Article pubs.acs.org/JAFC
Proteomics Investigation Reveals Cell Death-Associated Proteins of Basidiomycete Fungus Trametes versicolor Treated with Ferruginol Yu-Han Chen,† Ting-Feng Yeh,† Fang-Hua Chu,† Fu-Lan Hsu,‡ and Shang-Tzen Chang*,† †
School of Forestry and Resource Conservation, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan Taiwan Forestry Research Institute, Council of Agriculture, Executive Yuan, 53 Nan-Hai Road, Taipei 100, Taiwan
‡
S Supporting Information *
ABSTRACT: Ferruginol has antifungal activity against wood-rot fungi (basidiomycetes). However, specific research on the antifungal mechanisms of ferruginol is scarce. Two-dimensional gel electrophoresis and fluorescent image analysis were employed to evaluate the differential protein expression of wood-rot fungus Trametes versicolor treated with or without ferruginol. Results from protein identification of tryptic peptides via liquid chromatography−electrospray ionization tandem mass spectrometry (LC−ESI-MS/MS) analyses revealed 17 protein assignments with differential expression. Downregulation of cytoskeleton βtubulin 3 indicates that ferruginol has potential to be used as a microtubule-disrupting agent. Downregulation of major facilitator superfamily (MFS)−multiple drug resistance (MDR) transporter and peroxiredoxin TSA1 were observed, suggesting reduction in self-defensive capabilities of T. versicolor. In addition, the proteins involved in polypeptide sorting and DNA repair were also downregulated, while heat shock proteins and autophagy-related protein 7 were upregulated. These observations reveal that such cellular dysfunction and damage caused by ferruginol lead to growth inhibition and autophagic cell death of fungi. KEYWORDS: antifungal activity, autophagy, basidiomycete, ferruginol, proteomics, Trametes versicolor
■
INTRODUCTION Ferruginol, a phenolic diterpene, is present in resins/exudates and heartwood of many plants, particularly coniferous trees such as Calocedrus macrolepis var. formosana,1 Cryptomeria japonica,2 Taiwania cryptomerioides,3 and Thuja plicata.4 In addition, an increasing number of reports revealed that ferruginol has excellent antifungal activities against fungi belonging to the family of basidiomycetes.3,5,6 The general mechanisms of antifungal actions reviewed by Odds et al.7 indicated that antifungal agents interfered with the cellular integrity of fungi via several targets including cell wall, cell membrane, microtubule assembly, sterol synthesis, protein synthesis, and DNA/RNA synthesis. Moreover, Ramsdale8 reported that antifungals/fungicides can induce cell death via programmed cell death (PCD) pathways in pathogenic fungi. These findings revealed that antifungal agents can target specific cellular molecules and further mediate cell-death pathways to inhibit the growth of fungi. Chang et al.3 examined the antifungal activities of five compoundsferruginol, helioxanthin, savinin, taiwanin C, and hinokiolisolated from T. cryptomerioides heartwood. They reported that ferruginol has the best antifungal performance against the basidiomycete fungus Trametes versicolor. However, specific study on the antifungal actions of ferruginol against basidiomycete fungus is limited. Proteomics is a research tool for surveying comprehensive information on molecular networks within the living cells. Fungal proteomics has been intensively studied in the last 10 years, and many fungal genomes have been sequenced.9,10 This tool can be further employed to identify the proteins that participate in regulation networks upon treatment with antifungal reagents or stress conditions.11 Cagas et al.12 investigated the proteomic response of an antifungal agent © XXXX American Chemical Society
(caspofungin) against ascomycete fungus Aspergillus fumigatus. Shimizu et al.13 used a proteomic differential display technique to identify the responses of basidiomycete fungus Phanerochaete chrysosporium to the addition of vanillin. These findings revealed that many cellular enzymes and metabolic shifts were regulated by specific stimulation. The objective of this study is to assess the regulation of cellular molecules at the proteome level of basidiomycete fungus T. versicolor upon addition of ferruginol. A proteomic differential display technique using two-dimensional electrophoresis (2DE) was adopted to profile the mechanism.
