Glutathione S-transferase P1 suppresses iNOS protein stability in RAW264.7 macrophage-like cells after LPS stimulation.

Glutathione S-transferase P1 (GSTP1) is a ubiquitous expressed protein which plays an important role in the detoxification and xenobiotics metabolism. Previous studies showed that GSTP1 was upregulated by the LPS stimulation in RAW264.7 macrophage-like cells and GSTP1 overexpression downregulated lipopolysaccharide (LPS) induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Here we show that GSTP1 physically associates with the oxygenase domain of iNOS by the G-site domain and decreases the protein level of iNOS dimer. Both overexpression and RNA interference (RNAi) experiments indicate that GSTP1 downregulates iNOS protein level and increases S-nitrosylation and ubiquitination of iNOS. The Y7F mutant type of GSTP1 physically associates with iNOS, but shows no effect on iNOS protein content, iNOS S-nitrosylation, and changes in iNOS from dimer to monomer, suggesting the importance of enzyme activity of GSTP1 in regulating iNOS S-nitrosylation and stability. GSTM1, another member of GSTs shows no significant effect on regulation of iNOS. In conclusion, our study reveals the novel role of GSTP1 in regulation of iNOS by affecting S-nitrosylation, dimerization, and stability, which provides a new insight for analyzing the regulation of iNOS and the anti-inflammatory effects of GSTP1.


Introduction
Glutathione S-transferases (GSTs) are a well-studied superfamily of detoxifi cation enzymes and catalyze the nucleophilic attack of the sulfur atom of glutathione (GSH) on electrophilic groups of substrate molecules [1]. According to their biochemical, immunologic, and structural properties, mammalian cytosolic GSTs are divided into six major classes: Alpha,Mu,Pi,Omega,Theta,and Zeta [2,3]. Among them, GSTP1 (GST π ) is the most ubiquitous in mammalian cells. There has been considerable interest in the properties of GSTP1, because it plays an important role in susceptibility to cancer and other diseases [4,5]. Adler et al. fi rst demonstrated that GSTP1 participates in the regulation of stress signaling and protects cells against apoptosis by mechanisms via its noncatalytic, ligand-binding activity. GSTP1 acts as an endogenous inhibitor of c-Jun N-terminal kinase (JNK) by interacting with the C-terminal of this kinase; oxidative stress could cause the dissociations of GSTP1 -JNK complex and then, enhance the JNK activity [6]. Our previous research demonstrated that GSTP1 can regulate tumor necrosis factor (TNF)-α -induced signaling and inhibit cell apoptosis by forming ligand-binding interactions with TNF receptorassociated factor 2 (TRAF2) [7]. It has been reported that GSTP1 may act as a nitric oxide (NO) carrier under diff erent for iNOS S-nitrosylation and ubiquitination. GSTM1, another member of GSTs, did not work in the same manner as GSTP1 in regulation of iNOS. Our study indicates the novel mechanism by which GSTP1 regulates iNOS protein stability.

DNA constructs
pcDNA3-HA-GSTP1 has been described previously [6]. pcDNA3-His was a generous gift from Dr. Zhenguo Wu (University of Science & Technology, HK), pFLAG-CMV5 α -GSTM1 was a gift from Kwang Je Kim (Korea University), and pcDNA3-iNOS was from Dr. Solomon H. Snyder (the Johns Hopkins University School of Medicine, USA). pET-28a-His6-hGSTP1 (Y7F) plasmid was constructed as previously described [10] and Xpresstagged GSTP1 (Y7F) were made by PCR from His6-hGSTP1 and cloned into pcDNA3 vectors. The His-Xpress-tagged GSTP1 and truncated GSTP1 constructs were made by PCR from pcDNA3-HA-GSTP1 and cloned into pcDNA3 vectors. The Myc-tagged iNOS and truncated iNOS constructs were made by PCR from pcDNA3-iNOS and cloned into pcDNA3 vectors.
Human GSTP1 (hGSTP1) are encoded by a single gene, hGSTP1 , while mice have two such genes, mGstp1 and mGstp2 . Since the degree of protein homology is striking, monoclonal antibodies against GSTP1 can recognize hGSTP1 and mGSTP1. However, in this experiment, we designed two diff erent GSTP1 RNA interference (RNAi) to interfere hGSTP1 and mGSTP1 . pRNA-U6.1/ neo-hGSTP1 RNAi and pRNA-U6.1/neo-mGSTP1 RNAi were constructed into pRNA-U6. All expression vectors were sequenced for confi rmation and purifi ed using the Endofree Plasmid Preparation Kit (Qiagen).

