S-nitrosylation of fatty acid synthase regulates its activity through dimerization
2016; Elsevier BV; Volume: 57; Issue: 4 Linguagem: Inglês
10.1194/jlr.m065805
ISSN1539-7262
AutoresMin Sik Choi, Ji-Yong Jung, Hyoung-June Kim, Mi Ra Ham, Tae Ryong Lee, Dong Wook Shin,
Tópico(s)Hormonal Regulation and Hypertension
ResumoNO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys1471 and Cys2091, but not at Cys1127, increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis. NO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys1471 and Cys2091, but not at Cys1127, increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis. FAS, which is highly expressed in adipose tissue, liver, and lactating mammary glands, catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA in the presence of NADPH (1Loftus T.M. Jaworsky D.E. Frehywot G.L. Townsend C.A. Ronnett G.V. Lane M.D. Kuhajda F.P. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors.Science. 2000; 288: 2379-2381Crossref PubMed Scopus (832) Google Scholar, 2Hu Z. Cha S.H. Chohnan S. Lane M.D. Hypothalamic malonyl-CoA as a mediator of feeding behavior.Proc. Natl. Acad. Sci. USA. 2003; 100: 12624-12629Crossref PubMed Scopus (223) Google Scholar, 3Wakil S.J. Fatty acid synthase, a proficient multifunctional enzyme.Biochemistry. 1989; 28: 4523-4530Crossref PubMed Scopus (682) Google Scholar). 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Therefore, under physiological conditions, S-nitrosylation can affect a number of cellular signaling pathways by inducing conformational changes of the protein and affecting protein-protein interactions and protein functions (36Nakamura T. Tu S. Akhtar M.W. Sunico C.R. Okamoto S-I. Lipton S.A. Aberrant protein s-nitrosylation in neurodegenerative diseases.Neuron. 2013; 78: 596-614Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 37Nakamura T. Lipton S.A. Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases.Cell Death Differ. 2011; 18: 1478-1486Crossref PubMed Scopus (174) Google Scholar, 38Ryan S.D. Dolatabadi N. Chan S.F. Zhang X. Akhtar M.W. Parker J. Soldner F. Sunico C.R. Nagar S. 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Almog O. Kovsan J. Bashan N. Benhar M. Rudich A. Increased adipocyte S-nitrosylation targets anti-lipolytic action of insulin: relevance to adipose tissue dysfunction in obesity.J. Biol. Chem. 2011; 286: 30433-30443Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and to enhance adipogenesis in primary human preadipocytes (42Hemmrich K. Gummersbach C. Paul N.E. Goy D. Suschek C.V. Kröncke K-D. Pallua N. Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes.Cytotherapy. 2010; 12: 547-553Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). However, little is known about the target proteins of S-nitrosylation during adipogenesis. In the present study, we showed that FAS is S-nitrosylated during adipogenesis and that the S-nitrosylation of FAS increases its activity by enhancing dimerization, indicating that S-nitrosylation of FAS contributes to adipogenesis. The NO scavengers, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) (P5084) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (C221), were obtained from Sigma-Aldrich (St. Louis, MO). The NO donor, diethylamine diazeniumdiolate diethylammonium salt (DEA-NONOate) (D5431), and a NO synthase (NOS) inhibitor, Nω-nitro-L-arginine (N5501), were also obtained from Sigma-Aldrich. S-nitrosocysteine (SNOC) was prepared as described previously (43Lei S.Z. Pan Z-H. Aggarwal S.K. Chen H-S.V. Hartman J. Sucher N.J. Lipton S.A. Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.Neuron. 