■
MATERIALS AND METHODS
Compound and Fungal Strain. Ferruginol was isolated from ethanolic heartwood extracts of a 40-year-old Taiwania cryptomerioides Hayata. The wood samples were harvested from the Experimental Forest of National Taiwan University in Nan-Tou County. Air-dried heartwood samples were soaked in 95% ethanol for 7 days. Crude extract was filtered, concentrated, and then lyophilized (yield 6.3%, based on dry weight of heartwood). The ethanolic extracts were fractionated by liquid−liquid partition to yield n-hexane-soluble fraction (50.8%), ethyl acetate-soluble fraction (27.0%), n-butanolsoluble fraction (1.6%), and water-soluble fraction (9.5%). The nhexane-soluble fraction was divided into nine subfractions (H1−H9) by open-column chromatography (230−400-mesh silica gel; Merck, Darmstadt, Germany) and then eluted with a stepwise gradient of ethyl acetate/n-hexane from 0% to 100% (v/v). According to our previous study,14 ferruginol was purified from subfraction H2 via a HPLC system equipped with a semipreparative silica column (mobile Received: September 29, 2014 Revised: November 26, 2014 Accepted: December 1, 2014
A
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
phase ethyl acetate/n-hexane = 5/95; flow rate 4 mL/min; retention time = 9.8 min). Trametes versicolor (Linnaeus: Fries) Pilat (BCRC 35253), a woodrot test strain in JIS K 1517, ASTM D1413, and EN113, was obtained from the Bioresource Collection and Research Center (BCRC) of Food Industry Research and Development Institute in Taiwan. Antifungal Assay. Agar dilution test was employed to evaluate the antifungal activity of ferruginol. Ferruginol was dissolved in 150 μL of 99.5% methanol and added into sterilized potato dextrose agar (PDA, 15 mL) in a 9 cm Petri dish with final concentrations of 25, 50, 100, 200, and 400 μg/mL. Methanol served as a control treatment. A 3 mm plug of mycelium was transferred to tested dishes and then incubated at 25 ± 2 °C and 70% relative humidity (RH). When the mycelia of the control group reached the border of the plate, the growth diameter was recorded to calculate the antifungal index (AI) as a percentage:
and stained with fluorescent dye (SYPRO Ruby), and the gel images were analyzed with ImageMaster software. For all tested groups, 300 μg of protein was mixed with rehydration solution (urea buffer, 2% ampholyte, and trace bromophenol blue) and then rehydrated with immobilized pH gradient (IPG) strips (17 cm, nonlinear gradient, BioRad) for 12 h. The voltage of the isoelectric focusing (IEF) was programed from 100 to 8000 V to reach a total of 40000 V·h (50 μA for each strip). After IEF, the strips were immediately equilibrated with reduction solution [2% (w/v) DTT in equilibration buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), and a trace of bromophenol blue] for 15 min, followed by the alkylation solution [2.5% (w/v) iodoacetamide (IAA) in equilibration buffer] for 15 min. The second-dimension separation was performed by SDS−12% polyacrylamide gel electrophoresis (PAGE). After the 2DE separation, all gels were washed three times with deionized water to remove the residue of SDS. Gels were stained with SYPRO Ruby (Invitrogen) and digitalized with Typhoon 9400 scanner (Amersham Bioscience) at 200 μm pixel size. The images were analyzed with ImageMaster Platinum software v4.01 (Amersham Bioscience). Spot detection, matching, background subtraction (average on boundary), and image normalization were performed by this software. Only spots present in at least two biological replicates were taken as true signals. The volume of spots was normalized to the total volume of all spots in the analysis to compensate for slight differences in sample loading and gel staining. Protein spots with significantly changed levels (t-test, p < 0.05) were selected for protein identification. Identification of Proteins and Database Search. Selected spots from both control and treatment gels were excised manually and washed three times with double-distilled H2O. Gels were destained with 50% acetonitrile (ACN)/25 mM ammonium bicarbonate (37 °C), reduced with 10 mM DTT/25 mM ammonium bicarbonate (56 °C, 1 h in dark), and alkylated with 55 mM IAA/25 mM ammonium bicarbonate (room temperature, 45 min in dark). Gels were dehydrated with 100% ACN and rehydrated with trypsin (Promega, Madison, WI) (37 °C, 16 h). Oligopeptides were extracted twice in 5% formic acid (FA)/50% ACN with agitation (on ice, 20 min) and then dried via a centrifugal evaporator. Tryptic peptides were analyzed on a nano-HPLC system (LC Packing, Netherlands) with a 75-μm innerdiameter/15 cm C18 microcapillary column (Agilent Technologies). The HPLC system was connected with an ion-trap mass spectrometer (LCQ DECA XP plus, Thermo Finnigan, San Jose, CA) equipped with an electrospray ionization source (spraying voltage 1.8 kV). Elution conditions included a 40 min linear gradient from 100% to 40% buffer A (buffer A, 5% ACN/0.1% formic acid; buffer B, 80% ACN/0.1% formic acid; flow rate, 0.2 mL/min). Spectra were acquired by a full scan (m/z 450−2000). Mascot software (www.matrixscience.com) was used for protein identification and comparison with the National Center for Biotechnology Information (NCBI) nonredundant protein sequence database and Swiss-Prot database against fungi taxonomy. Two missed tryptic cleavages per peptide and variable modification of carboxymethylation on cysteine and lysine, oxidation on methionine, and deamidation on asparagine or glutamine residues were permitted during the identification process. The identified proteins whose MOWSE (molecular weight search) scores exceeded 51 (NCBI) or 35 (Swiss-Prot) were taken as significant hits (p < 0.05). Sequences of identified proteins are listed in Tables S1 and S2 in Supporting Information.
