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Protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification

Matthew B. West^*,1, Bradford G. Hill^*,1, Yu-Ting Xuan and Aruni Bhatnagar^,2

^* Department of Biochemistry and Molecular Biology and

Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, USA

²Correspondence: Division of Cardiology, Department of Medicine, Delia Baxter Bldg., 580 S. Preston St., Rm. 421F, University of Louisville, Louisville, KY 40202, USA. E-mail: aruni{at}louisville.edu

SPECIFIC AIMS

Under basal conditions, most cells maintain a small subset of proteins that are covalently bound to glutathione (GSH). The origins of glutathiolated proteins, however, remain unclear, and the significance of glutathiolation as a unique, temporally regulated mode of post-translational protein modification remains to be fully evaluated. This study was undertaken to determine whether exogenous NO or NO generated by endothelial or inducible NO synthases is a physiological regulator of protein glutathiolation.

PRINCIPAL FINDINGS

1. Exogenous NO induces saturable and reversible glutathiolation of specific proteins
The abundance of glutathiolated proteins in COS-7 and rat aortic smooth muscle (RASM) cells in culture was measured using monoclonal antibodies that recognize protein-glutathione mixed disulfides (PSSG). Exposure to the NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) led to robust glutathiolation of specific proteins corresponding to 37, 50, and 75 kDa. The extent of glutathiolation depended on the time of exposure and SNAP concentration. Prolonged incubation of the cells led to a spontaneous decrease in protein glutathiolation that was accelerated by removing SNAP from the medium, indicating that NO-mediated glutathiolation is reversible. Immunoprecipitation and SDS-PAGE separation of glutathiolated proteins revealed increased immunoaffinity of two proteins in SNAP-treated RASM cells. These proteins were identified by matrix assisted laser desorption-time-of-flight mass spectrometry (MALDI-TOF/MS) analysis to be heat shock protein 70 (HSP70) and ßbeta;-actin. Taken together, these data suggest that exogenous NO induces a reversible and a saturable increase in glutathiolation of a specific subset of proteins.

2. NO-dependent vascular relaxation induces protein glutathiolation
To assess the role of eNOS, we examined protein glutathiolation in arterial rings during acetylcholine (ACh)-induced relaxation. For this, rat aortic rings were preincubated in the presence or absence of the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) hydrochloride and then precontracted with phenylephrine. The rings were then treated with ACh to stimulate eNOS. Representative tracings are shown in Fig. 1 A. Sections from untreated vessels displayed relatively low levels of glutathiolated proteins as assessed by immunostaining with anti-PSSG antibodies (Fig. 1B ). A significant increase in staining with the anti-PSSG antibody (Ab) was observed in ACh-treated vessels, which was abolished in L-NAME-pretreated vessels (Fig. 1C ). ACh-induced increase in protein glutathiolation was also evident in immunoblots prepared from total protein extracts of arterial rings (Fig. 1D ). Protein glutathiolation by ACh was prevented in rings pretreated with L-NAME and was abolished by ßbeta;-mercaptoethanol (Fig. 1D , inset), suggesting that glutathiolation was induced by eNOS.

View larger version (49K): [in this window] [in a new window]   Figure 1. Stimulation of eNOS increases protein glutathiolation. A) Vascular tone of isolated rings prepared from rat aorta precontracted by phenylephrine (PE, 1 µM) and stimulated by ACh (ACh, 1 µM) in the absence or the presence of 100 µM L-NAME. Control rings were equilibrated in buffer alone. B) Photomicrographs of untreated rings (Control) or rings treated with ACh or ACh + L-NAME). Rings were fixed and stained with the anti-PSSG Ab. C) Relative intensity of anti-PSSG Ab staining in rings treated with ACh ± L-NAME. Staining intensity was quantified using Metamorph software and normalized to that of buffer-incubated rings. D) Intensity of immunoblots from homogenates of aortic rings that were either left untreated (Control) or treated with ACh ± L-NAME. Glutathiolated proteins were detected by immunoblotting with anti-PSSG antibodies. Inset shows images of blots developed using anti-PSSG Ab after treatment with ßbeta;-mercaptoethanol (BME). E) Two-dimensional Western blots of aortic rings precontracted with PE and relaxed with ACh in the absence (a) or presence (b) of L-NAME. Whole tissue extracts were subjected to 2D electrophoresis, and glutathiolated proteins were detected by anti-PSSG antibodies. a, Inset) Same blot after stripping and reprobing for smooth muscle actin. Proteins 1–6 were excised from corresponding Sypro Ruby-stained gels and identified by MALDI-TOF/MS. Data are representative of 4 individual experiments. Bars are mean ± SE; *P < 0.05 vs. control; **P < 0.01 vs. ACh.

