Proteomic Analysis of Cellular Response to Microcystin in Human Amnion FL Cells Wen-yu Fu,† Li-hong Xu,*,† and Ying-nian Yu*,‡ Department of Biochemistry and Molecular Biology, Zhejiang University, School of Medicine, Hangzhou, China, and Department of Pathophysiology, Zhejiang University, School of Medicine, Hangzhou, China Received September 27, 2005
Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Image analysis of silver-stained 2-dimensional gels revealed that 89 proteins showed significant differential expression in MC-RR treated cells compared with control, and 8 proteins were unique to MC-RR treated cells and 8 proteins were only detected in control cells. Sixty-six proteins were further identified with high confidence by peptide mass fingerprinting. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis, signal transduction, and cytoskeleton alteration which are consistent with the literature. The functional implications of alterations in the levels of these proteins were discussed. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, this work will provide new insight into the mechanism of MC-RR toxicity. Keywords: microcystin • proteome • two-dimensional gel electrophoresis • apoptosis • PP2A
1. Introduction The outbreak of the cyanobacterial (specifically Microcystin aeruginosa) blooms due to eutrophication has been a worldwide threat since the late 1970s, it exerts severe adverse health effects on human and livestock,1-4 by virtue of their ability to produce the heptapeptide toxin, microcystin.5 The general structure of microcystin (MC) is cyclo-(D-Ala-L-X-D-MeAsp-LZ-Adda-D-Glu-Mdha), where MeAsp stands for erythro-βmethylaspartic acid, Mdha for N-methyldehydroalanine, Adda for 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid, and X and Z are two variable amino acids. The toxins are named according to the two variable acids and other minor structural modifications. Till now, over 60 structural variants have been recognized. The XZ variable amino acids for MC-LR, MC-RR and MC-YR are leucine (L), arginine (R), and tyrosine (Y), which are the three most predominant microcystins. Using tritium-labled MC, it has been demonstrated that the liver is the prime target organ affected, but to some extent, MC * To whom correspondence should be addressed. Prof. Ying-nian Yu, Tel: 86-571-87217149. E-mail:
[email protected]. Prof. Li-hong Xu, Tel: 86-571-87217056. Fax: 86-571-86967920. E-mail:
[email protected]. † Department of Biochemistry and Molecular Biology. ‡ Department of Pathophysiology. 10.1021/pr050325k CCC: $30.25
2005 American Chemical Society
also concentrated in the intestine and the kidneys.6 The toxic effects of MC on different types of mammalian cells including hepatocytes, embryo kidney cells, fibroblast, endothelial, and epithelial cells or lymphocytes, were observed.7-9 Recently, it has been reported that MC could accumulate on the eggs of the shrimps except in the hepatopancreas and gonads, indicating that it can be transferred to offspring from their adults.10 MCs are poisonous to higher levels of the food web due to the bioaccumulation of MCs in aquatic animals of natural waters. Therefore, the toxicology of MC is cause for concern. Microcystins are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promotion activity have not been fully elucidated. One of the most extensively studied mechanisms is that microcystins are potent inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), leading to increased protein phosphorylation.1,11 The hyperphosphorylation of proteins have been attributed to the destruction of cytoskeleton which related to its cytotoxicity,12,13 moreover, which also attributed to its tumor promotion activity.14,15 Some previous result showed that MC can initiate apoptosis in a variety of cell types characterized by cell membrane blebbing, cytoplasmic shrinkage, nuclear chromatin condensaJournal of Proteome Research 2005, 4, 2207-2215
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research articles tion,DNAfragmentation,andformationofapoptoticbodies.16-18Although it has been implicated that protein phosphorylation and caspases8 may play an important role in MC-induced apoptosis, and the induction of oxidative stress and mitochondrial alteration17,19,20 were involved in this process, the exact mechanisms of MC-induced apoptosis are still unknown. The increased distribution of MCs has become a global issue, and these toxins are shown to have been associated with acute, usually lethal toxicity in various species of domestic animals and wildlife in addition to cases of illness in humans.21 However, the MCs response machinery have not been studied completely and elucidated till now. A person’s risk of developing an illness as a result of an environmental exposure might be dependent on the efficiency of their own unique set of environmental response genes.22 Therefore, toxicogenomics, which combines global genomic-scale mRNA and protein expression patterns, and bioinformatics, will aid to understand the role of gene-environment interactions in disease and the biological responses machinery followed exposure to toxins. Although screening of gene expression at the transcriptional level, using methods such as mRNA differential display and cDNA microarray, can provide valuable information about which specific genes might participate in toxins induced biochemistry alteration, they cannot provide direct information of how proteins are regulated at the translational or posttranslational levels. Since the biological processes are directly executed by proteins, which are dynamically modified and processed at multiple levels during or after their maturation, the state of the cell is essentially reflected in its proteome rather than genome, which is much more stable.23 Currently, proteomics analysis combining 2-DE and mass spectrometry is becoming a popular method of choice to detect differentially expressed proteins between profiles after exposure to toxicants by allowing the simultaneous study of thousands of proteins at a time. A number of studies which investigated individual proteins attempted to explain effects observed at the cell level in response to MC.8,24 In contrast, proteomic analysis allows simultaneous monitoring of the expression of hundreds and even thousands of proteins in a sample following exposure to toxicant. In our present studies, 2-DE combining with matrixassisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) was undertaken to identify differentially expressed proteins following exposure to MC-RR. Following, western blot analysis was employed to validate the deregulated expression of proteins. The implications of the identified proteins in terms of their involvement in the process of MC-RR induced biochemical alteration were discussed, which will aid our understanding of the molecular mechanisms of MC-RR toxicity as well as provide the possibility for the finding of novel biomarkers of exposure to toxins.