AI = (1 − Dt /Dc) × 100 Dt is the mycelium diameter of test groups, and Dc is the mycelium diameter of the control. Liquid Culture of Fungal Cells for Proteomic Analysis. For proteomic analysis, T. versicolor was incubated in a liquid medium [700 mL of potato dextrose broth (PDB) in a 1-L Erlenmeyer flask] at 25 ± 2 °C under air. Cultures were incubated on an orbital shaker at 100g in darkness. After a 7-day preincubation, ferruginol in ethanol (1500 μL) and Tween 20 (350 μL) was added into the liquid medium with a final concentration of 100 μg/mL. For the control, only ethanol (1500 μL) and Tween 20 (350 μL) were added in the culture. At the 11th incubation day, cultured mycelia were filtered and weighed (100 °C dry weight). Extraction and Quantification of Fungal Proteins. T. versicolor was treated with ferruginol for 4 days, and its mycelia were filtered, washed with chilled ultrapure water, and frozen in liquid nitrogen. The frozen mycelia (2 g, fresh weight) were ground into a fine powder in liquid nitrogen with a mortar and pestle and extracted with precooled (−20 °C) buffer containing 10% trichloroacetic acid and 20 mM dithiothreitol (DTT) in acetone. The mixture was precipitated at −20 °C for 50 min and centrifuged at 6000g (4 °C) for 30 min. Pellets were washed with precooled acetone containing 20 mM DTT, washed twice with 100% precooled acetone, and then dried in a freeze-dryer. The lyophilized pellets (500 mg) were solubilized by urea buffer containing 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and 1% (w/v) DTT, and the supernatant was collected by centrifugation at 12000g (25 °C) for 10 min. The protein concentration was quantified by the Bradford method15 with bovine serum albumin (BSA) and protein assay dye reagent (Bio-Rad Laboratories, Inc.). Two-Dimensional Electrophoresis. Figure 1 illustrates the experimental process of proteomic differential analysis. Proteins from T. versicolor were separated by two-dimensional electrophoresis (2DE)
■
RESULTS AND DISCUSSION Antifungal Ability of Ferruginol. An agar dilution test was performed to assess the antifungal activity of ferruginol against Trametes versicolor (white-rot fungus) at concentrations of 25, 50, 100, 200, and 400 μg/mL. Results (Figure 2A) show that the antifungal indexes were 26%, 34%, 41%, 42%, and 43%, respectively. The antifungal activity of ferruginol reached a maximum at the dosage of 100 μg/mL and then leveled off.
Figure 1. Workflow for studying differential protein expression of T. versicolor treated with or without ferruginol (100 μg/mL). B
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 3. Two-dimensional electrophoretic analysis of T. versicolor proteins (300 μg) extracted from control and ferruginol-treated (100 μg/mL) groups. Proteomics analysis was carried out with (A) widerange (pH 3−10) IPG strips and (B) narrow-range (pH 4−7) IPG strips. pI, isoelectric point.
Figure 2. (A) Antifungal activity of ferruginol against T. versicolor on agar plate tests. (B) Dry weight of mycelium of T. versicolor on liquid culture test treated with (■) or without (□) ferruginol (100 μg/mL). Results are mean ± SD (n = 4).
Downregulated Proteins upon Ferruginol Treatment. Exposure of T. versicolor cells to ferruginol showed decreased expression volumes in manganese ion homeostasis-related protein (spot 473) (Figure 5 and Table 1). Manganese homeostasis in the budding yeast Saccharomyces cerevisiae is under intensive investigation.16,17 Reports indicated that manganese is both an essential trace element to living cells and a cofactor for metalloproteins. These findings suggest that an array of metalloproteins, such as oxidoreductases, DNA/ RNA polymerases, peptidases, kinases, decarboxylases, and sugar transferases, of T. versicolor may function abnormally in transcription, glycolysis, and sugar transformation regulation upon ferruginol treatment. To survive in natural environments, microorganisms possess two major transporter proteins, ATP-binding cassette (ABC) and major facilitator superfamily (MFS), for active secretion of endogenous and exogenous toxicants. Nelissen et al.18 carried out a computer-aided genome analysis of 186 MFS transporters from the budding yeast S. cerevisiae, and their results showed that a total of 28 proteins confer a role in multidrug-efflux pumps with multiple drug resistance (MDR) function. Remarkably, the potential MFS−MDR transporter (spot 584) of T. versicolor was downregulated after ferruginol treatment (Figure 5 and Table 1). Thus, it was hypothesized that the firstline defense barrier, active secretion of toxins, of T. versicolor cells was reduced or dysfunctional. This might cause further damage within T. versicolor cells. Del Sorbo et al.19 also suggested that the inhibitory activity of the two types of transporters (ABC and MFS) could be developed as an antifungal strategy. Taken together, these results suggest that downregulation of MFS−MDR transporter is an antifungal strategy of ferruginol against T. versicolor. Removal of hydrogen peroxide, alkyl hydroperoxides, and various oxidative stresses by peroxiredoxin TSA1 (spot 503), a thiol-specific antioxidant enzyme (thioredoxin peroxidase), had
