Proteomics Characterization of the Cytotoxicity Mechanism of Ganoderic Acid D and Computer-automated Estimation of the Possible Drug Target Network
2008; Elsevier BV; Volume: 7; Issue: 5 Linguagem: Inglês
10.1074/mcp.m700259-mcp200
ISSN1535-9484
AutoresQing‐Xi Yue, Zhiwei Cao, Shuhong Guan, Xiaohui Liu, Lin Tao, Wanying Wu, Yixue Li, Pengyuan Yang, Xuan Liu, De‐an Guo,
Tópico(s)Fungal Biology and Applications
ResumoTriterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of G. lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50 value of 17.3 ± 0.3 μm. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G2/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, two-dimensional gel electrophoresis was performed, and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore these possible GAD target-related proteins were evaluated by an in silico drug target searching program, INVDOCK. The INVDOCK analysis results suggested that GAD could bind six isoforms of 14-3-3 protein family, annexin A5, and aminopeptidase B. The direct binding affinity of GAD toward 14-3-3 ζ was confirmed in vitro using surface plasmon resonance biosensor analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly or associate with each other via only one intermediate protein from previous protein-protein interaction experimental results. When the network was expanded to a further interaction outward, all 21 proteins could be included into one network. In this way, the possible network associated with GAD target-related proteins was constructed, and the possible contribution of these proteins to the cytotoxicity of GAD is discussed in this report. Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of G. lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50 value of 17.3 ± 0.3 μm. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G2/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, two-dimensional gel electrophoresis was performed, and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore these possible GAD target-related proteins were evaluated by an in silico drug target searching program, INVDOCK. The INVDOCK analysis results suggested that GAD could bind six isoforms of 14-3-3 protein family, annexin A5, and aminopeptidase B. The direct binding affinity of GAD toward 14-3-3 ζ was confirmed in vitro using surface plasmon resonance biosensor analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly or associate with each other via only one intermediate protein from previous protein-protein interaction experimental results. When the network was expanded to a further interaction outward, all 21 proteins could be included into one network. In this way, the possible network associated with GAD target-related proteins was constructed, and the possible contribution of these proteins to the cytotoxicity of GAD is discussed in this report. Ganoderma lucidum is a medicinal mushroom known to the Chinese as “Lingzhi.” It has been used as a home remedy in traditional Chinese medicine (TCM) 1The abbreviations used are: TCM, traditional Chinese medicine; GAD, ganoderic acid D; SPR, surface plasmon resonance; RU, response unit(s); eIF5A, eukaryotic translation initiation factor 5A; PRDX3, thioredoxin-dependent peroxide reductase mitochondrial precursor; 14-3-3E, 14-3-3 ε; EB1, microtubule-associated protein RP/EB family member 1; AHA1, activator of heat shock 90-kDa protein ATPase homolog 1; PDI, protein-disulfide isomerase; 2-DE, two-dimensional gel electrophoresis; 3-D, three-dimensional; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PMF, peptide mass fingerprint; PPI, protein-protein interaction. 