Cell culture and transfection
Murine macrophage-like RAW264.7 cells and human embryonic kidney (HEK 293) cells were obtained from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, PR, China), and were cultured in Dulbecco ' s modifi ed Eagle ' s medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μ g/ml streptomycin) at 37 ° C in an atmosphere of 5% CO2. All experiments with RAW264.7 and HEK293 cells were performed below 10 passages. Transient transfection was performed with a modifi ed calcium phosphate method or by the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer ' s instructions. In all cases, the total amount of DNA was normalized by the empty control plasmids.

Stable transfection of GSTP1, hGSTP1 RNAi, and mGSTP1 RNAi
Clones of RAW264.7 cells stably expressing GSTP1 has been described previously [9]. pcDNA3 plasmids with or without Xpress-tagged GSTP1 were transfected into HEK293 cells using Lipofectamine 2000. pRNA-U6.1 plasmid with or without hGSTP1 RNAi and mGSTP1 RNAi were transfected into HEK293 and RAW264.7 cells using Lipofectamine 2000, too. 72 hours after transfection, the cells were incubated in fresh medium containing 500 μ g/ml of G418 for 4 weeks. Subsequently, cell colonies resistant to G418 were isolated and screened by limited dilution. These stable expression clones and control clones were selected for further studies.

Immunoprecipitation and immunoblotting analysis
Cells were lysed on ice in a lysis buff er containing 20 mM Tris (pH: 7.5), 135 mM NaCl, 2 mM ethylenediaminetetraacetic acid or EDTA, 2 mM dithiothreitol (DTT), 25 mM β -glycerophosphate, 2 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, and complete protease inhibitor cocktail for 20 min. Nonreducing sample buff er lacking DTT but SDS was added. Lysates were centrifuged (12,500 ϫ g) at 4 ° C for 15 min. Proteins were immunoprecipitated for 2 h with the indicated antibodies. The precleared Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were incubated with immunocomplexes for another 2 h and washed four times with the lysis buff er. The immunoprecipitates were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene difl uoride or PVDF, or nitrocellulose membrane at 350 mA for 100 min on ice. After transferring, the membranes were blocked in Tris-buff ered saline and Tween 20 or TBST-5% skim milk for 45 min and then were incubated overnight at 4 ° C with primary antibodies (1:1000). The membranes were washed and incubated with infrared dye coupled secondary antibodies (1:5000) for another 1 h. The antibody -antigen complexes were visualized by the LI-COR Odyssey Infrared Imaging System according to the manufacturer ' s instruction (LI-COR Biosciences, Lincoln, NE). Quantification was directly performed on the blot using the LI-COR Odyssey analysis software. Aliquots of whole cell lysates were subjected to immunoblotting to confi rm the appropriate expression of proteins.

Docking
The crystal structure of GSTP1 and iNOS used for molecular modeling was obtained from Protein Data Bank or PDB. Docking of GSTP1 to iNOS was performed with ZDOCK3.0 [17]. With rigid-body docking, 2000 poses were generated. All complexes were analyzed and scored by Zrank [18]. The complex with the lowest Z-rank score was regarded as the most possible native-like model.