To investigate the mechanisms of ferruginol on inhibition of T. versicolor growth, a liquid culture test was performed at the concentration of 100 μg/mL (Figure 2B). The dry weights of nontreated mycelia (control) increased slightly for 5 incubation days (lag phase, about 5 mg), increased linearly from the fifth to the 11th day of incubation (log phase), and exhibited stationary-phase fungal growth after the 11th day of incubation (about 90 mg). However, the dry weights of treated-mycelia decreased to lower than 20 mg after treatment with ferruginol at the seventh day. To analyze the protein expression patterns of ferruginol-treated T. versicolor, the mycelia were harvested at the end of the growth log phase (11th day) for further electrophoretic analysis. Two-Dimensional Electrophoresis and Differential Display Analysis. Total proteins were extracted from mycelia with or without ferruginol treatments and used for quantitative comparison in the 2DE analysis (Figure 3). The average fluorescence intensity of each protein spot was measured to determine the protein expression level. The gels showed major spots between pH 4 and 7 in the wide-range (pH 3−10) immobilized pH gradient (IPG) strips (Figure 3A). To further separate the overlapping spots, narrow-range (pH 4−7) IPG strips were used, and the results revealed more detectable spots on a 2D gel (Figure 3B). Approximately 360 spots were visualized in each 2D gel separated from the pH 4−7 IPG strip. The protein spot volumes from the control and treatment groups were statistically compared (t-test). A total of 34 protein spots, including 19 spots from nontreated (control) samples and 15 spots from ferruginol-treated samples, showed differential protein expression levels (p < 0.05) (Figure 4). A total of 17 nonredundant proteins and two not-identified proteins were identified among these proteins: 12 were downregulated (Table 1) and 7 were upregulated (Table 2) upon ferruginol treatment. C
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
further result in the nonintegrity of T. versicolor cellular molecules such as cytoskeleton, polypeptide, and DNA. The downregulated DNA polymerase ζ catalytic subunit (spot 494) of ferruginol-treated T. versicolor cells also corroborates the aforementioned damage situations. DNA polymerase ζ is an error-prone polymerase and comprises both Rev3 (catalytic subunit) and Rev7 (accessory subunit) in S. cerevisiae.23 Downregulation of DNA polymerase ζ catalytic subunit (Figure 5 and Table 1) might result in dysfunction of DNA polymerase ζ. Lawrence24 reviewed that the cellular functions of DNA polymerase ζ are participating in an extension step as a mismatch extender in translesion synthesis (TLS). For severe injuries caused by DNA damage, TLS is an important damage tolerance system and rescues cells for survival.25−27 Hence, these findings suggest that the damage tolerance system of T. versicolor cannot function normally to assist cells in recovery from DNA damage caused by ferruginol. In this study, β-tubulin 3 (spot 529) of T. versicolor was identified with a decrease in expression volume (Figure 5 and Table 1), suggesting that the ferruginol-treated T. versicolor cells might reduce cell motility, arrest the cell cycle, and inhibit cell growth/division.28−30 Kiso et al.31 screened antifungal agents and their results revealed that 2,5-dihydrophenylalanine (DHPA), a phenylalanine analogue, induced selective loss of both α- and β-tubulins in the filamentous fungus Aspergillus nidulans. According to the liquid culture test of T. versicolor treated with ferruginol, the growth of T. versicolor cells was inhibited. Average dry weights of treated mycelia were significantly lower than that of nontreated one (control) (Figure 2B). These findings suggest that ferruginol may be a type of microtubule-disrupting antifungal agent. In addition, this study identified two downregulated proteins with uncertain functions, including hypothetical protein NCER_101469 (spot 491) and protein kinase C substrate (spot 305) of ferruginol-treated T. versicolor cells. The ratios of expression volumes (treatment/control) are 0.4 and 0.1, respectively (Table 1). Their functions await further investigation. Among the 12 downregulated proteins, spots 596, 332, 598, and 214 were identified as phosphoenolpyruvate carboxykinase AcuF, nascent polypeptide-associated complex subunit β, mitochondrial protein involved in sorting of proteins in the mitochondria, and DNA damage checkpoint protein Rad24,
Figure 4. Two-dimensional gel images of (A) nontreated (control) and (B) ferruginol-treated T. versicolor protein expression patterns.
been demonstrated.20,21 Iraqui et al.22 further indicated that TSA1 maintains genome stability and avoids cell death of S. cerevisiae. Taken together, the downregulations of MFS−MDR transporter and peroxiredoxin TSA1 suggested that ferruginol reduced the defensive capabilities of T. versicolor cells. This may
Table 1. Downregulated Proteins Identified in T. versicolor Mycelia as Differentially Expressed in Response to Ferruginol spot no.a
expression ratiob
473 584 503 494 529 491 505 305 596 332 598
0.7 0.6 0.6 0.6 0.5 0.4 0.3 0.1 ctrl only ctrl only ctrl only
214
ctrl only
Mowse score
cov (%)/pepc
theor mol mass (kDa)
pI
XP_569373 XP_719316 XP_001382622 XP_754928 AAM92170 EEQ81917 d EEQ92115 XP_002375147 A5DF06 XP_002493683
70 75 126 52 629 61
3/2 2/2 9/2 1/2 27/10 4/2
46.9 61.8 21.8 188.1 43.1 32.9
9.44 8.25 4.92 6.64 5.81 8.50
65 51 36 53
2/2 2/2 7/2 2/1
62.8 66.5 16.5 39.6
5.20 5.87 5.54 7.66
XP_001837506
125
16/4
28.9
4.76
protein name
accession no.