1The abbreviations used are: TCM, traditional Chinese medicine; GAD, ganoderic acid D; SPR, surface plasmon resonance; RU, response unit(s); eIF5A, eukaryotic translation initiation factor 5A; PRDX3, thioredoxin-dependent peroxide reductase mitochondrial precursor; 14-3-3E, 14-3-3 ε; EB1, microtubule-associated protein RP/EB family member 1; AHA1, activator of heat shock 90-kDa protein ATPase homolog 1; PDI, protein-disulfide isomerase; 2-DE, two-dimensional gel electrophoresis; 3-D, three-dimensional; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PMF, peptide mass fingerprint; PPI, protein-protein interaction. for over 2000 years (1Yun T.K. Update from Asia. Asian studies on cancer chemoprevention.Ann. N. Y. Acad. Sci. 1999; 889: 157-192Crossref PubMed Scopus (116) Google Scholar). In TCM, it was believed to preserve the human vitality and to promote longevity. More recently, it has been used for the prevention or treatment of a variety of diseases including cancer. And in Western countries, the dried powder of G. lucidum is also popularly used as a dietary supplement (2Sliva D. Cellular and physiological effects of Ganoderma lucidum (Reishi).Mini-Rev. Med. Chem. 2004; 4: 873-879Crossref PubMed Scopus (99) Google Scholar). Among the reported biological/pharmacological properties of G. lucidum, their antitumor activities are of particular interest. Investigations into the anticancer activity of G. lucidum have been performed in both in vitro and in vivo studies, supporting its application for cancer treatment and prevention (for reviews, see Refs. 3Yuen J.W. Gohel M.D. Anticancer effects of Ganoderma lucidum: a review of scientific evidence.Nutr. Cancer. 2005; 53: 11-17Crossref PubMed Scopus (193) Google Scholar and 4Sliva D. Ganoderma lucidum in cancer research.Leuk. Res. 2006; 30: 767-768Crossref PubMed Scopus (92) Google Scholar). Polysaccharides and triterpenes are two major categories of the bioactive ingredients from G. lucidum, and it has been found previously that polysaccharides exert their anticancer effect mainly via an immune-modulatory mechanism, whereas triterpenes directly suppress growth and invasive behavior of cancer cells (5Yeung W.H. Lu Q.L. Zhang Q. Go V.L.W. Chemical and biochemical basis of the potential anti-tumor properties of Ganoderma lucidum.Curr. Top. Nutraceutical Res. 2004; 2: 67-77Google Scholar). Triterpenes were reported to be able to inhibit growth, induce apoptosis, and cause cell cycle arrest of cancer cells (6Lin S.B. Li C.H. Lee S.S. Kan L.S. Triterpene-enriched extracts from Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C, activating mitogen-activated protein kinases and G2-phase cell cycle arrest.Life Sci. 2003; 72: 2381-2390Crossref PubMed Scopus (217) Google Scholar, 7Yang H.L. Ganoderic acid produced from submerged culture of Ganoderma lucidum induces cell cycle arrest and cytotoxicity in human hepatoma cell line BEL7402.Biotechnol. Lett. 2005; 27: 835-838Crossref PubMed Scopus (39) Google Scholar, 8Kimura Y. Taniguchi M. Baba K. Antitumor and antimetastatic effects on liver triterpenoid fractions of Ganoderma lucidum: mechanism of action and isolation of active substance.Anticancer Res. 2002; 22: 3309-3318PubMed Google Scholar, 9Min B.S. Gao J.J. Nakamura N. Hattori M. Triterpenes from the spores of Ganoderma lucidum and their cytotoxicity against meth-A and LLC tumor cells.Chem. Pharm. Bull. 2000; 48: 1026-1033Crossref PubMed Scopus (236) Google Scholar). However, the cytotoxicity mechanism of Ganoderma triterpenes is still far from clear. In the present study, ganoderic acid D (GAD), a main component of Ganoderma triterpenes, with purity greater than 99% was used. We checked the GAD-mediated response on the proliferation of HeLa human cervical carcinoma cells. Then for a comprehensive analysis of the molecular targets of GAD, a proteomics approach was used for identifying proteins altered in steady-state levels after exposure of HeLa cells to GAD for 48 h. 2-DE was conducted, and then differentially expressed proteins were identified by MALDI-TOF MS/MS and further confirmed by Western blot analysis. Moreover a computational program, INVDOCK, was applied to verify the possible direct targets of GAD. The predicted binding between GAD and 14-3-3 ζ was then confirmed by using surface plasmon resonance (SPR) biosensor analysis. And finally a