2. Materials and Methods 2.1. Reagents and Materials. MC-RR was generously provided by Prof. Ken-ichi Harada (Faculty of Pharmacy, Meijo University, Japan). Dimethyl sulfoxide (DMSO), trypsin (sequencing grade), R-cyano-4-hydroxy-trans-cinnamic (CHCA) were from Sigma (St. Louis. MO). DTT, iodoacetamide, urea, CHAPS, acrylamide, Bis-acrylamide, Tris-base, SDS, ammonium persulfate and TEMED were from Promega. Pharmalyte (47), IPG Strips (4-7 L) were from Amersham Pharmacia. Rabbit polyclonal anti-ERK antibody, horseradish peroxidase-conjugated anti-rabbit were from Cell Signaling Technology (Beverly, 2208
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MA). Mouse monoclonal anti-human antibody was from Calbiochem (San Diego, CA). All of other chemicals were of the highest grade available from commercial sources. 2.2. Cell Culture and Sample Preparation. Human amnion epithelial FL cells were maintained in Eagle’s Minimal Essential Medium supplemented with 10% newborn bovine serum. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in air. MC-RR was dissolved in DMSO as 0.25, 1, 2 mM stock solution respectively and stored at -20 °C. For MC-RR treatment, a 10 µL stock solution was added into 10 mL serum-free medium to get a final concentration of 0.25, 1, 2µM respectively when cells reached 70% confluence. Equal volume of DMSO was used as vehicle control. After 2.5 h incubation, the treatment medium was removed completely. And the cells were incubated in fresh medium for another 12 h before harvest. After exposure, the cells were washed twice with ice cold phosphate-buffered saline (PBS) and harvested with 0.02% EDTA and 0.025% trypsin, rinsed three times in PBS, then pelleted in microtubes by centrifugation. The cell pellets were dissolved in lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris, 65 mM DTT, 2% Pharmalyte) containing protease inhibitors (10 µg/mL Aprotinin, 10 µg/mL Leupeptin and 1 mmol/L PMSF) and put on ice for 1 h. The cells were then sonicated and centrifuged at 40 000 × g for 40 min at 4 °C to remove debris. Protein concentrations were determined using the Bradford assay.25 All samples were stored at -70 °C prior to electrophoresis. 2.3. 2-DE and Silver Staining. Isoelectric focusing (IEF) was performed following the manufacturer’s instructions (Amersham Biosciences) using IPGphor Isoelectirc focusing system (Amersham Biosciences). Briefly, 300 µg of total proteins was mixed with a rehydration solution containing 8 M urea, 2% CHAPS, 0.5% Pharmalyte (pH 4-7), 18 nM DTT, and a trace of bromophenol blue, to a total volume of of 450 µL and applied to 24 cm IPG strip (pH 4-7, linear). Rehydration and IEF were carried out on the IPGphor platform automatically at 20 °C, and the total Vh of IEF is 68 000-72 000 Vh. After IEF separation, the gels were equilibrated for 15 min in an equilibration buffer (50 mM Tris-Hcl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue) containing 1% DTT and subsequently in the same buffer except DTT was replaced with 5% iodoacetamide for another 15 min. The equilibrated gel strip was subjected to 12.5% SDS-PAGE and sealed with 0.5% agarose. SDS-PAGE was performed at constant power of 2.5 W/gel for 30 min, and switched to 15 W/gel until the bromophenol blue frontier reached the bottom of the gel. The separated proteins were visualized by MS-compatible silver staining as described previously.26 Three separate gels were conducted for each concentration in order to minimize the contribution of experimental variations. 2.4. Image Acquisition and Statistical Analysis. The silverstained 2-dimensional gels were digitized at an optical resolution of 400 dpi with PowerLook 1000 scanner (Umax, Willich, Germany). The digitized images were analyzed with Phoretix 2D 6.01 Analysis software (Nonlinear Dynamic, Durham, NC). Following spot detection, a matchset including all three batches of gels was built. One batch of gels from one experiment included the gels from control, 0.25, 1, 2 µM MC-RR, respectively. A reference gel was selected from the control groups and unmatched protein spots were automatically added to the reference gel. The amount of a protein spot was expressed as the volume of the spot, which was defined as the sum of the intensities of all the pixels that make up the spot. To correct
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Cellular Response to Microcystin
for variations due to silver staining and to reflect the quantity of protein spots, the individual spot volume was normalized as a percentage of the total volume of all the spots detected on the gel. Statistical analysis was performed using one-way analysis of variance (ANOVA). Differences were considered significant at P < 0.05. 2.5. In-Gel Digestion. All the proteins differentially expressed with statistical difference were excised from the gel and in-gel digestion with trypsin was performed according to published procedures with slight modification (Fernandez et al., 1998; Gharahdaghi et al., 1999). The gel pieces were washed twice with water and destained in a 1:1 solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate until the brownish color disappeared, then they were rinsed three more times in distilled water to stop the reaction. Next, the gel pieces were equilibrated in 100 mM ammonium bicarbonate for 10 min, dehydrated with acetonitrile and dried in a Savant Speed-Vac system (Thermo Savant, Holbrook, NY). Subsequently, the gels were rehydrated in 10-20 µL of proteomics grade trypsin solution (20 µg/mL in 40 mM ammonium bicarbonate in 9% acetonitrile) and incubated overnight at 37 °C. The peptides were extracted twice with 50% acetonitrile, 5% trifluoroacetic acid at RT and dried in a vacuum centrifuge. The peptide extracts were reconstituted in 10 µL of 50% acetonitrile solution with 0.1% trifluoroacetic acid. This protocol often gave a good signal-to-noise ratio in MALDI-TOF spectra. If not, peptides were treated with ZipTips (Millipore) prior to being applied onto the sample plate. 