To identify glutathiolated proteins, 2D Western blots were developed from extracts of aorta treated with ACh in the absence or presence of L-NAME using anti-PSSG antibodies. Immunopositive spots were excised from parallel Sypro Ruby stained gels and proteins were identified by MALDI-TOF/MS analysis. As shown in Fig. 1E , a, multiple immunopositive spots were observed by Western blotting, the intensity of which was much less in L-NAME pretreated rings (Fig. 1E, b ), indicating NO-specificprotein modification. MALDI-TOF/MS analysis of the NO-responsive spots led to the detection of six proteins, four of which were identified as -actin. The presence of actin in these spots was confirmed by Western analysis using anti-actin antibodies (Fig. 1E , inset). Other spots were assigned to vimentin and HSP70.

3. Actin is glutathiolated by S-nitrosoglutathione or by the addition of reduced GSH to S-nitrosylated protein
Because actin was glutathiolated by NO in cultured cells and in isolated aortic rings, we examined the mechanism of protein glutathiolation using actin as a model protein. Purified skeletal muscle actin was reduced by DTT and incubated with 1 mM S-nitrosoglutathione (GSNO) for 1 h at 25°C. ESI⁺/MS analysis of the modified preparation revealed molecular masses of 41,887 and 42,193 Da (Fig. 2 A, a), which were ascribed to native actin and actin bound to a single molecule of GSH (+305 Da). In contrast, incubation with 1 mM oxidized GSH (GSSG) did not cause actin glutathiolation (Fig. 2A, b ). Preferential glutathiolation of actin by GSNO was also demonstrated by Western analysis, which showed intense anti-PSSG Ab positive bands with GSNO- but not GSSG-treated actin (Fig. 2B ). No nitrosylation was observed when actin was treated with GSNO. To ensure that this was not due to the breakdown of the actin-NO bond during mass spectrometry, reduced actin was incubated with the nonthiol NO donor spermine NONOate (NOC-22). As shown in Fig. 2A, c , NOC-22 induced extensive nitrosylation of actin as indicated by multiple +29 Da peaks. These data indicate that instead of S-nitrosylation, the reaction of actin with GSNO leads to S-glutathiolation of the protein. Significantly, GSNO was more potent in glutathiolating actin than GSSG.

View larger version (11K): [in this window] [in a new window]   Figure 2. Mechanisms of glutathiolation of actin in vitro. A) ESI-mass spectra of rabbit skeletal muscle incubated with GSNO (a), GSSG (b), or NOC-22 (c). Purified rabbit skeletal muscle actin (7 µM) was incubated with 1 mM of the indicated reagents, desalted, and analyzed by ESI/MS. B) Western blots of actin incubated with the indicated concentrations of GSNO and GSSG using anti-PSSG antibodies. C) Immunoblot analysis of glutathiolated actin. Purified actin was incubated with NOC-22, and after desalting, nitrosylated actin was incubated without (+NOC-22) or with GSH (NOC-22+GSH). As a control, naive actin was incubated with GSH. Samples were treated with 50 mM NEM before electrophoresis and Western blotting with the anti-PSSG Ab.

To determine whether glutathiolated proteins could be generated by reaction of reduced GSH with S-nitrosylated proteins, actin was nitrosylated by incubating with NOC-22, and then treated with GSH. Treatment with GSH alone did not induce protein glutathiolation; however, nitrosylated actin was readily glutathiolated by GSH (Fig. 2C ).