Biotin switch
The biotin switch assay was carried out according to the strategy developed by Jaff rey and Snyder [19]. Briefl y, the protein samples were treated with 20 mM MMTS and 2.5% SDS at 50 ° C for 20 min with frequent vortexing. MMTS was then removed by precipitation with two volumes of Ϫ 20 ° C acetone. 1 mM sodium ascorbate solution and 100 μ M biotin-N- [6-(biotinamido)hexyl]-3 ' -(2 'pyridyldithio)propionamide (HPDP) were added after resuspending the proteins in HENS buff er. The samples were incubated for 2 h at 25 ° C in the dark and then biotinylated nitrosothiols were precipitated with two volumes of Ϫ 20 ° C acetone. The proteins were centrifuged, and resuspended in HENS buff er. After that, two volumes of neutralization buff er and Streptavidin-agarose were added and the mixtures were incubated for 1 h at room temperature. The agarose was washed 3 -4 times with HENS buff er. Bound proteins were then eluted in SDS loading buff er.

Statistical analysis
Data were represented as mean Ϯ SD. We performed statistical comparison by Student ' s t-test. A value of P Ͻ 0.05 was considered statistically signifi cant. Statistical calculations were performed by SPSS 13.0 software.

Overexpression of GSTP1, but not GSTM1 and GSTP1 (Y7F) mutant, reduces endogenous iNOS protein level
Our previous studies have demonstrated that intraperitoneal administration of GSTP1 protein to mice signifi cantly decreased mortality of endotoxic shock and inhibited acute lung injury and peritonitis [10]. We also found that GSTP1 could inhibit LPS-induced iNOS increase and NO production in RAW264.7 cells. In the present study, overexpression of GSTP1 in RAW264.7 cells inhibited elevation of LPS-induced iNOS protein level in a dosedependent manner ( Figure 1A). Similarly, iNOS protein levels in stable GSTP1-overexpressing cells were lower than those in the control cells after LPS stimulation ( Figure 1B). In order to confi rm the eff ect of GSTP1 on iNOS, HEK293 cells were cotransfected with pcDNA3-iNOS and various concentrations of Xpress-GSTP1 or pFLAG-CMV5 α -GSTM1, and 36 h after transfection, the cells were subjected to immunoblotting analysis. Results showed that overexpression of GSTP1, but not GSTM1, another member of GSTs, reduced iNOS protein level in a dose-dependent manner ( Figure S1A to be found at online http://informahealthcare.com/doi/abs/ 10.3109/ 10715762.2015.1085978 and Figure 1C). The same result was obtained from G418 -selected, GSTP1-stable transfected HEK293 clones (Figure S1B to be found at online http://informahealthcare.com/doi/abs/ 10.3109/10715762. 2015.1085978). These results suggested the specifi city of GSTP1 in regulation of iNOS protein level. In order to evaluate if the enzyme activity of GSTP1 was involved in iNOS regulation, we transfected pcDNA3-iNOS and various concentrations of Xpress-GSTP1 (Y7F) plasmids into HEK293 cells and observed the eff ect of overexpressive GSTP1 (Y7F) on iNOS protein level reduction. Western blot results showed that GSTP1 Y7F mutant did not aff ect iNOS protein level, suggesting that the enzyme activity of GSTP1 is very important for such iNOS regulation ( Figure 1D).
The above data indicated that GSTP1 overexpression could decrease both endogenous and exogenous iNOS protein level. To further confi rm the eff ect of endogenous GSTP1 on iNOS, we utilized RNA interference (RNAi) technology to knock down endogenous mGSTP1 protein expression in RAW264.7 cells and endogenous mGSTP1 was successfully downregulated by RNAi ( Figure 2A). As expected, iNOS level was obviously higher in mGSTP1 downregulated cells than in pRNA-U6.1 transfected control cells after LPS stimulation (Figure 2A). LPS stimulation also led to elevation of iNOS protein level, in RAW264.7 cells in which mGSTP1 was stably knocked down, than that in the control cells ( Figure 2B). The same results were obtained in HEK293 cells ( Figure S2 to be found at online http://informahealthcare.com/doi/abs/ 10.3109/10715762.2015.1085978).