2.6. Matrix-Assisted Laser Desorption/Ionization-Time-ofFlight Mass Spectrometry (MALDI-TOF-MS) Analysis. Peptide mixtures (1µL) were mixed an equal volume of 10 mg/mL CHCA saturated with 50% acetonitrile in 0.05% trifluoroacetic acid and applied to a target disk and allowed to air-dry. All of the samples were analyzed using Voyager-DE STR MALDI-TOF mass spectrometer (ABI Applied System, Framingham, MA). Mass/charge ratios were measured in the reflector/delayed extraction mode with an accelerating voltage of 20 kV, grid voltage of 64.5% and delayed time of 100 ns. Monoisotopic peptide masses were assigned and TOF spectra were collected over the mass range of 800-4000 Da. External calibration was performed using des-Argl-bradykinin and ACTH in the same series as the samples to be measured. Moreover, the autodigested peaks of trypsin were served as internal standards for mass calibration. 2.7. Protein Identification and Database Searching. Protein identification using peptide mass fingerprinting (PMF) was performed by the MASCOT search engine (http://www.matrixscience.com) against the NCBI protein database. The search parameters were defined as follows: homo sapiens, trypsin digest (allowed up to 1 missed cleavage), cysteines modified by carbamidomethylation, methionine modified by oxidization, maximal mass tolerance of 100 ppm. Proteins matching more than four peptides and with a MASCOT score higher than 63 were considered significant (P < 0.05). 2.8. Western Blot Analysis. Aliquots from supernatant containing 50 µg of proteins were mixed with equal volume of 2×c1AÄ sample buffer. The samples were boiled for 5 min and subjected to 12.5% SDS-PAGE. After electrophoresis, the resolved proteins were transferred to nitrocellulose membrane. Membranes were blocked at room temperature for 1 h in TBST (50 mmol/L Tris-Cl, pH 7.6, 150 mmol/L NaCl, 0.1% Tween 20) containing 5% nonfat milk to prevent nonspecific binding of reagents, and then incubated with anti-ERK(1:1000 dilution)
or anti-p53 (1:10 dilution) at 4 °C overnight. The membranes were washed in TBST for 15 min and incubated with HRP conjugated secondary antibody (1:2000 dilution) for 1h at room temperature. The membranes were washed three times in TBST. Target protein was detected by ECL. Equal protein loading was confirmed by exposure of the membranes to the anti-GAPDH antibody.
3. Results 3.1. Differential Protein Expression in FL Cells after MCRR Treatment. Image analysis of the entire match set consisted of 12 gels from three independent experiments was performed by comparison of control gels and gels loaded with samples from cells exposed to three different concentrations of MCRR. Figure 1 showed the 2-D pattern obtained after separation of 300 µg of protein sample from control FL cells in comparison to 2 µM MC-RR treated cells. The dose of 0.25 and 1, 2 µM MC-RR treated samples yielded very similar results. The 2 µM MC-RR treated sample relative to the control was shown as an example because the most differentially expressed protein spots were found in this treatment. Figure 2. showed some zoom image of differentially expressed protein. On the basis of image analysis, 1221 ( 9 protein spots were detected in control FL cells, whereas 1175 ( 65, 1235 ( 65 and 1205 ( 60 protein spots were detected in 0.25, 1, 2 µM of MC-RR treated FL cells, respectively. One-hundred ten protein spots were found to have their relative volumes changed significantly in three doses of MC-RR treated FL cells compared with the control totally (Table 1). Importantly, there are some differentially expressed protein spots which were found in all three doses of MC-RR treated cells compared with control, whereas the others were detected only in one or two doses of MC-RR treatment. Therefore, a summation of all differentially expressed protein spots from three doses was 89. The numbers of up-regulation or downregulation spot and the spot unique to control (which mean the spot only detected exclusively in controls but not MC-RR treated cells), the spot unique to the MC-RR (which mean the spot induced by the MC-RR treated cells and not found in the controls) were shown in the Table 1. 3.2. Proteins with Altered Expression Identified by PMF. The differentially expressed protein spots were excised from the sliver-stained gels, followed by in-gel digestion with trypsin and identified by MALDI-TOF analysis and database search. Table 1 showed a summary of the proteins that were identified by MALDI-TOF-MS and database thus far. Sixty-six proteins were successfully identified with high confidence based on high scores and sequence coverage (Table 2). Among them, three of spots were the protein mixture including two proteins (spot No. 217, 500, and 1102). The remaining 21 protein spots were not identified by PMF or produced no spectrum. The proteins identified can be assigned into several functional groups, such as signal transducer, enzyme and enzyme regulator, structure molecule and transporter et al., rather they take part in a wide variety of cellular processes. 3.3. Verification of Differential Expressed Proteins by Western Blot Analysis. From the candidates, ERK and p53 were selected for Western blot analysis as shown in Figure 3 and Figure 4. The expression change of the selected protein was consistent with the 2-DE and silver-staining results. Such result demonstrated that the proteomics analysis of cellular response to MC-RR was convincing. Journal of Proteome Research • Vol. 4, No. 6, 2005 2209
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Figure 1. Differentially expressed 2-D pattern in the 2 µM MC-RR treated FL cells compared with control. The global cellular proteins extracted from the FL cells were separated on a pH 4-7 linear IPG strip, followed by 12.5% SDS-PAGE and silver staining. The circles indicate proteins only detected in the control cells. The rectangles indicate proteins which is unique to the MC-RR treatment. Upwardor downward-arrows indicate proteins which up-regulated or down-regulated by MC-RR treatment, respectively. The number around the spot was the index no. in the reference gel. Each experiment was conducted independently at least three times, and the image from one representative experiment was shown.