4. Overexpression of inducible NOS in the heart increases protein glutathiolation
To examine the role of inducible NOS (iNOS) in protein glutathiolation, we used cardiac-specific iNOS transgenic mice (NOS-TG). Western analysis iNOS-TG hearts showed enhanced protein immunoreactivity with anti-PSSG antibodies compared with homogenates of wild-type (WT) littermates. Intense staining was observed with protein bands of 30, 50, 120, and >250 kDa. MALDI-TOF/MS analysis led to the identification of the adenine nucleotide translocator (ANT) and the -subunit of the F₁F₀-ATPase, both of which displayed peptides with a 305 Da increase in mass, indicating adduction with a single GSH molecule. These results show that elevation of iNOS expression in the heart induces glutathiolation of mitochondrial proteins.

CONCLUSIONS AND SIGNIFICANCE

The results of this study show that NO is a critical regulator of protein glutathiolation. In several types of cells and tissues, treatment with NO donors or increasing endogenous NO production increased protein glutathiolation, suggesting that this is a general autocrine and paracrine response to NO. Our data show that NO-induced protein glutathiolation was saturable and reversible and therefore unlikely to be an inevitable consequence of NO reacting indiscriminately with protein thiols. These findings support the notion that NO facilitates the addition of GSH to reactive protein cysteines.

Glutathiolated proteins have been detected under a variety of physiological and pathological conditions, but the mechanism(s) by which GSH binds to proteins is unclear. Previous studies showing that protein glutathiolation is increased under oxidative stress suggest that glutathiolated proteins could arise from thiol transfer reactions between GSSG [or its sulfenic acid form (GSOSG)] and an ionized cysteine. The role of GSOSG as an endogenous glutathiolating agent, however, is unclear. Our results indicate that proteins are preferentially glutathiolated by NO, which could promote glutathiolation by two distinct mechanisms: 1) direct GSNO-mediated protein modification or 2) denitrosylation of S-nitrosylated proteins by GSH or both (Fig. 3 ).

View larger version (61K): [in this window] [in a new window]   Figure 3. Proposed mechanism of NO-mediated glutathiolation. NO generated from eNOS or iNOS S-nitrosylates proteins (PSH), and reduced GSH could assist in the denitrosylation and the glutathiolation of specific proteins. Alternatively, GSNO may directly glutathiolate target proteins. Glutathiolation could regulate protein function and/or protect protein thiols from irreversible oxidation.

Mechanism 1 is supported by observations that GSNO is the most abundant biological nitrosothiol and that it readily glutathiolates proteins in vitro. We found that actin, the most abundantly glutathiolated protein in NO-exposed cells, was stoichiometrically glutathiolated by GSNO. Under the conditions used, GSNO was a more potent glutathiolating agent than GSSG, suggesting that in vivo proteins are more likely to be glutathiolated by GSNO than GSSG. Nevertheless, it remains unclear whether the bulk concentration of GSNO in cells is high enough to induce protein glutathiolation or whether local increases in GSNO are sufficient.

Mechanism 2 is supported by our novel observation that S-nitrosylated actin is glutathiolated by GSH, indicating that proteins could be glutathiolated by GSH-assisted denitrosylation and that glutathiolation is a significant fate of S-nitrosylated proteins. Nitrosylation of proteins, however, could compete with GSNO formation. Although the relative contribution of these two pathways remains to be assessed, the observation that proteins such as actin, vimentin, and HSP70 could be either nitrosylated or glutathiolated suggests that the two modifications are inter-related.

Regardless of specific pathways, our data suggest that NO is an effective regulator of protein glutathiolation in vivo. This is supported by the observation that physiological stimulation of eNOS or increased expression of iNOS in the heart increases protein glutathiolation suggesting a regulatory mechanism common to both NO synthases. Although the full extent of this regulatory axis remains unknown, the small subset of glutathiolated proteins identified here suggests that this modification may be functionally significant. Glutathiolation of actin and vimentin could contribute to cytoskeletal changes caused by NO. Glutathiolation of HSP70 could convert it to an active chaperone, and glutathiolation of ANT could affect its interaction with cyclophilin D, thereby regulating mitochondrial permeability transition. Additional studies are required to ascertain the functional significance of NO-mediated protein glutathiolation reactions.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5843fje

¹ These authors contributed equally to this work.

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