1992; 8: 1087-1099Abstract Full Text PDF PubMed Scopus (686) Google Scholar). Lipofectamine 2000 (11668019) and Lipofectamine LTX with Plus reagent (15338030) were obtained from Life Technologies (Carlsbad, CA). Anti-nitrosocysteine (ab94930) and anti-FAS (ab22759) antibodies were purchased from Abcam (Cambridge, UK), mouse monoclonal anti-FLAG (F3166) antibody from Sigma-Aldrich, and anti-GAPDH (SC-25778) from Santa Cruz Biotechnology (Dallas, TX). HEK293 cells were grown in DMEM (Life Technologies) containing 10% FBS at 37°C under 5% CO2/95% air. Human adipose-derived stem cells (ADSCs) were purchased from Lonza (Walkersville, MD). Confluent ADSCs were incubated in ADSC differentiation medium containing 1 μg/ml insulin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM troglitazone (referred to as IDXT), and 10% FBS. The culture medium was changed every 2 days. Insulin (I5500), IBMX (I5879), dexamethasone (D8893), and troglitazone (T2573) were purchased from Sigma-Aldrich. Flash-frozen human subcutaneous adipose tissue (T-SQFX-FF) was purchased from Zen-Bio (Research Triangle Park, NC). For quantitative determination of the NO level, a NO assay kit (Colorimetric, ab65328) from Abcam was used. Total nitrate and nitrite levels were measured in a two-step process. The first step converted nitrate to nitrite with nitrate reductase. The second step used Griess reagents to convert nitrite to a deep purple azo compound. The amount of azochromophore reflected the amount of nitric oxide in the samples. The absorbance at 550 nm was immediately recorded and compared with the absorbance of a freshly prepared standard curve of sodium nitrite. Adipocytes were fixed for 1 h in 10% formalin in PBS followed by permeabilization in 0.1% Triton X-100 for 10 min. Blocking was performed in PBS containing 10% normal goat serum for 1 h. The fixed cells were incubated using anti-FAS or anti-nitrosocysteine overnight in the blocking solution. The cells were washed and incubated for 1 h with either Alexa-Fluor-488-conjugated goat anti-mouse antibody (A10680) or Alexa-Fluor-594-conjugated goat anti-rabbit antibody (A11012) (Molecular Probes, Carlsbad, CA). After washing, the cells were mounted in a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). Cells were observed using an EVOS FL cell imaging system (Life Technologies). Formalin solution (HT501128), goat serum (G9023), and Fluoroshield mounting medium with DAPI (F6057) were purchased from Sigma-Aldrich. A plasmid encoding human FAS (SC127829, NM_004104) was obtained from Origene (Rockville, MD) and subcloned into the pcDNA3.1 vector (Life Technologies). Point mutations were introduced to change each cysteine in the FAS gene (Cys161, Cys1127, Cys1471, or Cys2091) to an alanine. The primer sequences for PCR-directed mutagenesis of FAS Cys161 to Ala were: forward, 5′-CGCACTGGACACAGCCGCCTCCTCCAGCCTGATGGC-3′ reverse, 5′-GCCATCAGGCTGGAGGAGGCGGCTGTGTCCAGTGCG-3′. For mutagenesis of FAS Cys1127 to Ala, the primer sequences were: forward, 5′-CCACACGGAGGAGGGGGCCCTGTCTGAGCGCGCTG-3′ reverse, 5′-CAGCGCGCTCAGACAGGGCCCCCTCCTCCGTGTGG-3′. For mutagenesis of FAS Cys1471 to Ala, the primer sequences were: forward, 5′-CGGGAACCGCCTCCGGGCTGTGCTGCTCTCCAACC-3′ reverse, 5′-GGTTGGAGAGCAGCACAGCCCGGAGGCGGTTCCCG-3′. For mutagenesis of FAS Cys2091 to Ala, the primer sequences were: forward, 5′-CCAGCGCATGGCGTCCGCCCTGGAGGTGCTGGACC-3′ reverse, 5′-GGTCCAGCACCTCCAGGGCGGACGCCATGCGCTGG-3′. Adipocyte differentiation was assessed using an Oil Red O stain (O0625, Sigma-Aldrich) as an indicator of intracellular lipid accumulation. After ADSC differentiation to adipocytes, cells were washed twice with PBS, fixed with 10% formalin in PBS for 1 h, and then washed with 60% isopropanol, before being allowed to dry completely. Adipocytes were stained with 0.2% Oil Red O reagent for 10 min at room temperature and washed with water four times. Each sample was eluted with 100% isopropanol for 10 min and absorbance was measured at 500 nm using a spectrophotometer. To visualize the nucleus, adipocytes were counterstained with a hematoxylin reagent (H3136, Sigma-Aldrich) for 2 min and washed twice with water. The level of adipocyte differentiation was observed using an inverted phase microscope. Cell lysates or adipose tissue lysates were prepared in HENTS buffer [100 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine, 1% Triton X-100, and 0.1% SDS (pH 7.4)]. The biotin-switch assay was carried out as described previously (44Jaffrey S.R. Snyder S.H. The biotin switch method for the detection of S-nitrosylated proteins.Sci. STKE. 2001; 2001: pl1Crossref PubMed Google Scholar) with a slight modification. Briefly, free thiols in the sample were blocked by incubation with 10 mM methyl methanethiosulfonate (MMTS) at 50°C for 15 min. The MMTS was then removed by acetone precipitation and the pellet was resuspended in HENS buffer [100 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine, and 0.1% SDS (pH 7.4)]. S-nitrosothiols were selectively reduced with 20 mM ascorbate and the reformed free thiols were labeled with 1 mM N-[6-(biotinamido)hexyl]3′-(2′-pyridyldithio)-propionamide (HPDP-biotin) for 1 h at room temperature. The biotinylated proteins were then collected on avidin agarose beads, which were then washed three times with neutralization buffer [20 mM HEPES-NaOH (pH 7.4), 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100]. Proteins were eluted from the beads by SDS-PAGE loading buffer and subjected to immunoblot analysis. Neocuproine (N1501), MMTS (64306), and ascorbate (A7506) were purchased from Sigma-Aldrich. EZ-Link HPDP-biotin and NeutrAvidin agarose beads were from Thermo Scientific (Waltham, MA). The FAS activity assay was performed as previously described (45Kelley D.S. Nelson G.J. Hunt J.E. Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes.Biochem. J. 1986; 235: 87-90Crossref PubMed Scopus (127) Google Scholar) with minor modifications. In brief, cell lysates were mixed with acetyl-CoA and NADPH in 0.2 M potassium phosphate buffer and 0.4 mM EDTA (pH 7.0), and incubated at 30°C for 10 min. The enzymatic reaction was initiated by adding 20 μl of malonyl-CoA solution (0.2 mM), and the decrease in optical density (OD) was measured every 1 min for 30 min via kinetic measurements obtained on a microplate reader set at 340 nm. Based on the results, the overall FAS enzyme activity was estimated by calculating the NADPH oxidation, using ε = 6,220 M−1cm−1. Acetyl-CoA (A2056), NADPH (N1630), and malonyl-CoA (M4263) were purchased from Sigma-Aldrich. The statistical analyses of the data were analyzed with the Student's t-test or by one-way ANOVA. Data are expressed as mean ± SD of at least three independent experiments. P < 0.05 was considered statistically significant. To investigate whether NO is involved in the process of adipocyte differentiation, we first evaluated NO production during adipocyte differentiation (Fig. 1A). The production of NO during adipocyte differentiation was estimated by measuring the total concentration of its oxidation metabolites, nitrite and nitrate, using a NO assay kit. Compared with the early differentiation period of adipocytes (days 0–2), there was a significant increase in total NO level in the supernatants of cells taken at the later differentiation period (days 6–8) (Fig. 1A). We next investigated whether protein S-nitrosylation, a major posttranslational modification by NO, might have a unique role in adipogenesis. Protein S-nitrosylation can occur even at low levels of NO and can affect the activity and stability of specific proteins (36Nakamura T. Tu S. Akhtar M.W. Sunico C.R. Okamoto S-I. Lipton S.A. Aberrant protein s-nitrosylation in neurodegenerative diseases.Neuron. 2013; 78: 596-614Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 37Nakamura T. Lipton S.A. Redox modulation by S-nitrosylation contributes to protein misfolding, mitochondrial dynamics, and neuronal synaptic damage in neurodegenerative diseases.Cell Death Differ. 2011; 18: 1478-1486Crossref PubMed Scopus (174) Google Scholar, 38Ryan S.D. Dolatabadi N. Chan S.F. Zhang X. Akhtar M.W. Parker J. Soldner F. Sunico C.R. Nagar S. Talantova M. et al.Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.Cell. 2013; 155: 1351-1364Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 39Jeon G.S. Nakamura T. Lee J-S. Choi W-J. Ahn S-W. Lee K-W. Sung J-J. Lipton S.A. Potential effect of S-nitrosylated protein disulfide isomerase on mutant SOD1 aggregation and neuronal cell death in amyotrophic lateral sclerosis.Mol. Neurobiol. 2014; 49: 796-807Crossref PubMed Scopus (47) Google Scholar). Immunofluorescence assay using an antibody against nitroso-cysteine showed that protein S-nitrosylation was higher in differentiated adipocytes (day 9) than in undifferentiated preadipocytes (Fig. 1B). SNOC, an NO donor, demonstrated a biphasic effect: it increased adipogenesis at a low concentration (50 μM) and decreased adipogenesis at a high concentration (200 μM) (Fig. 1C). cPTIO and PTIO, well-known NO scavengers, inhibited adipogenesis in a concentration-dependent manner (Fig. 1D, E). The better NO scavenger, cPTIO showed slightly more efficient inhibition of adipogenesis than PTIO at the same concentrations (Fig. 1D, E). To test the effect of NOS inhibitor on adipogenesis, we performed the same experiments with Nω-nitro-L-arginine (NNA), a broad NOS inhibitor. We found that there was no effect of NNA on adipogenesis (supplementary Fig. 1A). In agreement with a previous report (46Elizalde M. Rydén M. van Harmelen V. Eneroth P. Gyllenhammar H. Holm C. Ramel S. Olund A. Arner P. Andersson K. Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans.J. Lipid Res. 2000; 41: 1244-1251Abstract Full Text Full Text PDF PubMed Google Scholar), we could not see the expression of inducible NOS (iNOS) and neuronal NOS (nNOS) during adipogenesis, whereas the endothelial NOS (eNOS) protein band was detectable only at the later differentiation stage (day 14) (supplementary Fig. 1B). Following a previous report on fatty acid metabolism and S-nitrosylation (40Doulias P-T. Tenopoulou M. Greene J.L. Raju K. Ischiropoulos H. Nitric oxide regulates mitochondrial fatty acid metabolism through reversible protein S-nitrosylation.Sci. Signal. 2013; 6: rs1Crossref PubMed Scopus (175) Google Scholar), we analyzed the levels of FAS, one of the essential proteins for lipid droplet formation during adipogenesis, in differentiated adipocytes. We confirmed that the levels of nitrosocysteine and FAS in more differentiated adipocytes (day 12) were higher than those in less differentiated adipocytes (day 2) (Fig. 2A). The yellow color in the merged image of nitrosocysteine (green) and FAS (red) images implied that FAS was likely to be S-nitrosylated (SNO-FAS) (Fig. 2A). We also performed immunostaining for nitrosocysteine with negative controls to reaffirm antibody and detection specificity (supplementary Fig. 2). To confirm that the biotin-switch assay allowed detection of SNO-FAS, the lysate of undifferentiated adipocytes was subjected to the assay after incubation in SNOC with or without the assay components, including MMTS, ascorbate, and HPDP-biotin (Fig. 2B). In addition, we tested whether FAS in adipose tissue homogenates could be S-nitrosylated. We eliminated SNOC by treatment with UV-light and used this sample for negative control. We found that FAS was nitrosylated in human adipose tissue and the SNO-FAS in lysates was eliminated by UV-light (Fig. 2C).Fig. 2Increased S-nitrosylation and dimerization of FAS in differentiated adipocytes. A: Immunofluorescence to detect expression of FAS (red) and S-nitrosylated proteins (green) in differentiated adipocytes (on days 2 and 12). Merged images (yellow) demonstrate the overlap between the localization of FAS and S-nitrosylated proteins. DAPI staining (blue) was used to identify cell nuclei. Scale bar = 100 μm. B: To confirm the S-nitrosylation of FAS and that the biotin-switch assay detects SNO-FAS properly, the lysate of undifferentiated ADSCs was subjected to a biotin-switch assay with or without the components of the assay, as described in Materials and Methods, and immunoblotting (IB) following incubation with the NO donor SNOC or old SNOC. C: The lysates of human adipose tissue (200 mg) were irradiated with UV-light or incubated in the dark for 10 min. Then, the lysates were subjected to a biotin-switch assay with or without ascorbate. UV-irradiated sample was used as a negative control by eliminating SNOC. Data are representative of three individual experiments. D: The lysates of cells at different stages of adipocyte differentiation (days 0–14) were subjected to a biotin-switch assay followed by immunoblotting for SNO-FAS (top), total FAS (middle), and GAPDH (bottom), and the ratio of SNO-FAS/total FAS expression was quantified. Data were normalized by setting day 0 as 1 (mean ± SD). *P < 0.05 compared with day 0 by t-test. E: Immunoblotting of cell lysates carried out under nondenaturing conditions shows the dimerization of FAS during adipogenesis (days 3 and 9). The ratio of FAS dimer/FAS monomer was quantified. Data were normalized by setting day 3 as 1 (mean ± SD). *P < 0.05 compared with day 3 by t-test. Data are representative of three individual experiments.View Large Image Figure ViewerDownload Hi-res
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