Figure 2. Zoom image of differentially expressed protein of p53, 14-3-3. ERK, and tubulin. 2210
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Cellular Response to Microcystin Table 1. Differentially Expressed Protein Spot Nos. in MC-RR Treated FL Cells compared with the control cells
0.25 µM MC-RR 1 µM MC-RR 2 µM MC-RR
unique to control
unique to MC-RR
upregulation
downregulation
1 2 5
0 2 6
6 11 22
15 17 23
4. Discussion In contrast to conventional biochemical approaches that monitor one or a few specific proteins at a time, proteomics is the large-scale study of gene expression at the protein level, which will ultimately provide direct measurement of protein expression levels and insights into the activity state of all relevant proteins.27 Toxicoproteomics, the application of proteomics in toxicology offers the prospect of identifying new toxic mechanisms and provides the possibility of screening or predictive toxicology.28 2-DE is a suitable technique of choice to separate and quantify thousands of proteins simultaneously and to determine differentially expressed proteins following exposure of cells to toxicants in toxicoproteomics. Sixty-six proteins have been identified with high confidence using 2-DE and PMF in this study. Western blot analysis of ERK and p53 validated that the differential expression of proteins obtained from proteomics analysis were convincing. The differentially expressed proteins that have been identified can be assigned into several groups such as apoptosis, signal transducer and cytoskeleton associated proteins according to their characteristics and functions. The groups of intriguing differentially expressed proteins may relate to the known mechanism or will provide new insight into the unknown mechanism of MC-RR. In our previous studies, we have found that low concentrations of MC-LR (0.1∼1µM) that have no obvious cytotoxicity can induce apoptotic alteration including phosphatidylserine externalization, caspase-3 activation when treated for 24 h. Further studies showed that MC-LR (0.1∼1µM) can alter the expression of apoptotic proteins Bcl-2, Bax, and p53 when treated for 1, 2, and 4 h in vitro and in vivo.29 Considering, the IC50 of MC-LR on PP2A was 0.50 µg‚L-1, whereas that of MCRR was 0.95 µg‚L-1.30 Therefore, 0.25, 1, 2 µM of MC-RR was administrated on the FL cells to investigate the biological responses machinery followed exposure to toxicants. In study of responses to toxins, dose-related responses should be well considered. However, different doses belong to nontoxic, subtoxic or toxic ranges may actually induce dosedependent or completely different responses. Interestingly, among the proteins that have been identified unambiguously in our present study, most of the alterations of the proteins were dose independent, and there was a little dose-dependent manner could be observed in a few proteins. The results were similar with previous reports, in which Moller et al. found that treatment with daunorubicin led to a significant up-regulation of most of the proteins, independent of the concentration used.23 Therefore, the biological responses of high dose exposure cannot be considered as only an amplification of low dose response although changes can occur after low and high dose exposure.31 This might partly due to the toxicity mechanisms of MC-RR is very complex and the effect on the function and biochemistry in cells is abroad. The cellular responses to MCRR were diverse in different concentration of MC-RR administrated to the cells. Moreover, it was might because that MCRR may activate the independent pathway to regulate the cell response machinery.