GSTP1 decreases the stability of iNOS protein
To further investigate how GSTP1 infl uences iNOS level, HEK293 cells were cotransfected with pcDNA3-iNOS and Xpress-GSTP1 plasmids or empty vectors and pretreated with cycloheximide (CHX), an inhibitor of protein synthesis for the indicated times. As shown in Figure 3A, under CHX treatment iNOS protein level was lower in GSTP1overexpressing cells than that in empty-vector-transfected cells. We then checked the change in iNOS at transcriptional level using real-time PCR in RAW264.7 cells. The result showed that overexpression of GSTP1 did not infl uence iNOS at mRNA level ( Figure 3B). These results suggested that GSTP1 could enhance the degradation of iNOS.

GSTP1 physically interacts with iNOS
As shown above, GSTP1 was able to downregulate the level of iNOS protein; we thus probed whether GSTP1 could be directly targeting on iNOS. RAW264.7 cells stably expressing GSTP1 and control pcDNA3 were treated with LPS for 12 h and harvested. Cell lysates were subjected to immunoprecipitation with anti-Xpress or anti-iNOS antibody followed by immunoblotting analysis. Results showed that endogenous GSTP1 specifi cally associated with iNOS ( Figure 4A). We further investigated the interaction of GSTP1 with iNOS in HEK293 cells under overexpression conditions and obtained the same results ( Figure 4B and C). In order to determine if the enzyme activity of GSTP1 was related with this process, HEK293 cells were cotransfected with Xpress-GSTP1 (Y7F) and Myc-iNOS. Figure 4D showed that GSTP1 (Y7F) also interacted with iNOS in HEK293 cells suggesting that the enzyme activity was not necessary for GSTP1 -iNOS interaction. Figure 4E showed that GSTM1 did not interact with iNOS in HEK293 cells.

Mapping of iNOS and GSTP1 domains required for interaction with each other
The above data indicated that GSTP1 could bind to iNOS. Besides, iNOS comprises two catalytic units: a C-terminal reductase domain and an N-terminal oxygenase domain. In order to determine the region of iNOS responsible for the binding with GSTP1, Xpress-GSTP1 and Myc-iNOS (full), Myc-iNOS (1-500), or Myc-iNOS (501-1145) were cotransfected into HEK293 cells and then the cell lysates  were immunoprecipitated with Xpress-specifi c antibody followed by Western blot analysis. The results revealed that the oxygenase domain (1-500), but not the reductase domain (501-1145), of iNOS was required for the binding with GSTP1 ( Figure 5A).
Both the GSH-binding site (G-site) and the xenobiotic substrate-binding site (H-site) exist in all GST family members [20]. To fi nd the region of GSTP1 responsible for binding with iNOS, diff erent Xpress-tagged GSTP1truncated fragments were constructed. HEK293 cells were cotransfected with indicated Xpress-tagged GSTP1 fragments and Myc-tagged iNOS, respectively, followed by co-immunoprecipitation with Myc-specifi c antibody and Western blotting with anti-Xpress antibody. The results showed that the G-site, but not the H-site, domain of GSTP1 was required for the specifi c association of GSTP1 with iNOS ( Figure 5B). Taken together, GSTP1 was physically associated with the oxygenase domain of iNOS by the G-site domain. The above results coincided with the prediction of protein docking software ZDOCK3.0 [17]. As shown in Figure 5C-E, the amino acids involved in the interaction between GSTP1 and iNOS might be Tyr 7,Phe 8,Val 32,Val 33,Glu 36,Lys 190,and Asn 200 in GSTP1 and Tyr 78,Arg 80,Lys 97,and Glu 154 in iNOS.