Mn ion homeostasis-related protein potential MFS−MDR transporter peroxiredoxin TSA1 DNA polymerase ζ catalytic subunit β-tubulin 3 hypothetical protein NCER_101469 d protein kinase C substrate phosphoenolpyruvate carboxykinase AcuF nascent polypeptide-associated complex subunit β mitochondrial protein involved in sorting of proteins in the mitochondria DNA damage checkpoint protein Rad24
a
Protein identified against NCBI, except spot 332 against Swiss-Prot. bRatio of treatment/control upon addition of ferruginol. cCoverage of protein sequence/matched peptide number. dNot identified. D
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Table 2. Upregulated Proteins Identified in T. versicolor Mycelia as Differentially Expressed in Response to Ferruginol spot no.a
expression ratiob
protein name
accession no.
Mowse score
cov (%)/pepc
theor mol mass (kDa)
pI
624 633 389 388 364 358 390
1.6 2.0 2.2 2.5 2.7 3.0 3.8
d ATP-dependent RNA helicase eIF4A heat shock protein heat shock protein HSS1 tpis_emeni triosephosphate isomerase autophagy-related protein 7 heat shock protein 70
d Q2UPY3 XP_567978 Q01877 XP_664504 Q5AWA2 ACT22525
106 117 1017 62 41 534
6/2 3/2 25/16 5/2 1/2 8/5
47.3 71.4 70.5 27.1 73.4 72.1
5.39 5.57 5.13 5.89 6.74 5.63
a
Protein identified against NCBI, except spots 633 and 358 against Swiss-Prot. bRatio of treatment/control upon addition of ferruginol. cCoverage of protein sequence/matched peptide number. dNot identified.
damage checkpoint protein Rad24 of T. versicolor cells strongly suggest that ferruginol is potentially involved in DNA damage or mutation, which might account for DNA nonintegrity and further cell death. Upregulated Proteins upon Ferruginol Treatment. Six proteins are upregulated, including ATP-dependent RNA helicase eIF4A (spot 633), heat shock protein (spot 389), heat shock protein HSS1 (spot 388), tpis_emeni triosephosphate isomerase (spot 364), autophagy-related protein 7 (spot 358), and heat shock protein 70 (spot 390, Table 2). Eukaryotic translation factor 4A (eIF4A) participates in the initial step of mRNA translation. This ATP-dependent RNA helicase can unwind the secondary structure of mRNA at the 5′ untranslated region (UTR).38 It is a critical stage of translational control. Upregulation of eIF4A suggests that T. versicolor cells appear to rescue cells by increasing their protein synthesis ability. However, the correct folding structure of protein is essential to such function. In this study, the nascent polypeptide-associated complex subunit β (spot 332, Table 1) and mitochondrial protein involved in sorting of proteins in the mitochondria (spot 598, Table 1) of T. versicolor cells were downregulated. These findings suggest that T. versicolor cells attempt to enhance the translation processes but the actions of protein sorting and folding cannot function normally. Taken together, T. versicolor cells might produce dysfunctional proteins and cause a massive stress. Among these upregulated proteins, there are three heat shock proteins (HSPs). The ratios of expression volumes (treatment/control) of HSP (spot 389), HSS1 (spot 388), and HSP70 (spot 390) are 2.2, 2.5, and 3.8, respectively (Table 2). HSPs are highly conserved from bacteria to animals and function as molecular chaperones against stress. Jolly and Morimoto39 reviewed the functions of HSPs and indicated that HSPs may control signal transduction at multiple checkpoints in the apoptosis pathways (type I programmed cell death, PCD). Moreover, among these HSPs, the small HSPs and HSP70 possess antiapoptotic activity for promoting cell survival. Matsuzaki et al.40 investigated the proteomic responses of the white-rot fungus Phanerochaete chrysosporium to exogenous benzoic acid, and their results exhibited a similar stress response that the HSP70 was upregulated. These findings suggest that exposure of T. versicolor cells to ferruginol activates a defensive response via induction of HSPs. Triosephosphate isomerase (spot 364) regulates the equilibrium of triosephosphates between dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate in glycolysis metabolism. Organisms convert glucose into pyruvate via glycolysis and release high-energy compounds, including ATP and NADH. In this study, the ratio of expression volumes (treatment/control) of triosephosphate isomerase was 2.7
Figure 5. Comparison of expression patterns from nontreated (control) and ferruginol-treated T. versicolor protein by statistical analysis. Results are mean ± SE (n = 3). Protein expressions are significantly different at the level of p < 0.05 according to the student’s paired t-test.