Among a few proteins which showed dose-dependent manner, p53 (spot no. 572) was up-regulated, whereas tubulin (spot no. 806) was down-regulated by three doses of MC-RR. Most of the alterations of the proteins were dose-independent such as ERK (spot no. 352) and peroxiredoxin 2 (spot no. 968), 143-3 (spot no. 1072) were up-regulated by 2µM MC-RR. The functional implication of these interest proteins which show differential expression were discussed as follows. Regulation of the Ras-Raf-MAPK pathway was involved in the cellular processes responded to stimuli. Extracellular signalregulated kinase (ERK) is known to act as anti-apoptotic molecule by transducing survival signals.32 In contrast, there was study showed that ERK could act as a positive regulator of apoptosis. Treatment of cell with the PP2A inhibitor, okadaic acid, causes activation of both MAP/ERK kinase (MEK) and ERK.33,34 In our present study, the expression level of ERK1 and MKK3 was increased in MC-RR treated FL cells. Moreover, the expression of adaptor protein Grb2 is up-regulated by MC-RR, which can recruit two Ras exchange factors, SOS and RasGRP1, sequentially activating Ras. So it is possible that MC-RR can regulate the expression level of the key proteins involved in the Ras-Raf-MAPK pathway, consequently modulate the signal transduction that regulates the cellular processes. p53 is a multi-faceted nuclear phosphoprotein induced in response to cellular stress, functioning as a transcriptional transactivator in DNA repair, apoptosis, and tumor suppression pathways. Apoptotic cell death is suppressed if p53 gene is mutated and its function is damaged. The up-regulation of p53 as observed in this study may attribute to the induced expression of its coding genes and/or prolonged half-life of p53 protein.35,36 In the present study, both the 2D analysis and western blot demonstrate that MC-RR can up-regulate the p53 expression in all the three doses used in the study compared with control. Furthermore, the up-regulation of p53 in MC-RR treated FL cells observed in this study is in agreement with our previous study of increased expression of p53 both in vivo and in vitro29 after MC administration and suggest that upregulation of p53 play a key role in the cellular responses to MC-RR. And there was a dose-dependent up-regulation of p53 in the present and the previous studies. So p53 may be used as the candidate biomarker for monitoring the exposure to MCRR. Moreover, it was noteworthy that heat shock protein 70 and retinoblastoma binding protein encoded by p53-targeted gene hsp70 and retinoblastoma binding protein showed differential expression. The binding of these proteins may affect the biological functions of p53 and aid to understand pathways by which p53 affects cellular outcomes.37 The significance of these two p53-targeted proteins should be studied further. 14-3-3 proteins are a family of homologous eukaryotic molecules with seven distinct isoforms in mammalian cells. Every isoform of 14-3-3 proteins can interact with Bad, a proapoptotic member of the Bcl-2 family which discovered as a heterodimeric partner for Bcl-2 and Bcl-XL, and inhibit Bad’s proapoptotic activity.38 Whether or not such binding of Bad to 14-3-3 is determined by the phosphorylation status of Bad. In its phosphorylated form, Bad associates in the cytoplasm with 14-3-3 protein. Free Bcl-XL is then capable of preventing apoptosis.39 Therefore, as a potent PP1 and PP2A inhibitor, MCRR can hyperphosphorylate Bad which mediate binding of Bad Journal of Proteome Research • Vol. 4, No. 6, 2005 2211
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Table 2. Differentially Expressed Proteins Identified Using MALDI-TOF-MS Analysis with High Confidence index no.
accession no.
mass
cov. (%)
pepetide match
MOWSE score
572
NP•000537
43653
38
14(43)
129
1072
NP•647539
27951
40
13(30)
120
301
NP•001485
41014
34
8(46)
87
352
NP•002737
43135
30
13(34)
110
476 528
NP•005198 XP•113967
33756 36292
46 31
15(40) 10(50)
98 73
703
NP•002063
49671
21
7(18)
86
889
NP•002747
36148
59
21(48)
127
914 947 971 1053
NP•002077 NP001337 NP•001168 NP•783865
25132 86175 20404 24154
49 32 32 25
12(37) 12(40) 8(15) 6(28)
97 88 86 73
1146
NP•057607
21191
40
8(40)
72
193
NP•002264
53641
48
26(45)
282
217
NP•149052
86452
46
30(58
141
486 500 500
NP•733821 NP•000217 NP•006112
57401 62072 65978
26 28 17
11(36) 12(27) 8(27)
110 112 65
525 692 740
NP•000993 NP•001382 NP•872578
34252 65114 45794
49 24 45
10(38) 10(29) 12(37)
102 74 91
806 810 837
NP•821133 NP•058518 NP•001531
41441 25413 22313
35 47 43
11(40) 8(46) 9(22)
100 82 114
998
NP•005548
51206
45
17(54)
96
1056 1173
NP•937818 NP•004598
19641 12847
44 25
7(24) 5(13)
76 97
1
NP•001866
166012
33
47(147)
102
217
NP•872423
64190
51
25(58)
141
287
NP•006249
76316
29
16(53)
122
483 522 814 879
NP•001340 NP•002645 NP•060599 NP•001010969
57156 57942 29969 51855
43 32 27 18
17(43) 16(42) 7(43) 9(45)
87 91 97 94
968 993 1065 1102 1129