comprehensive network analysis was conducted to mine the functional association between the experimentally defined proteins. GAD was isolated and purified from G. lucidum by the laboratory of TCM chemistry, Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences as reported before (10Wang X.M. Yang M. Guan S.H. Liu R.X. Xia J.M. Bi K.S. Guo D.A. Quantitative determination of six major triterpenoids in Ganoderma lucidum and related species by high performance liquid chromatography.J. Pharm. Biomed. Anal. 2006; 41: 838-844Crossref PubMed Scopus (74) Google Scholar). The structure of GAD (including the chemical structure and 3-D structure) is shown in Fig. 1. GAD was identified by spectral analyses, primarily NMR and MS, and comparison with previous literature (11Komoda Y. Nakamura H. Ishihara S. Uchida M. Kohda H. Yamasaki K. Structures of new terpenoid constituents of Ganoderma lucidum (Fr.) Karst (Polyporaceae).Chem. Pharm. Bull. 1985; 33: 4829-4835Crossref Scopus (102) Google Scholar). After identification, it was further purified by HPLC to yield authorized compound with a purity of at least 99%. The result of spectral analyses and HPLC analysis of GAD is shown in supplemental Figs. Fig. 1, Fig. 2, Fig. 3, Fig. 4. All reagents used in 2-DE were purchased from Bio-Rad. Other chemicals, except where specially noted, were purchased from Sigma-Aldrich.Fig. 2Effect of GAD on HeLa cell viability, cell cycle arrest, and apoptosis.A, HeLa cells were treated with 1, 5, 10, 20, and 50 μm GAD for 24, 48, and 72 h, and cell viability was determined by MTT assay. B, DNA histograms of HeLa cells obtained by flow cytometry analysis. Accumulation in G2/M phase was observed in 10 and 50 μm GAD-treated cells after 24-h treatment. An increase in the percentage of apoptotic cells was observed in GAD-treated cells after 48-h treatment. C, morphological change induced by 10 and 50 μm GAD in HeLa cells after 48-h treatment (×600 magnification). Typical apoptotic morphological change in GAD-treated cells was observed. D, DNA fragmentation induced by 10 and 50 μm GAD in HeLa cells after 48-h treatment. Typical apoptotic DNA fragmentation (DNA ladder) was observed in HeLa cells treated with 50 μm GAD. Shown are representative results of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3The proteome maps (2-DE images) of GAD-treated HeLa cells.A, panels a and b, are 2-DE images with better separation of higher molecular weight proteins of control and GAD-treated HeLa cells, respectively. Panels c and d are 2-DE images with better separation of lower molecular weight proteins of control and GAD-treated HeLa cells, respectively. GAD-treated HeLa cells were treated with 10 μm GAD for 48 h. The gel pair is the representative gel of nine replicate gels collected from three independent experiments. Differentially expressed spots are shown by the arrows. B, the expanded region of differentially expressed protein spots. The proteins within the circles are the differentially expressed proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4The result of the MALDI-TOF MS/MS analysis of protein that marked as spot 9 in Fig. 1. The protein was identified to be human 14-3-3E by protein database search. A, peptide mass fingerprint of the tryptic digest of spot 9. * indicates unique peptides further identified by MS/MS. B, MS/MS profile of the peptide with a mass of 1819.95 Da. C, MS/MS profile of the peptide with a mass of 1384.70 Da. D, MS/MS profile of the peptide with a mass of 1256.61 Da. y-ions resulting from fragmentation of the peptides and amino acids they represent are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The HeLa human cervical carcinoma cell line (CCL-2) was obtained from the American Type Culture Collection (Manassas, VA), and cells were cultured in minimum essential medium (Invitrogen) with 2 mm l-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mm non-essential amino acids, 1.0 mm sodium pyruvate, and 10% fetal bovine serum. Antibiotics