GSTP1 regulates iNOS monomer/dimer level
The excessive production of NO by dimeric form of iNOS has been well documented [21,22]. We thus investigated whether GSTP1 regulated iNOS monomer/dimer level. RAW264.7 cells were transiently transfected with diff erent concentrations of Xpress-GSTP1 or control vectors and 18 h after transfection, the cells were treated with 100 ng/ml of LPS. Monomer/dimer iNOS were detected by nonreducing Western blotting. As shown in Figure 6A, the dimerized iNOS decreased under GSTP1 overexpression condition. Similar results were also obtained in RAW264.7 cells stably overexpressing GSTP1 ( Figure 6B). On the contrary, in RAW264.7 cells in which  RAW264.7 cells stably expressing GSTP1 and the control pcDNA3 were treated with LPS (100 ng/ml) for 12 h and cell lysates were subjected to immunoprecipitation with anti-Xpress or anti-iNOS antibody. B and C, HEK293 cells were transiently transfected with Xpress-GSTP1 and Myc-iNOS, and cell lysates were subjected to immunoprecipitation with anti-Xpress (B) or anti-Myc (C). D and E, HEK293 cells were transiently transfected with Xpress-GSTP1 (Y7F) (D) or pFLAG-CMV5 α -GSTM1 (E) and Myc-iNOS and 36 h after transfection, the cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-Xpress, and the precipitates were analyzed by immunoblotting with anti-Myc and anti-Xpress antibodies. mGSTP1 were stably knocked down, LPS induced higher iNOS dimer level than that in pRNA-U6.1 transfected cells ( Figure 6C).
To further prove the eff ect of GSTP1 on iNOS monomer/dimer, GSTP1 and iNOS were cotransfected in HEK293 cells and cell lysates were subjected to Western blot. The results showed that GSTP1 overexpression obviously reduced the amount of dimerized iNOS and increased monomer form of iNOS in a dose-dependent manner ( Figure S3A to be found at online http://informa healthcare.com/doi/abs/ 10.3109/10715762.2015.1085978). Both in GSTP1 stably overexpressed and hGSTP1 RNAi HEK293 cells, similar results were also obtained ( Figure S3B to be found at online http://informahealthcare.com/doi/abs/ 10.3109/10715762.2015.1085978 and C). As expected, data showed that GSTM1 and GSTP1 (Y7F) have no eff ect on monomer/dimer form of iNOS ( Figure S3D to be found at online http://informahealthcare.com/doi/abs/ 10.3109/10715762.2015.1085978).

GSTP1 increases S-nitrosylation and ubiquitination of iNOS, but has no eff ect on Tyr phosphorylation of iNOS
A variety of post-transcriptional mechanisms regulate the activity of iNOS, which mainly include phosphorylation, S-nitrosylation, and ubiquitination [23,24]. iNOS can be phosphorylated, although its biological signifi cance is unclear [25]. Thus, we fi rst explored the eff ect of GSTP1 on Tyr phosphorylation of iNOS. HEK293 cells were transiently transfected with Myc-iNOS or cotransfected with Xpress-GSTP1 and Myc-iNOS, and after 36 h cells were incubated with 400 mM Na 3 VO 4 for 30 min before being harvested. Cell lysates were then subjected to immunoprecipitation with anti-Myc, and the precipitates were analyzed by immunoblotting with anti-p-Tyr and anti-Myc antibodies. Results showed that the level of p-Tyr iNOS increased after Na 3 VO 4 was added, but overexpression of GSTP1 showed no obvious eff ect on Tyr phosphorylation of iNOS ( Figure 7A).
We next investigated the eff ect of GSTP1 on iNOS S-nitrosylation. RAW264.7 cells stably expressing GSTP1 were simulated with 500 ng/ml of LPS for 12 h. After using nonreducing lysate to harvest cells, we subjected the samples to the biotin switch procedure, and then affi nity purifi ed them by immobilized streptavidin. The proteins were separated by SDS-PAGE, electroblotted to nitrocellulose, and probed with antibody of iNOS. The results showed that GSTP1 increased iNOS S-nitrosylation level in RAW264.7 cells ( Figure 7B). Furthermore, we utilized the same method to explore the eff ect of exogenous expression of GSTP1 on iNOS S-nitrosylation level in HEK293 cells. Our data showed that exogenous overexpression of GSTP1 also increased iNOS S-nitrosylation level in HEK293 cells ( Figure 7C). The same results were observed in HEK293 cells under stable overexpression and knockdown of GSTP1 conditions ( Figure 7D). In addition, Figure 7E demonstrated that GSTM1 and GSTP1 (Y7F) have no eff ect on the regulation of iNOS S-nitrosylation level.
It has been reported that the ubiquitination of iNOS is required for its degradation [24]. To further determine whether GSTP1 could also aff ect iNOS ubiquitination, we employed proteasome inhibitor MG132 to terminate the protein degradation through proteasome pathway. The results of co-immunoprecipitation revealed that GSTP1 facilitates iNOS ubiquitination in HEK293 cells ( Figure 7F).