NP•859427 BAC04923 NP•003611 NP•001012750 NP•066953
21878 24049 66667 21000 17870
66 48 45 72 68
12(47) 8(18) 25(67) 22(59) 16(37)
104 90 113 131 92
1153
NP•003339
17127
49
9(29)
105
1196
NP•203747
14786
49
6(11)
85
1130
NP•057392
22208
36
9(25)
87
286
XP•533670
47337
43
14(38)
94
700 836
XP•376841 NP•068810
94251 60219
60 32
50(87) 13(44)
134 93
842
NP•775951
59714
24
10(39)
96
874
NP•115540
64216
25
10(31)
87
1102
NP•775753
56873
55
29(59)
131
2212
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expression change protein description
0.25 µM
1 µM
2 µM
Apoptosis Cellular tumor antigen p53 1.68 1.91 2.67 (Tumor suppressor p53) 14-3-3 protein beta/alpha 2.71 Signal Transducer Rab GDP dissociation 1.67 inhibitor beta Mitogen-activated protein 2.24 kinase 3 (p44-ERK1) CRKL 1.68 PREDICTED: similar to 0.64 0.59 0.66 Rab12 protein Guanine nucleotide-binding 1.42 protein G(q), alpha subunit mitogen-activated protein 1.93 2.16 kinase kinase (EC 2.7.1.-) 3 [similarity] GRB2 [Homo sapiens] 2 Diacylglycerol kinase, gamma induced ADP-ribosylation factor-like 1 1.71 RAB37, member RAS 1.38 oncogene family lectin-like receptor F1, 0.53 splice variant 1 KLRF1-s1 Cytoskeleton Keratin, type II cytoskeletal 1.87 8 (Cytokeratin 8) microtubule-associated 1.53 protein 6 isoform 1 lamin A protein 0.59 keratin 9 0.73 0.81 Keratin, type II cytoskeletal 1 0.73 0.81 (Cytokeratin 1) (67 kDa cytokeratin) PREDICTED: ribosomal protein P0 0.47 dystrobrevin isoform DTN-2 induced 28S ribosomal protein S9, 1.62 mitochondrial precursor (S9mt) (MRP-S9) Tubulin beta-2 chain 0.54 0.49 0.48 microtubule-associated protein tau repressed heat shock protein 27 0.54 [Homo sapiens] keratin 16, type I, cytoskeletal 0.58 - human NM23-H1 [Homo sapiens] 1.84 tubulin-specific chaperone a 1.37 Metabolism carbamoyl-phosphate 0.65 0.61 synthetase 1 variant xenobiotic/medium-chain fatty 1.53 acid:CoA ligase cGMP-dependent protein kinase 1.92 type I alpha aspartyl-tRNA synthetase 1.48 Pyruvate kinase 3, isoform 1 repressed pyridoxine-5′-phosphate oxidase 1.64 cytochrome P450, family 4, induced subfamily A, polypeptide 22 [Homo sapiens] peroxiredoxin 2 isoform b 1.86 unnamed protein product induced Protein phosphatase 1D 0.53 dCMP deaminase isoform a 0.49 Chain F, Cypa Complexed 2.02 With Hvgpia ubiquitin-conjugating enzyme 1.25 E2N (UBC13 homolog, yeast) dual specificity phosphatase 0.43 KIAA1725 protein UMP-CMPK [Homo sapiens] induced Transcription PREDICTED: similar to 26S 3.88 protease regulatory subunit 6B (MIP224) (TAT-binding protein-7) (TBP-7) unnamed protein product 0.74 Transcription factor p65 (Nuclear 0.59 1.57 factor NF-kappa-B p65 subunit) MGC33584 protein 0.58 0.67 repressed [Homo sapiens] zinc finger protein 394 1.7 [Homo sapiens] unnamed protein product 0.49
up-/down-a
v v v v v V v v v U v v V v v V V V V U v V U V V v v V V v v U v U v U V V v v V U v
V Vv V v V
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Cellular Response to Microcystin Table 2. (Continued) index no.
accession no.
mass
cov. (%)
pepetide match
MOWSE score
481
NP•003748
39479
66
19(37)
137
137
NP•006576
59583
23
13(46)
88
453
NP•003305
33505
46
11(33)
100
559
NP•002145
78945
51
39(49)
125
265 479 847 967
NP•006391 NP•958930 NP•659464 NP•659464
44204 37581 17786 17786
34 49 63 62
13(49) 16(40) 7(38) 7(32)
104 79 78 77
501
NP•066358
75290
23
12(26)
93
1011
NP•057671
19708
41
8(21)
106
49 79 234
NP•006830 NP•444295 XP•373513
83497 59183 62398
35 17 24
24(63) 9(13) 9(43)
118 95 98
243 254
NP•064530 NP•002884
48585 52282
25 35
12(43) 16(40)
126 85
310 879 1014
NP•057542 XP•028810 XP•379665
42103 53001 11534
46 25 51
17(41) 13(36) 12(18)
108 106 74
1084 1161 870
NP•055070 CAH71105 NP•056226
20639 25070 33284
41 41 34
8(11) 6(27) 11(38)
80 87 102
expression change protein description
0.25 µM
Translation eukaryotic translation initiation factor 3, subunit 2 beta, 36kDa Chaperone chaperonin containing TCP1, subunit 8 (theta) tetratricopeptide repeat domain 1 heat shock protein 70 Motor Activity DCTN2 protein unnamed protein product OTTHUMP00000016350 OTTHUMP00000016350 Nucleic Acid Binding Zinc finger protein 14 (Zinc finger protein KOX6) (Gonadotropin inducible transcription repressor-4) HP1Hs-gamma Unknown proliferation-inducing gene 4 ubiquilin 1 PREDICTED: hypothetical protein XP•373513 KIAA1848 protein retinoblastoma binding protein 7 Brain protein 16 RP5-1054A22.3 [Homo sapiens] PREDICTED: hypothetical protein XP•379665 TMEM4 [Homo sapiens] novel protein [Homo sapiens] SUMF2 protein [Homo sapiens]
1 µM
2 µM
up-/down-a
0.62
V
1.7
v
0.56
V
0.61
0.58
0.56
V
2.07 1.41
2.45
2.45
v v V v
0.66 1.57 1.36
0.47
v
0.47
2.07
v V U
0.55 induced
repressed 0.65 1.5
V
0.67 1.94
V v
repressed
U V v
0.62
V v U
1.77 induced
a Upward arrow indicates up-regulation, downward arrow indicates downregulation, upward and downward arrow indicate the change of protein in the different doses of MC-RR is different, U indicates the protein is unique to control or unique to MC-RR treatment.