added were 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The cytotoxicity of GAD was determined by a calorimetric tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) assay as reported before (12Liu X. Fan X.L. Zhao Y. Luo G.R. Li X.P. Li R. Le W.D. Estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor-α and estrogen receptor-β in microglia.J. Neurosci. Res. 2005; 81: 653-665Crossref PubMed Scopus (93) Google Scholar). Briefly cells were plated in 96-well flat bottomed plates at a density of 1 × 103 cells/well in complete medium and incubated overnight. Then the media were changed into fresh media containing various amounts of GAD for 24, 48, or 72 h. At the end of the incubation, 20 μl of the dye MTT (5 mg/ml) was added to each well, and the plates were incubated for 3 h at 37 °C. Then 100 μl of lysis buffer (20% SDS in 50% N,N-dimethylformamide containing 0.5% (v/v) 80% acetic acid and 0.4% (v/v) 1 n HCl) was added to each well and incubated overnight (16 h). Cell viability was evaluated by measuring the mitochondria-dependent conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolic active cells. The optical density (proportional to the number of live cells) was assessed with a Bio-Rad 550 microplate reader at 570 nm. Each experiment was performed in triplicate. Results of three independent experiments were used for statistical analysis. IC50 value (half-maximal inhibitory concentration) was calculated by the Logit method. Flow cytometric analysis of cell cycle was conducted as reported before (13Liu X. Zhu X.Z. Roles of p53, c-Myc, Bcl-2, Bax and caspases in glutamate-induced neuronal apoptosis and the possible neuroprotective mechanism of basic fibroblast growth factor.Brain Res. Mol. Brain Res. 1999; 71: 210-216Crossref PubMed Scopus (62) Google Scholar). Briefly adherent and detached cells were harvested with trypsin, washed with PBS three times, and then fixed in ice-cold 70% ethanol at 4 °C for 2 h. After centrifugation at 100 × g for 2 min, cells were resuspended in propidium iodide stain buffer (0.1% Triton X-100, 10 μg/ml DNase-free RNase A, and 50 μg/ml propidium iodide in PBS) for 30 min in the dark. Flow cytometric analysis was conducted using a BD Biosciences FACStar Plus flow cytometer. To detect morphological changes in the apoptotic process, nuclear staining was performed as reported before (13Liu X. Zhu X.Z. Roles of p53, c-Myc, Bcl-2, Bax and caspases in glutamate-induced neuronal apoptosis and the possible neuroprotective mechanism of basic fibroblast growth factor.Brain Res. Mol. Brain Res. 1999; 71: 210-216Crossref PubMed Scopus (62) Google Scholar). Briefly after treatment with GAD (10 or 50 μm) for 48 h, cells were washed with PBS, and then fixed with 4% paraformaldehyde (pH 7.4) for 30 min at room temperature. After PBS washes, cells were stained with a 0.5 mg/ml solution of 4,6-diamido-2-phenylindole hydrochloride in PBS for 10 min at room temperature. The cells were washed twice with PBS and photographed using an Olympus UV light fluorescence microscope. The integrity of the genomic DNA of the cells was assessed by agarose gel electrophoresis. Briefly after treatment with GAD (10 or 50 μm) for 48 h, cells were washed with PBS and then collected by scraping. The cell genomic DNA was extracted using DNAzol (Invitrogen) and then loaded on 2% agarose gels for electrophoresis. The gels were stained with ethidium bromide (0.5 mg/l) and photographed under UV illumination. For sample preparation, cells were cultured in 75-cm2 flasks at a density of 2 × 105 cells/flask. Cells at 70% confluency were incubated for 48 h with medium containing 0.1% DMSO (solvent control) or in addition with 10 μm GAD. Subsequently cells were washed three times with ice-cold PBS and then scraped off with a cell scraper. Cells of two flasks were combined and subsequently centrifuged for 10 min at 2500 × g. The supernatant was discarded, and cell pellets were dissolved in 200 μl of lysis buffer containing 7 m urea, 2 m thiourea, 2% CHAPS, 1% DTT, 0.8% Pharmalyte, and protease inhibitor (all from Bio-Rad). Homogenization of the cells was achieved by ultrasonication (10 strokes, low amplitude) on ice. The lysed cells were centrifuged at 15,000 × g for 30 min at 4 °C, and the supernatant containing the solubilized proteins was used directly or stored at −80 °C. Protein samples from at least three independent experiments were collected for 2-DE assay. 