respectively. They were detected in nontreated (control) T. versicolor cells only. Phosphoenolpyruvate carboxykinase AcuF (spot 596) is a key enzyme associated with gluconeogenesis for producing hexose sugars of filamentous fungi via the tricarboxylic acid cycle to metabolize carbon sources.32 This result suggests that ferruginol-treated T. versicolor cells cannot regulate glucose content normally via the processes of gluconeogenesis and tricarboxylic acid cycle. Simultaneous observations of a drastic decrease in nascent polypeptide-associated complex subunit β (spot 332) and mitochondrial protein involved in sorting of proteins in the mitochondria (spot 598) (Figure 5 and Table 1) suggested that ferruginol-treated T. versicolor cells diminished the flux of nascent polypeptides and the capacity for correct translation. Nascent polypeptide-associated complex (NAC) is a heterodimer composed of α- and β-subunits and has a chaperone role in cells.33,34 It also has a central role as a proteostasis sensor to control the folding state of the cellular proteome.35 These results suggest that the proteostasis of ferruginol-treated T. versicolor cells is not under control. In this study, expression of the DNA damage checkpoint protein Rad24 (spot 214) of T. versicolor cells was observed only in nontreated control (Figure 5 and Table 1). This finding supports the idea that ferruginol-treated T. versicolor cells might arrest the processing of the cell cycle and result in functionally defective DNA replication. Hartwell and Weinert36 reported that eliminating the checkpoint control functions of eukaryotic cells may result in chromosome transmission infidelity. Aylon and Kupiec37 further indicated that Rad24 plays a critical role in coordination of the response to repair chromosomal damage in S. cerevisiae. Taken together, the downregulation of peroxiredoxin TSA1, DNA polymerase ζ catalytic subunit, and DNA E
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
■
(Figure 5 and Table 2). This upregulation of glycolytic protein suggests that T. versicolor cells appear to enhance energy production against stress conditions. Parente et al.41 investigated the proteomic responses of dimorphic fungus Paracoccidoides brasiliensis under iron deprivation, and their results exhibited a similar response that glycolytic proteins were upregulated against stress. Upregulation of these proteins seems to indicate that T. versicolor cells can activate defense and rescue responses against ferruginol. However, the current results showed upregulation of a key autophagy-related protein (Atg7, spot 358, Table 2) of T. versicolor cells upon addition of ferruginol, inferring that those rescue reactions may be insufficient to reduce the toxic effects caused by ferruginol. This idea is consistent with the liquid culture test result of T. versicolor that the growth of mycelium was inhibited after treatment with ferruginol (Figure 2B). Autophagy (self-eating, type II PCD) leads aberrant organelles and misfolded proteins for breakdown in autophagosomes, and the cells recycle these molecules for energy generation in response to aging or starvation conditions.42 Nevertheless, excessive autophagy will induce a total collapse of cellular functions due to autophagic-type cell death. In this study, the ratio of expression volumes (treatment/control) of Atg7 is 3.0 (Figure 5 and Table 2). Atg7 is a unique ubiquitinactivating enzyme involved in the ubiquitin-like conjugation systems for processing the formation of autophagosomes.43 This evidence demonstrates that T. versicolor cells had massive autophagy. Garrido et al.44 further indicated that HSP70 is a decisive regulator that blocks the mitochondrial apoptotic pathway (type I PCD). In summary, upregulation of both HSP70 and Atg7 supports the idea that ferruginol induces T. versicolor cells toward cellular demise via type II PCD. In this study, the downregulation of DNA damage checkpoint protein Rad24 (spot 214, Table 1) and DNA polymerase ζ catalytic subunit (spot 494, Table 1) suggests that the cell cycle of T. versicolor was arrested and the DNA damage cannot be completely repaired. In addition, the downregulation of peroxiredoxin TSA1 (Table 1) also suggests that T. versicolor cells suffered an abnormal accumulation of reactive oxygen species, contributing to irreversible damage that might cause cell death. Similar evidence that catalase, a key antioxidant enzyme, was selectively eliminated during autophagic cell death was reported.45 Hence, ferruginol caused T. versicolor cells to move toward autophagic cell death in the current study. In conclusion, antifungal processes of ferruginol against the basidiomycete T. versicolor at the proteome level were investigated. Results indicated that the defensive capabilities of T. versicolor cells were reduced. DNA and nascent polypeptides of T. versicolor cells might be attacked. The current results suggest that ferruginol induced destruction of vital cell organelles or proteins and caused cellular dysfunction. This process may further contribute to cell death of T. versicolor.
■
Article
AUTHOR INFORMATION
Corresponding Author
* Phone +886-2-33664626; fax +886-2-23654520; e-mail
[email protected]. Funding
This work was financed by the Ministry of Science and Technology from Taiwan (NSC 96-2628-B-002-023-MY3). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Dr. Keng-Hao Hsu for technical assistance and suggestions. We thank the Experimental Forest of National Taiwan University for providing experimental materials. We appreciate the excellent technical assistance of Technology Commons, College of Life Science, National Taiwan University, with the image analyzer (Typhoon 9400), and we thank the Instrument Center of National Cheng Kung University, National Science Council of Taiwan, for LC-MS/ MS analyses.