to overexpressed 14-3-3 protein as observed in this study and lead to cytoplasm retention of Bad and inhibit its proapoptotic activity. Furthermore, it was observed that MC-RR can induce the differential expression of the cytoskeleton and its associated proteins in this study. The primary mechanism of action of MCs is a very potent inhibition of the ubiquitous PP1 and PP2A, leading to rapid reorganization of all three major cytoskeletal components, microfilaments (MFs), microtubules (MTs), and intermediate filaments (IFs).40 Some cells lose MTs after MC treatment except the reorganization and aggregation of MTs. Ding et al. showed that binding of MC directly to the cysteine residues found on the tubulin subunits could have resulted in more free tubulin in the cytoplasm, thereby reducing the stability of the tubulin mRNA and a decrease in cellular tubulin.41 The decreased expression of tubulin is accordance with our present study of down-regulation of tubulin in each MC-RR treated FL cells. Nm23 has high homology to nucleotide diphosphate kinase, which may affect the state of tubulin polymerization.42 Increased expression of nm23-H1 (spot No. 1056) in 1µM of MC-RR treated FL cells is consistent with the previous report of nm23 as a putative granulocyte differentiation inhibitor and apoptosis inducer43 indicate its role in apoptosis because MC could induce apoptosis in a variety of cell types. One of the most extensively studied mechanisms is that MCs are potent inhibitors of PP1 and PP2A, thereby influencing regulation of cellular protein phosphorylation. The specific dephosphorylation of Ser/Thr residues plays an essential role in control of the cell, with more than 97% of protein phospho-
rylation occurring at Ser/Thr residues.44 Therefore, as a potent inhibitor of PP1 and PP2A, MCs can regulate the phosphorylation level of a variety of proteins, consequently affecting some cellular processes. Surprisingly, all of the proteins with differential expression mentioned above including p53,44-46 143-3,47 mitogen-activated protein kinase 3,33,34 tubulin,41 mitogenactivated protein kinase kinase 3 (spot no. 889),48 microtubuleassociated protein tau (spot no. 810)49,50 can be regulated by PP2A. So there was the possibility that MC may not only regulate the phosphorylation status but also the expression levels of the key proteins simultaneously, consequently affecting the cellular processes. The further studies are needed to investigate the effects of MC-RR on the phosphorylation status of these proteins. In this study, some molecules such as peroxiredoxin 2 other than PP1 and PP2A was observed to be modulate by MC-RR. Peroxiredoxins belong to an ubiquitous family of antioxidant proteins, act as antioxidants and catalyze elimination of hydrogen peroxides with the help of reducing systems, such as thioredoxin/thioredoxin reductase.51 Peroxiredoxin responded differently to different concentrations of MC-RR. At high concentration (2 µM) of MC-RR, a 1.86-fold increase in peroxiredoxin 2 was observed whereas there were no significant differential expressions in other two concentrations. In the latest publication, it was reported that peroxiredoxin 2 was upregulated in response to MC-LR treatment52 and could be potential biomarker for hepatotoxicological mechanisms. Because peroxiredoxins have been shown to inhibit apoptosis induced by p5353 and by hydrogen peroxide in a level upstream of Bcl-2,54 it was conceivable that the up-regulation of peroxJournal of Proteome Research • Vol. 4, No. 6, 2005 2213
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Fu et al.
Figure 3. Effect of MC-RR upon the ERK expression in FL cells. Western blot analysis of ERK was performed as described in section 2.8. A representative autoradiograph is shown (A). Intensities of protein bands were quantified by densitometry. Equal protein loading was confirmed by exposure of the membranes to the anti-GAPDH antibody. Data were normalized using the GAPDH signal (B). Spot intensity attained from the silver staining 2-D gels (C). Data were indicated as means ( SD from three independent experiments. ** value significantly differ from the control (P < 0.01).
Figure 4. Effect of MC-RR upon the p53 expression in FL cells. Western blot analysis of p53 was performed as described in section 2.8. A representative autoradiograph is shown (A). Intensities of protein bands were quantified by densitometry. Equal protein loading was confirmed by exposure of the membranes to the anti-GAPDH antibody. Data were normalized using the GAPDH signal (B). Spot intensity attained from the silver staining 2-D gels (C). Data were indicated as means ( SD from three independent experiments. ** value significantly differ from the control (P < 0.01).
iredoxin 2 was the cell response to toxin exposure, protecting the cell from destruction by apoptosis. On the other hand, the cell was burdened with reactive oxygen species in high concentration of MC-RR and the cellular response to MC-RR may involve mitochondrial damage which was agreement with that the induction of oxidative stress and mitochondrial alteration17,19,20 were implicated in the cellular response machinery. Meanwhile, it also suggested that the cellular response to MCRR was diverse in different concentration of MC-RR.
or protein synthesis such as pyruvate kinase 3 (spot no. 522), pyridoxine-5′-phosphate oxidase (spot no. 814), cytochrome P450 (subfamily A) (spot no. 880), aspartyl-tRNA synthetase (spot no. 483) and UMP-CMPK (spot no. 1130) are all regulated by MC-RR, offering the prospect of identifying new toxic mechanisms.