2-DE was carried out similarly to that described by Roberts et al. (14Roberts K. Bhatia K. Stanton P. Lord R. Proteomic analysis of selected prognostic factors of breast cancer.Proteomics. 2004; 4: 784-792Crossref PubMed Scopus (16) Google Scholar) using a Bio-Rad 2-DE system following the Bio-Rad handbook (15Garfin D. Heerdt L. 2-D Electrophoresis for Proteomics: a Methods and Product Manual. Bio-Rad Laboratories, Richmond, CA2001: 25-27Google Scholar). Briefly a 150-μg protein sample was applied for IEF using the ReadyStrip IPG strips (17 cm, pH 4–7; Bio-Rad). The strips were placed into a Protean IEF cell (Bio-Rad) and were rehydrated at 50 V for 12 h, and then the proteins were separated based on their pI according to the following protocol: 250 V with linear climb for 30 min, 1000 V with rapid climb for 60 min, 10,000 V with linear climb for 5 h, and 10,000 V with rapid climb until 60,000 V-h was reached. After IEF, the IPG strips were equilibrated for 15 min in a buffer containing 50 mm Tris-HCl, pH 8.8, 30% glycerol, 7 m urea, 2% SDS, and 1% DTT followed by further treatment in a similar buffer (but containing 4% iodoacetamide instead of DTT) for 15 min and then directly applied onto 12% homogeneous SDS-PAGE gels for electrophoresis using a Protean II xi cell system (Bio-Rad). Furthermore two kinds of electrophoresis conditions, which were suitable for the separation of proteins with higher molecular weight (10 mA/gel for 30 min followed by 30 mA/gel for 5.5 h) and for the separation of proteins with lower molecular weight (10 mA/gel for 30 min followed by 20 mA/gel for 8 h), respectively, were both used. The gels were then silver-stained using Bio-Rad Silver Stain Plus kit reagents (Bio-Rad) according to the manufacturer's instructions. The silver-stained gels were scanned using a GS-800 densitometer (Bio-Rad) and then analyzed using PDQuest software (Bio-Rad). Paired (control and GAD-treated) protein samples from three independent experiments were analyzed by 2-DE. And for each pair of protein samples, triplicate electrophoreses were performed to ensure reproducibility. Comparisons were made between gel images of protein profiles obtained from the GAD-treated group and control group. The individual protein spot quantity was normalized as follows: the raw quantity of each spot in a member gel was divided by the total quantity of the valid spots in the gel, and normalized spot intensities were expressed in ppm. Quantitative analysis was performed using the Student's t test between protein gels from the control and GAD-treated group. The significantly differentially expressed protein spots (p < 0.05) with 2-fold or more increased or decreased intensity between the control and GAD-treated group were selected and subjected to further identification by MALDI-TOF MS/MS. Proteins of interest were excised from the gels with an EXQuest spot cutter (Bio-Rad) and placed into a 96-well microtiter plate. MS analysis was performed at the Institutes of Biomedical Sciences, Fudan University, Shanghai, China (16Shen H. Cheng G. Fan H. Zhang J. Zhang X. Lu H. Liu C. Sun F. Jin H. Xu X. Xu G. Wang S. Fang C. Bao H. Wang Y. Wang J. Zhong H. Yu Z. Liu Y. Tang Z. Yang P. Expressed proteome analysis of human hepatocellular carcinoma in nude mice (LCI-D20) with high metastasis potential.Proteomics. 