4, 25). Thus NO and derived species could potentially exert important regulatory control on mesangial cell function via S-nitrosylation of proteins involved in a variety of cellular processes. To identify proteins uniquely sensitive to S-nitrosylation, we incubated mesangial cell lysates with the NO donor GSNO. GSNO is present in the circulation and may represent an endogenous pool of NO groups for S-nitrosylation of proteins (26). After incubation with the NO donor, the extracts were subjected to the S-nitrosylation assay and proteomic analysis. The effectiveness of this assay relates in part to the stability of S-nitrosothiol linkages, which might vary among proteins and specific assay conditions, and the potential action of specific denitrosylating activities (11, 27). In general the S-nitrosylated proteins can be segregated into five groups: signaling proteins, cytoskeletal and cell matrix proteins, receptors and membrane proteins, cytoplasmic proteins, and transcription factors. Many of these proteins are likely to undergo S-nitrosylation in other cell types. Indeed three of the proteins we identified to be S-nitrosylated in mesangial cells, glyceraldehyde-3-phosphate dehydrogenase, caspase-6, and dimethylarginine dimethylaminohydrolase, have been reported to be S-nitrosylated in other tissues and cell types (8, 23). We confirmed that PPAR, uroguanylin, and NADPH-cytochrome P450 oxidoreductase were S-nitrosylated by NO endogenously produced in mesangial cells (Fig. 2).
Several cell signaling proteins were found to be S-nitrosylated. Guanylyl cyclase is a classic target of NO action, and other GTP-regulated proteins, such as p21ras (28), have been reported to be S-nitrosylated. We identified the GTP-binding protein -subunit G
i2 as a novel target for S-nitrosylation. G
i2 is involved in diverse cell signaling events. G
i2 was found to be activated by NO (29) underscoring the role of NO in modulating the signal transduction through G-protein-coupled receptor. Farnesylation plays a fundamental role in activating G-proteins, Ras, Rho, lamin, and peroxisome farnesylated protein (30). Farnesylated protein(s) mediates IL-1ß induction of iNOS (30). It seems likely that NO activates some of these farnesylated proteins as a positive feedback. Uroguanylin belongs to the natriuretic peptide family and is present in the stomach, kidney, lung, pancreas, and intestine. It binds to the membrane guanylate cyclase C and signals through cGMP as a second messenger (31). Mesangial cells are a primary target of natriuretic peptides, and these peptides are thought to play a role in regulating glomerular filtration rate. Protein-tyrosine phosphatase, non-receptor type 11, or SHP-2, a member of a small subfamily of cytosolic protein-tyrosine phosphatases, was also S-nitrosylated. SHP-2 is ubiquitously expressed and is involved in the cellular response to growth factors, hormones, cytokines, and cell adhesion molecules. Recent studies showed that bradykinin inhibits mesangial cell proliferation by a novel mechanism involving a direct protein-protein interaction between the bradykinin B2 receptor and SHP-2 (32). Defects in myotonic dystrophy (DM-kinase) result in myotonic dystrophy. NO synthase activity is required for the transcription factor MyoD to induce DM-kinase (33). Since this kinase is localized to gap junctions (34), S-nitrosylation of DM-kinase may play a role in cell-cell communication.
The release of NO seems to protect a significant number of proteins from ubiquitination and degradation mediated by ubiquitin ligase A3 (30). The mechanism of this NO-mediated protection is unclear, but it presumably requires S-nitrosylation (35). Our evidence of S-nitrosylation of ubiquitin ligase A3 could account for this protective action. Conversely iNOS itself is subject to ubiquitin-dependent degradation (36).
In this report, we show that a K+ channel-interacting protein KchIP4A (37) is S-nitrosylated. A recent report indicates the activation of K+ channels by NO (38). Three proteins involved in phospholipid metabolism and cell signaling, choline kinase, phospholipase A2-activating protein, and otoconin-95, were also S-nitrosylated in mesangial cells. Early reports indicated that IL-1 partially inhibits the choline kinase activity in tumors (39), while it induces the synthesis of phospholipase A2-actiavting protein (40). The role of otoconin-95 in the kidney has not yet been identified. The protein is a homologue of the phospholytic enzyme phospholipase A2, constitutes the major protein of the utricular and saccular otoconia, and is expressed by various non-sensory cell types (41).
Mitosin is a nuclear protein that associates with the mitotic apparatus, especially the kinetochore, during mitosis (42). Nitrosylation of mitosin could explain its signaling role in NO-mediated cell cycle progression. Proteins 14-3-3, an emerging family of proteins and protein domains that bind to serine/threonine-phosphorylated residues, were found to be S-nitrosylated. These proteins regulate key proteins involved in various physiological processes such as intracellular signaling, cell cycling, apoptosis, and transcriptional regulation (e.g. FKHRL1, DAF-16, p53, TAZ, TLX-2, and histone deacetylase) (43). These proteins also act as adaptor molecules, stimulating protein-protein interactions, and regulate the subcellular localization of proteins (43, 44). Annexin V belongs to a family of calcium-binding and phospholipid-binding proteins and participates in multiple biological processes such as the regulation of calcium concentration and certain endothelial functions (44). This protein was also identified as an S-nitrosylated protein in mesangial cells. Recently GSNO was found to inhibit the activity of the related protein annexin II tetramer (45).