■
REFERENCES
(1) Chang, S. T.; Wang, S. Y.; Wu, C. L. Identification of exudates from the bark of Calocedrus macrolepis var. formosana (Florin). For. Prod. Ind. 2000, 19, 59−65 (in Chinese). (2) Imai, T.; Tanabe, K.; Kato, T.; Fukushima, K. Localization of ferruginol, a diterpene phenol, in Cryptomeria japonica heartwood by time-of-flight secondary ion mass spectrometry. Planta 2005, 221, 549−556. (3) Chang, S. T.; Wang, S. Y.; Wu, C. L.; Su, Y. C.; Kuo, Y. H. Antifungal compounds in the ethyl acetate soluble fraction of the extractives of Taiwania (Taiwania cryptomerioides Hayata) heartwood. Holzforschung 1999, 53, 487−490. (4) Sharp, H.; Latif, Z.; Bartholomew, B.; Bright, C.; Jones, C. D.; Sarker, S. D.; Nash, R. J. Totarol, totaradiol and ferruginol: three diterpenes from Thuja plicata (Cupressaceae). Biochem. Syst. Ecol. 2001, 29, 215−217. (5) Kusumoto, N.; Ashitani, T.; Murayama, T.; Ogiyama, K.; Takahashi, K. Antifungal abietane-type diterpenes from the cones of Taxodium distichum Rich. J. Chem. Ecol. 2010, 36, 1381−1386. (6) Ho, C. L.; Yang, S. S.; Chang, T. M.; Su, Y. C. Composition, antioxidant, antimicrobial and anti-wood-decay fungal activities of the twig essential oil of Taiwania cryptomerioides from Taiwan. Nat. Prod. Commun. 2012, 7, 261−264. (7) Odds, F. C.; Brown, A. J. P.; Gow, N. A. R. Antifungal agents: Mechanisms of action. Trends Microbiol. 2003, 11, 272−279. (8) Ramsdale, M. Programmed cell death in pathogenic fungi. Biochim. Biophys. Acta, Mol. Cell Res. 2008, 1783, 1369−1380. (9) Martin, F.; Cullen, D.; Hibbett, D.; Pisabarro, A.; Spatafora, J. W.; Baker, S. E.; Grigoriev, I. V. Sequencing the fungal tree of life. New Phytol. 2011, 190, 818−821. (10) Martinez, D.; Larrondo, L. F.; Putnam, N.; Gelpke, M. D. S.; Huang, K.; Chapman, J.; Helfenbein, K. G.; Ramaiya, P.; Detter, J. C.; Larimer, F.; Coutinho, P. M.; Henrissat, B.; Berka, R.; Cullen, D.; Rokhsar, D. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat. Biotechnol. 2004, 22, 695−700. (11) Kroll, K.; Pähtz, V.; Kniemeyer, O. Elucidating the fungal stress response by proteomics. J. Proteomics 2014, 97, 151−163. (12) Cagas, S. E.; Jain, M. R.; Li, H.; Perlin, D. S. Profiling the Aspergillus f umigatus proteome in response to caspofungin. Antimicrob. Agents Chemother. 2011, 55, 146−154. (13) Shimizu, M.; Yuda, N.; Nakamura, T.; Tanaka, H.; Wariishi, H. Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin-degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin. Proteomics 2005, 5, 3919−3931.
ASSOCIATED CONTENT
* Supporting Information S
Two tables listing sequences of identified down- and upregulated proteins. This material is available free of charge via the Internet at http://pubs.acs.org. F
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
(14) Wang, S. Y.; Wu, J. H.; Shyur, L. F.; Kuo, Y. H.; Chang, S. T. Antioxidant activity of abietane-type diterpenes from heartwood of Taiwania cryptomerioides Hayata. Holzforschung 2002, 56, 487−492. (15) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (16) Reddi, A. R.; Jensen, L. T.; Culotta, V. C. Manganese homeostasis in Saccharomyces cerevisiae. Chem. Rev. 2009, 109, 4722−4732. (17) Luk, E.; Jensen, L. T.; Culotta, V. C. The many highways for intracellular trafficking of metals. J. Biol. Inorg. Chem. 2003, 8, 803− 809. (18) Nelissen, B.; De Wachter, R.; Goffeau, A. Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae. FEMS Microbiol. Rev. 1997, 21, 113−134. (19) Del Sorbo, G.; Schoonbeek, H. J.; De Waard, M. A. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal Genet. Biol. 2000, 30 (1), 1−15. (20) Netto, L. E. S.; Chae, H. Z.; Kang, S. W.; Rhee, S. G.; Stadtman, E. R. Removal of hydrogen peroxide by thiol-specific antioxidant enzyme (TSA) is involved with its antioxidant properties: TSA possesses thiol peroxidase activity. J. Biol. Chem. 1996, 271, 15315− 15321. (21) Park, S. G.; Cha, M. K.; Jeong, W.; Kim, I. H. Distinct physiological functions of thiol peroxidase isoenzymes in Saccharomyces cerevisiae. J. Biol. Chem. 2000, 275, 5723−5732. (22) Iraqui, I.; Kienda, G.; Soeur, J.; Faye, G.; Baldacci, G.; Kolodner, R. D.; Huang, M.