In addition to the proteins which are related to known mechanisms consistent with the literature, there are some important proteins which have not been reported previously to be involved in cellular processes responded to MC-RR. Among these, protein phosphatase 1D (spot no. 1065), dual specificity phosphatase KIAA 1725 protein (spot no. 1196) and cGMP-dependent protein kinase (spot no. 287) showed the differentially expression, suggesting that MC-RR can affect some other phosphatases and kinases except inhibition PP1 and PP2A. Moreover, some proteins involved in metabolism 2214
Journal of Proteome Research • Vol. 4, No. 6, 2005
5. Concluding Remarks In this study, a proteomic approach was carried out to evaluate the cellular response to MC-RR in FL cells. Eightynine proteins show the differentially expressed followed MCRR treatment and 66 proteins were identified with high confidence which take part in a variety of cellular processes. Western blot analysis of selected protein confirmed the proteomic analysis result. Some of the identified differentially expressed proteins have clearly relationship with the process of apoptosis and cytoskeleton alteration which are consistent with the literature. However, most of which have not been reported previously to be involved in cellular processes responded to MC-RR. Therefore, more work is needed to evaluate
research articles
Cellular Response to Microcystin
the physiological relevance of particular proteins to the FL cellular responses to MC-RR. This work provides the new insight into the mechanisms of MC-RR and the possibility of new biomarkers for evaluating the exposure to MC-RR.
Acknowledgment. This work was supported by grants from the National Key Basic Research and Development Program (no. 2002CB512901), the National Nature Science Foundation of China (20137010) and Specialized Research Fund for the Doctoral Program of Higher Education (20020335078). We thank Professor Jun Yang for kindly help through the experiment and Run-liu Yu for MALDI-TOF-MS analysis. References (1) Carmichael, W. W. Sci. Am. 1994, 270, 78-86. (2) Pilotto, L. S.; Douglas, R. M.; Burch, M. D.; Cameron, S. et al. Aust. N. Z. J. Public Health 1997, 21, 562-566. (3) Pouria, S.; de Andrade, A.; Barbosa, J.; Cavalcanti, R. L. et al. Brazil Lancet. 1998, 352, 21-26. (4) Jochimsen, E. M.; Carmichael, W. W.; An, J. S.; Cardo, D. M. et al. N. Engl. J. Med. 1998, 338, 873-878. (5) Dawson, R. M. Toxicon. 1998, 36, 953-962. (6) Robinson, N. A.; Miura, G. A.; Matson, C. F.; Dinterman, R. E. et al. Toxicon. 1989, 27, 1035-1042. (7) McDermott, C. M.; Nhom, C. W.; Howard, W.; Holton, B. Toxicon. 1998, 36, 1981-1996. (8) Fladmark, K. E.; Brustugun, O. T.; Hovland, R.; Boe, R. et al. Cell Death Differ. 1999, 6, 1099-1108. (9) Mankiewicz, J.; Tarczynska, M.; Fladmark, K. E.; Doskeland, S. O. et al. Environ. Toxicol. 2001, 16, 225-233. (10) Chen, J.; Xie, P. Toxicon. 2005, 45, 615-625. (11) Yoshizawa, S.; Matsushima, R.; Watanabe, M. F.; Harada, K. et al. J. Cancer Res. Clin. Oncol. 1990, 116, 609-614. (12) Toivola, D. M.; Goldman, R. D.; Garrod, D. R.; Eriksson, J. E. J. Cell Sci. 1997, 110, 23-33. (13) Wickstrom, M. L.; Khan, S. A.; Haschek, W. M.; Wyman, J. F. et al. Toxicol. Pathol. 1995, 23, 326-337. (14) Nishiwaki-Matsushima, R.; Ohta, T.; Nishiwaki, S.; Suganuma, M. et al. J. Cancer Res. Clin. Oncol. 1992, 118, 420-424. (15) Humpage, A. R.; Hardy, S. J.; Moore, E. J.; Froscio, S. M. et al. J. Toxicol. Environ. Health A 2000, 61, 155-165. (16) McDermott, C. M.; Nhom, C. W.; Howard, W.; Holton, B. Toxicon. 1998, 36, 1981-1996. (17) Ding, W. X.; Shen, H. M.; Ong, C. N. Hepatology 2000, 32, 547555. (18) Fu, W. Y.; Li, M. W.; Chen, J. P.; Xu, L. H. Acta Hydrobiol. Sin. 2004, 28, 101-102. (19) Ding, W. X.; Shen, H. M.; Shen, Y.; Zhu, H. G. et al. Environ. Health Perspect. 1998, 106, 409-413. (20) Ding, W. X.; Shen, H. M.; Zhu, H. G.; Ong, C. N. Environ. Res. 1998, 78, 12-18. (21) Yu, S. Z. J. Gastroenterol. Hepatol. 1995, 10, 674-682. (22) Olden, K.; Wilson, S. Nat. Rev. Genet. 2000, 1, 149-153. (23) Moller, A.; Soldan, M.; Volker, U.; Maser, E. Toxicology 2001, 160, 129-138. (24) Ding, W. X.; Shen, H. M.; Ong, C. N. Biochem. Biophys. Res. Commun. 2002, 29, 321-331.
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PR050325K
Journal of Proteome Research • Vol. 4, No. 6, 2005 2215