Several proteins that regulate transcription were found to be S-nitrosylated. The human general transcription factor TFIIA is one of several proteins involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit of TFIID (46). PPAR is a member of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. PPARs play an important role in transcription of cells including glucose and lipid homeostasis, cell cycle progression, differentiation, inflammation (47), and extracellular matrix remodeling. PPAR
is known to be expressed in mesangial cells (48) and to be up-regulated in the presence of glomerular injury, including diabetic glomerular disease (49). PPAR
has also been shown to trans-repress iNOS transcription in murine macrophages (50, 51). It is intriguing to speculate about the existence of a feedback inhibition cycle in which NO derived from iNOS in activated mesangial cells and perhaps macrophages S-nitrosylates PPAR
, which, so modified, becomes competent to trans-repress iNOS gene expression. NO has been shown to inactivate zinc finger proteins (52), most likely by S-nitrosation of thiols in zinc-sulfur clusters leading to reversible disruption of zinc finger structures. Evidence of S-nitrosylation of the zinc finger protein 297B presented here could provide an addition molecular mechanism underlying the regulation of gene transcription. RFG (RET fused gene), a chimeric oncogene coactivator that results from a structural rearrangement between the ELE1 and the RETgenes, was also found to be S-nitrosylated. Transducin ß-like 1 protein, a ubiquitously expressed WD-40 repeat-containing protein, partners with nuclear receptor co-repressor and histone deacetylases to form the SMRT (silencing mediator of retinoid and thyroid hormone receptor) corepressor complex that directs diverse repression pathways (5355). Neither RFG nor transducin ß-like 1 protein has been reported to be expressed in mesangial cells.
A number of cell matrix and cytoskeletal proteins were S-nitrosylated. These include the actin-binding protein ß-tropomyosin, which is thought to stabilize actin filaments and influence aspects of F-actin, -internexin, a type IV intermediate filament protein, which may act as a scaffold for the formation of neuronal intermediate filaments during early development, drebrin, an F-actin-binding protein typically expressed in neuronal cells (56), and testase 3, a member of the ADAM (a disintegrin and metalloproteinase) family of metalloproteinases (57). ADAM-15 has been shown to participate in mesangial cell migration (58), but a similar role for testase 3 has not yet been demonstrated. Although members of the ADAM family have not been reported previously to be S-nitrosylated, matrix metalloproteinase-9 was recently shown to be activated by S-nitrosylation, resulting in the formation of a stable sulfinic or sulfonic acid with pathological activity in neurons (59). Melusin interacts with ß-integrin and presumably plays a role in protein-protein interactions, and having a cysteine-rich domain, it is a strong biological candidate for S-nitrosylation by NO. The ß-integrins play a role in mesangial cell adhesion, migration, survival, and proliferation (60). Collagen XI regulates the assembly of cartilaginous matrices by co-polymerizing with collagen II trimers. Collagen plays a critical role in thrombosis by enhancing platelet aggregation through its interaction with von Willebrand factor. S-Nitrosylation of collagen
1(XI), prothrombin (coagulation factor II), and annexin V may contribute to the ability of NO to inhibit platelet aggregation.
Mannose-binding lectin (MBL) is a serum protein of the innate immune system that circulates as a complex with a family of MBL-associated serine proteases (MASPs). Complexes of MBL·MASP-2 activate the complement system. MASP-3, which we show to be S-nitrosylated, down-regulates the C4 and C2 cleaving activity of MASP-2 (61). NADPH-cytochrome P450 oxidoreductase is an essential component of the microsomal P450 mixed function oxidase system, mediating electron transfer from NADPH to cytochrome P450s and several other acceptors. Thus, the ability of NO to S-nitrosylate this protein may impact a variety of signaling pathways.
Several factors may govern the specificity of S-nitrosylation for selected targets. Cysteine-rich proteins could be more susceptible to S-nitrosylation, and the spatial relationship of NO sources and targets within the cell has been shown to be important for the specificity of this post-translational modification (62). A Cys-based motif for S-nitrosylation has been proposed based on screenings of large databases: XHRKDECDE, where X can be any amino acid (63). The motif has been postulated to impart a degree of specificity for S-nitrosylation. However, other cysteine residues in each protein could also be candidates for nitrosylation based on tertiary structural conformation. The possibility also exists that S-nitrosylation prefers hydrophobic compartments so that hydrophobic tunnels within or between proteins may serve to channel NO to target thiols. Of the proteins identified in the present study, only PPAR has a perfect linear match of this motif.
Our results provide a stage from which mechanistic and biologic aspects of protein S-nitrosylation can be examined. Such studies should provide a comprehensive view of how NO regulates cellular processes. In addition to identifying novel targets of S-nitrosylation in mesangial cells, our results provide a more complete picture of protein targets of this modification that should facilitate future research to define common motifs of the protein targets, the determinants of specificity, and the functional outcomes of S-nitrosylation in cells and tissues.
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FOOTNOTES |
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Published, MCP Papers in Press, April 1, 2003, DOI 10.1074/mcp.M300003-MCP200
1 The abbreviations used are: NOS, nitric-oxide synthase; iNOS, inducible NOS; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; GSNO, S-nitrosoglutathione; PPAR, peroxisome proliferator activated receptor; biotin-HPDP, N-[6-(biotinamido)hexyl]-3''-(2'-pyridyldithio)propionamide; DM, dystrophia myotonica; 2D, two-dimensional; IL, interleukin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SHP-2, Src homology 2-containing phosphotyrosine phosphatase-2; TF, transcription factor; RFG, RET fused gene; ADAM, a disintegrin and metalloproteinase; MBL, mannose-binding lectin; MASP, MBL-associated serine protease.
* This work was supported by National Institutes of Health Grants RO1 DK-50745 and P50 GM-20529 (to B. C. K.), the "DREAMS" Center Grant from the Department of Defense (to B. C. K.), and endowment funds from The James T. and Nancy B. Willerson Chair (to B. C. K.).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Depts. of Internal Medicine and of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030. Tel.: 713-500-6873; Fax: 713-500-6890 or 6882: E-mail: Bruce.C.Kone{at}uth.tmc.edu
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REFERENCES |
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