2006; 6: 528-537Crossref PubMed Scopus (29) Google Scholar). Briefly gel pieces were destained with a solution of 15 mm potassium ferricyanide and 50 mm sodium thiosulfate (1:1) for 2 min at room temperature. Then the gel pieces were washed twice with deionized water and shrunk by dehydration in ACN. The samples were then swollen in a digestion buffer containing 25 mm ammonium bicarbonate and 12.5 ng/μl trypsin at 4 °C. After 30-min incubation, the gels were digested for more than 12 h at 37 °C. Peptides were then extracted twice using 0.1% TFA in 50% ACN. The extracts were dried under the protection of N2. For MALDI-TOF MS/MS, peptides were mixed with 0.7 μl of MALDI matrix (5 mg/ml α-cyano-4-hydroxycinnamic acid diluted in 0.1% TFA and 50% ACN) and spotted onto the 192-well stainless steel MALDI target plates. MS measurements were carried out on an ABI 4700 Proteomics Analyzer with delayed ion extraction (Applied Biosystems). PMFs and peptide sequence spectra were obtained using the settings presented in supplemental Tables 1 and 2. The first five precursor ions with highest intensity were selected for fragmentation. The accelerated voltage was operated at 20 kV, and the positive ion mass spectra were recorded. MS accuracy was internally calibrated with trypsin-digested peptides of horse myoglobin. Using the individual PMF spectra, peptides exceeding a signal-to-noise ratio of 20 that passed through a mass exclusion filter (supplemental Table 3) were submitted to fragmentation analysis. MS/MS accuracy was calibrated against the MS/MS fragments of m/z 1606.85, which is one of the peaks generated in myoglobin PMF. The parameters for peak matching were: minimum signal-to-noise ratio was 20, mass tolerance was 0.2 Da, minimum peaks to match reference masses was 4, and maximum outlier error was set to 100 ppm. The number of total shots for each PMF spectrum was 2000, whereas for MS/MS the total number of shots was 3000. All PMF and MS/MS peak list data were generated by GPS Explorer software 3.6 with parameter settings as summarized in supplemental Table 4. Data search files were generated according to the settings presented in supplemental Table 5 and submitted for protein homology identification by using the MASCOT 2.1 search engine (Matrix Science) against the Homo sapiens (human, 138,060 sequences) subset of the sequences in the National Center for Biotechnology non-redundant (NCBInr) database (updated on March 17, 2007 with 4,736,044 sequences; 1,634,373,987 residues). Peptide differential modifications allowed during the search were carbamidomethylation of cysteines and oxidation of methionines. The maximum number of missed cleavages was set to 1 with trypsin as the protease. Protein homology identifications of the top hit (first rank) with a relative score exceeding 95% probability and additional hits (second rank or more) with a relative score exceeding 98% probability threshold were retained. The probability-based score, assuming that the observed match is significant (p < 0.05), had to be more than 64 when submitting PMF data to the database and be more than 30 for individual peptide ions when submitting peptide sequence spectra. Proteins belonging to a protein family with multiple members were singled out based on the identification of unique and diagnostic peptides. As reported before (12Liu X. Fan X.L. Zhao Y. Luo G.R. Li X.P. Li R. Le W.D. Estrogen provides neuroprotection against activated microglia-induced dopaminergic neuronal injury through both estrogen receptor-α and estrogen receptor-β in microglia.J. Neurosci. Res. 2005; 81: 653-665Crossref PubMed Scopus (93) Google Scholar), cells were washed three times with cold TBS, harvested using a cell scraper, and lysed in 10 volume of cold lysis buffer (50 mm Tris-HCl, pH 7.2, 250 mm NaCl, 0.1% Nonidet P-40, 2 mm EDTA, 10% glycerol, 1 mm PMSF, 5 μg/ml aprotinin, and 5 μg/ml leupeptin) on ice. Lysates were centrifuged, and then the supernatant protein was denatured by mixing with an equal volume of 2× sample loading buffer and then boiling at 100 °C for 5 min. An aliquot (containing 50 μg protein) of the supernatant was loaded onto a 12% SDS gel, separated electrophoretically, and transferred to a PVDF membrane (Bio-Rad). After the PVDF membrane was incubated with 10 mm TBS with 1.0% Tween 20 and 10% dehydrated skim milk to block nonspecific protein binding, the membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies used were mouse anti-eIF-5A monoclonal antibody (1:1000; BD Biosciences), rabbit anti-14-3-3E polyclonal antibody (1:1000; Abgent, San Diego, CA), rabbit anti-PRDX3 polyclonal antibody (1:1000; Proteintech Group, Chicago, IL), mouse anti-EB1 monoclonal antibody (1:500; BD Biosciences), and mouse anti-actin monoclonal antibody (1:2000; Sigma). Blots were then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) or horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) for 1 h at room temperature at a 1:5000 dilution and then visualized using chemiluminescence (Pierce). Significances of difference between groups were determined by a non-paired Student's t test. For each variable three independent experiments were carried out. Data are given as the mean ± S.D. To verify the proteins related to possible GAD targets derived from the experimental results, a flexible ligand-protein inverse docking program, INVDOCK, was adopted that can predict proteins directly binding with a small molecule through an automatic search of every entry in a protein cavity database (17Chen Y. Z Zhi D.G. Ligand-protein inverse docking and its potential use in the computer search of protein targets of a small molecule.Proteins. 2001; 43: 217-226Crossref PubMed Scopus (320) Google Scholar). To save the computing time, a subset of the cavity database was derived from the 3-D structures of all the experimentally derived proteins beforehand. And this small dataset, instead of the huge cavity database derived from all Protein Data Bank entries, was used to run INVDOCK. Those proteins containing the cavities hit by the GAD molecule were predicted as possible protein targets of GAD. The binding affinity of GAD to 14-3-3 ζ in vitro was assayed by the Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences using an SPR-based Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) as reported before (18Ye F. Zhang Z.S. Luo H.B. Shen J.H. Chen K.X. Shen X. Jiang H.L. The dipeptide H-Trp-Glu-OH shows highly antagonistic activity against PPARγ: bioassay with molecular modeling simulation.Chembiochem. 2006; 7: 74-82Crossref PubMed Scopus (36) Google Scholar, 19Chen S. Chen L.L. Tan J.Z. Chen J. Du L. Sun T. Shen J.H. Chen K.X. Jiang H.L. Shen X. Severe acute respiratory syndrome coronavirus 3C-like proteinase N terminus is indispensable for proteolytic activity but not for enzyme dimerization.J. Biol. Chem. 2005; 280: 164-173Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Human recombinant GST-14-3-3 ζ protein expressed in Escherichia coli (molecular mass, 55 kDa; pI 5.36 in PBS) with a purity of more than 90% was bought from Calbiochem. The manufacturer indicated that it could be used in in vitro binding assays. Human recombinant GST expressed in E. coli (molecular mass, 27 kDa; pI 8.91 in PBS) was a gift from Prof. Jia Li (Shanghai Institute of Materia Medica, Chinese Academy of Sciences) and used as control in the SPR analysis. Both the GST-14-3-3 ζ and GST protein were dissolved in coupling buffer (15 μg/ml, in 10 mm sodium acetate, pH 4.36) and immobilized onto the same sensor chip but on different flow cells. The GST-14-3-3 ζ and GST protein were immobilized on a CM5 sensor chip as ligand in 8000 response units (RU) with N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide according to the standard primary amine-coupling procedures, and HBS-EP (10 mm HEPES, 150 mm NaCl, 3 mm EDTA, and 0.005% (v/v) surfactant P20, pH 7.4) was used as the running buffer. Equilibration of the base line was performed by a continuous flow of HBS-EP through the chip surface for 1–2 h. Biacore data were collected at 25 °C with HBS-EP as the running buffer at a constant flow of 30 μl/min. GAD was serially diluted into the r
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