Discussion
NO is a neutral free radical that can react fast with several targets such as other free radicals or metal centers in proteins and plays an important role in many physiological and diverse pathophysiological conditions [11,12,26]. Findings over the last decade have implicated that NO produced by aberrant expression of iNOS may result in many diseases including Alzheimer ' s disease, Parkinson ' s disease, asthma, infl ammatory bowel disease, arthritis, etc. [14,16,27,28]. iNOS is a Ca 2 ϩ -independent isoform of NOS and can produce cytotoxic levels of NO from L-arginine in response to infl ammatory mediators. iNOS can be regulated through its synthesis, stability, and catalytic activity [13,29,30]. The factors that aff ect iNOS stability have not been studied in detail. Our previous study suggests that GSTP1 plays an anti-infl ammatory role in response to LPS and inhibits LPS-induced iNOS protein increase and NO release [9]. Earlier report demonstrates that GSTP1 acts as a NO carrier [8]. However, up till now the detailed mechanism utilized by GSTP1 for regulating cellular iNOS protein level remains largely unclear.
Here, we used overexpression and RNAi technique to show that GSTP1 inhibited LPS-induced iNOS protein level in RAW264.7 cells. Since HEK293 cells lack NOS expression background, they are suitable for investigating exogenous iNOS expression. In HEK293 cells, we got the same results as those in RAW264.7 cells. In GST family, both GSTmu and GSTpi have a similar structure of C-terminal domain, which implies some of their similar properties, but diff erent protein binding features between these two enzymes have also been reported [31]. Although they share 25% -30% sequence identity, their substrate specifi city and diversity have been reshaped by gene duplication, genetic recombination, and an accumulation of mutations. In the present experiment, unlike GSTP1, GSTM1 protein posed no eff ect on iNOS protein level. This result suggests that among GSTs, GSTP1 is specifi c in regulating elevation of LPS-induced iNOS protein level. Our study also suggests that the enzyme activity of GSTP1 was necessary for GSTP1 to regulate iNOS protein level. Figure 6. GSTP1 regulates iNOS monomer/dimer level. A, RAW264.7 cells were transiently transfected with 0.5, 1, and 2 μ g of Xpress-GSTP1 or control vectors by Lipofectamine 2000 and 18 h after transfection, the cells were treated with or without 100 ng/ml LPS for 12 h before being harvested. B, The clones of control and RAW264.7 cells stably expressing GSTP1 were treated with 100 ng/ml of LPS for 12 hours. C, The control and mGSTP1 stably knockdown RAW264.7 clones were treated with 100 ng/ml of LPS for 12 hours. Cell lysates were then subjected to nonreducing Western blot using Inos, Xpress, or GSTP antibody. GAPDH expression was measured to confi rm the equal amount of protein. A representative Western blot for each treatment from three independent experiments is shown. Results are represented by means Ϯ S.E. ( n ϭ 3), * p Ͻ 0.05 compared with the cells treated with 100 ng/ml of LPS only.
Since real-time PCR experiments demonstrated that GSTP1 did not infl uence iNOS mRNA level, we focused the eff ect of GSTP1 on iNOS protein stability. We found that GSTP1 reduced the full length of iNOS protein level and both oxygenase and reductase domains of iNOS were involved in regulation by GSTP1. GSTP1 participates in the regulation of stress signaling via its noncatalytic, ligand-binding activity [31]. Through analyzing the interaction of GSTP1 with iNOS, we found that G-site domain of GSTP1 physically associated with the oxygenase domain of iNOS. Although GSTP1 (Y7F) did not aff ect iNOS stability, it could interact with iNOS, which indicated that the interaction of GSTP1 and iNOS depended on ligand-binding activity of GSTP1 and the enzyme activity of GSTP1 was necessary for regulating iNOS stability. This result suggested that GSTP1 regulated iNOS stability through modifying iNOS at post-translational level.