-E. Peroxiredoxin Tsa1 is the key peroxidase suppressing genome instability and protecting against cell death in Saccharomyces cerevisiae. PLoS Genet. 2009, 5, No. e1000524. (23) Nelson, J. R.; Lawrence, C. W.; Hinkle, D. C. Thymine-thymine dimer bypass by yeast DNA polymerase zeta. Science 1996, 272, 1646− 1649. (24) Lawrence, C. W. Cellular roles of DNA polymerase zeta and Rev1 protein. DNA Repair 2002, 1, 425−435. (25) Prakash, S.; Johnson, R. E.; Prakash, L. Eukaryotic translesion synthesis DNA polymerases: Specificity of structure and function. Annu. Rev. Biochem. 2005, 74, 317−353. (26) Murakumo, Y. The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2002, 510, 37−44. (27) Broomfield, S.; Hryciw, T.; Xiao, W. DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. DNA Repair 2001, 486, 167−184. (28) Xiang, X.; Plamann, M. Cytoskeleton and motor proteins in filamentous fungi. Curr. Opin. Microbiol. 2003, 6, 628−633. (29) Dumontet, C.; Durán, G. E.; Steger, K. A.; Murphy, G. L.; Sussman, H. H.; Sikic, B. I. Differential expression of tubulin isotypes during the cell cycle. Cell Motil. Cytoskeleton 1996, 35, 49−58. (30) Nogales, E.; Wolf, S. G.; Downing, K. H. Structure of the alpha beta tubulin dimer by electron crystallography. Nature 1998, 391, 199−203. (31) Kiso, T.; Fujita, K. I.; Ping, X.; Tanaka, T.; Taniguchi, M. Screening for microtubule-disrupting antifungal agents by using a mitotic-arrest mutant of Aspergillus nidulans and novel action of phenylalanine derivatives accompanying tubulin loss. Antimicrob. Agents Chemother. 2004, 48, 1739−1748. (32) Hynes, M. J.; Draht, O. W.; Davis, M. A. Regulation of the acuF gene, encoding phosphoenolpyruvate carboxykinase in the filamentous fungus Aspergillus nidulans. J. Bacteriol. 2002, 184, 183−190. (33) Beatrix, B.; Sakai, H.; Wiedmann, M. The alpha and beta subunit of the nascent polypeptide-associated complex have distinct functions. J. Biol. Chem. 2000, 275, 37838−37845. (34) Wegrzyn, R. D.; Deuerling, E. Molecular guardians for newborn proteins: Ribosome-associated chaperones and their role in protein folding. Cell. Mol. Life Sci. 2005, 62, 2727−2738.
(35) Kirstein-Miles, J.; Scior, A.; Deuerling, E.; Morimoto, R. I. The nascent polypeptide-associated complex is a key regulator of proteostasis. EMBO J. 2013, 32, 1451−1468. (36) Hartwell, L. H.; Weinert, T. A. Checkpoints: Controls that ensure the order of cell cycle events. Science 1989, 246, 629−634. (37) Aylon, Y.; Kupiec, M. The checkpoint protein Rad24 of Saccharomyces cerevisiae is involved in processing double-strand break ends and in recombination partner choice. Mol. Cell. Biol. 2003, 23, 6585−6596. (38) Rogers, G. W.; Komar, A. A.; Merrick, W. C. eIF4A: The godfather of the DEAD box helicases. Prog. Nucleic Acid Res. Mol. Biol. 2002, 72, 307−331. (39) Jolly, C.; Morimoto, R. I. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. JNCI, J. Natl. Cancer Inst. 2000, 92, 1564−1572. (40) Matsuzaki, F.; Shimizu, M.; Wariishi, H. Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid. J. Proteome Res. 2008, 7, 2342−2350. (41) Parente, A. F. A.; Bailão, A. M.; Borges, C. L.; Parente, J. A.; Magalhães, A. D.; Ricart, C. A. O.; Soares, C. M. A. Proteomic analysis reveals that iron availability alters the metabolic status of the pathogenic fungus Paracoccidioides brasiliensis. PLoS One 2011, 6, No. e22810. (42) Gozuacik, D.; Kimchi, A.; Gerald, P. S. Autophagy and cell death. Curr. Top. Dev. Biol. 2007, 78, 217−245. (43) Tsujimoto, Y.; Shimizu, S. Another way to die: Autophagic programmed cell death. Cell Death Differ. 2005, 12, 1528−1534. (44) Garrido, C.; Galluzzi, L.; Brunet, M.; Puig, P. E.; Didelot, C.; Kroemer, G. Mechanisms of cytochrome c release from mitochondria. Cell Death Differ. 2006, 13, 1423−1433. (45) Yu, L.; Wan, F. Y.; Dutta, S.; Welsh, S.; Liu, Z. H.; Freundt, E.; Baehrecke, E. H.; Lenardo, M. Autophagic programmed cell death by selective catalase degradation. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4952−4957.
G
dx.doi.org/10.1021/jf504717x | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