Dimerization of iNOS protein is essential for NO synthetic activity [21,22]. In our following study, we discovered that GSTP1 but not GSTM1 and GSTP1 (Y7F) obviously decreased the amount of iNOS dimer level, which suggested the eff ect of GSTP1 on iNOS protein dimerization. There are very few reports about post-translational modifi cation of iNOS. It has been reported that human Tyr 1055 in iNOS might be a target for Src-mediated phosphorylation and such post-translational modifi cation serves to stabilize iNOS half-time [32]. S-nitrosylation, a redoxbased post-translational modifi cation of proteins by NO, is recognized to regulate the activities of an increasing number of target proteins. S-nitrosylation is a ubiquitous regulatory reaction comparable to phosphorylation and is already considered a main form for NO to play its " second messenger " function [33 -35]. NO could also alter iNOS dimer stability through increasing the protein S-nitrosylation. S-nitrosylation of the zinc tetrathiolate cysteines in iNOS Figure 7. GSTP1 increases S-nitrosylation and ubiquitination of iNOS, but has no eff ect on p-Tyr of iNOS. A, HEK293 cells were transiently transfected with Xpress-GSTP1 and Myc-iNOS and 400 mM Na3VO4 was added for 30 min before the cells were harvested. B, The clones of control and RAW264.7 cells stably expressing GSTP1 were treated with 500 ng/ml of LPS for 12 hours. C, HEK293 cells were transiently transfected with 0.5 and 1 μ g of Xpress-GSTP1 and pcDNA-iNOS (1.5 μ g) or control vectors for 36 h before being harvested. D, The clones of control stably expressing GSTP1 and HEK293 cells with stable knockdown of GSTP1 were transfected with pcDNA-iNOS (1.5 μ g) for 36 hours. E, HEK293 cells were transiently transfected with 1 μ g of Flag-GSTM1, 1 μ g of Xpress-GSTP1 (Y7F), and 1 μ g of Xpress-GSTP1 or control vectors for 36 h before being harvested. These cells were then lysed and iNOS was immunoprecipitated using anti-iNOS and immobilized protein A/G (agarose beads). The samples were then subjected to the biotin switch procedure, and then affi nity purifi ed by immobilized streptavidin, washed, eluted, and separated by SDS-PAGE, electroblotted to nitrocellulose, and probed with antibody for iNOS. F, HEK293 cells were transiently transfected with Xpress-GSTP1 and Myc-iNOS, and MG132 (30 μ M) was added for additional 8 h. Cell lysates were subjected to immunoprecipitation with anti-Myc, and the precipitates were analyzed by immunoblotting with anti-Ub and anti-Myc antibodies. stimulates zinc release from the dimer interface, resulting in monomer formation and reduction in catalytic activity [12]. Monomer of iNOS is easy to be ubiquitinated and then degraded. Our laboratory has reported that COOH terminus of heat shock protein 70-interacting protein or CHIP facilitates ubiquitination of iNOS and promotes its proteasomal degradation [36]. Since GSTP1 can infl uence the amount of iNOS monomer/dimer and work as a NO carrier, it is reasonable to consider if GSTP1 regulates iNOS through S-nitrosylation. Our results indicated that GSTP1, but not GSTM1, increased S-nitrosylation and ubiquitination of iNOS. However, GSTP1 showed no eff ect on phosphorylation of iNOS. As GSTP1 (Y7F) mutant did not aff ect S-nitrosylation of iNOS, the enzyme activity of GSTP1 was demonstrated to play an important role in regulating S-nitrosylation of iNOS.
In summary, our results reveal a novel role of GSTP1 in regulation of iNOS and provide a new insight for investigating the anti-infl ammatory eff ects of GSTP1. Understanding this function of GSTP1 is benefi cial for developing the new therapeutic strategies aimed at modulating S-nitrosylation and ubiquitination of iNOS.