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Proteasomal degradation pathways in Trametes versicolor and Phlebia radiata
Enzyme and Microbial Technology 30 (2002) 537–541
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Proteasomal degradation pathways in Trametes versicolor and Phlebia radiata Magdalena Staszczak Department of Biochemistry, Maria Curie-Sklodowska University, Plac Marii Curie-Sklodowskiej 3, 20 – 031 Lublin, Poland
Abstract Possible involvement of the non-lysosomal (proteasome-mediated) pathway in the regulation of ligninolytic activities was studied. Proteasome activity was detected in mycelial extracts of the efficient lignin-degrading white rot fungi, Trametes versicolor and Phlebia radiata, by monitoring cleavage of a fluorogenic (7-amido-4-methyl-coumarin-linked) peptide substrate (SucLLVY-MCA). One of the more potent peptide aldehyde inhibitors of the proteasome, MG132 (CbzLLLal) was used to define a role for the proteasome-mediated protein degradation in their metabolism. Proteasome activity was assayed in cultures of T. versicolor and P. radiata following a nutritional shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. In carbon-starved cultures, addition of MG132 decreased intracellular and extracellular laccase and peroxidase activities whereas in nitrogen-starved cultures, addition of this proteasome inhibitor increased intracellular activities of these enzymes by 1.5 to 2 fold and extracellular activities by 4 to 7 fold in T. versicolor. Similar but less dramatic changes were observed with P. radiata. The results indicate that proteasomes play a role in the metabolism of these organisms. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction The growth of wood decaying fungi, especially under natural conditions, requires control of their nitrogen economy. This involves regulation of proteolytic activities for the intracellular protein turnover, extracellular digestion of protein sources, and modification of proteins through limited proteolysis. Continuous protein turnover is involved in basic cellular functions such as the modulation of the levels of structural, catalytic and regulatory proteins, adjustment to stress, as well as preferential removal of defective proteins. Rapid protein turnover was shown in the white-rot fungus Phanerochaete chrysosporium during the transition between depletion of medium nitrogen and the onset of ligninolytic activity [1]. Eukaryotic cells contain two major systems for protein degradation: lysosomal (vacuolar) and non-lysosomal (nonvacuolar). The lysosome was long believed to be the only site for protein breakdown in cells, but it is now clear that intracellular proteolysis is largely accomplished by a highly selective non-lysosomal pathway that requires ATP and a proteasome particle [2– 4]. The 26S proteasome consists of
Tel.: ⫹48-81-5375-775; fax: ⫹48-81-5375-102. E-mail address: [email protected].
a barrel-shaped proteolytically active core (20S proteasome) and 19S regulatory complexes. During the past decade, this multisubunit, multicatalytic complex of the cytosol and nucleus, was shown to have vital regulatory functions. It degrades many important proteins involved in signaling pathway, in cell cycle control, and in general metabolism, including transcription factors and key metabolic enzymes. In recent years, it has become clear that intracellular proteolysis plays an essential role in response to stress conditions such as high temperatures or nutrient deprivation [5]. Lignin-modifying enzymes of white-rot fungi are mainly expressed during the secondary phase of growth (i.e. idiophase), when the limitation of carbon, nitrogen and sulphur occurs [6,7]. It has been demonstrated for many eukaryotic organisms, including yeasts, that both the lysosomal (vacuolar) and non-lysosomal (non-vacuolar) proteolytic systems are activated by nutrient starvation [8]. Thus intracellular proteolytic enzymes are likely to be involved in the metabolic transition from primary growth (trophophase) to idiophase triggered in the white-rot fungi by nutrient deprivation. Even though the synthesis of intracellular and extracellular proteases is a common feature among fungi [9], comparatively little study has been devoted to proteases of wood-degrading Basidiomycetes [10 –13]. Our previous study showed that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 0 1 0 - 8
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M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
laccase and peroxidase activity in cultures of Trametes versicolor under nutrient limitation [14]. A major goal of the present study was to detect proteasome activity in the efficient lignin-degraders and to examine whether the specific agent (CbzLLLal, MG 132) that blocks the function of yeast and mammalian 26S proteasome [15], can affect the levels of ligninolytic enzymes of white-rot fungi. 2. Materials and methods 2.1. Culture conditions Mycelia of white-rot fungi Trametes versicolor (ATCC 44308, strain FCL 7) and Phlebia radiata (ATCC 64658, strain FCL 99a) were maintained, through periodic (every 7 days) inoculation with floating discs of mycelium (5 mm of the diameter) as surface cultures at 26°C, in scintillation flasks containing 10 ml of nutrient-rich growth medium [16] with glucose as a carbon source and L-asparagine as a nitrogen source. After the 7-day cultivation period, mycelia were transferred to fresh growth media deprived of glucose (carbon starvation) or L-asparagine (nitrogen starvation), and to trophophasic media. The trophophasic media contained glucose and asparagine of the same concentrations as those measured after seven days of fungal growth. Other conditions of starvation experiments were essentially those described previously [14]. Three independent experiments were performed in duplicate. 2.2. Effect of proteasome inactivation The proteasome inhibitor, MG132 (CbzLLLal) was added (to a final concentration of 40 M) at the time of transfer of mycelia to the nutrient-deprived or trophophasic media. An equivalent amount of solvent used (DMSO) was added to parallel sets of cultures (nutrient-deprived and trophophasic). At 6 h after exposure to the proteasome inhibitor, enzymatic activities were assayed by the methods described below. The results are expressed as a percentage of remaining activity. For each determination, the specific activity of samples from the parallel culture (carbon-deprived, nitrogen-deprived and trophophasic) without the inhibitor was taken to be 100%.
Potter’s homogenizer, in 2 ml of 0.05 M Tris-HCl buffer, pH 7.3. The homogenates were then centrifuged for 10 min at 10000⫻ g, at 4°C. The supernatants were desalted through a Sephadex G-25 column as described above. 2.5. Proteasome assays Proteasome activity was detected in mycelial extracts by cleavage of a fluorogenic peptide substrate (Suc-LLVYMCA) by the modified stopped assay procedure [17,18]. Briefly, assay mixtures containing 5–10 l of the mycelial extract, 100 M Suc-LLVY-MCA (in DMSO), and 100 mM Tris-HCl buffer, pH 8.0, were made up in a total volume of 100 l and then incubated at 37°C for 15 and 30 min. The fluorescent proteolysis product was quantified in a spectrofluorometer (FluoroMax-2, Instruments S.A., Inc., JOBIN YVON/SPEX Division, USA), at an emission wavelength of 440 nm, with excitation at 360 nm. Fluorescence units measured were converted to picomoles of MCA released by using a standard curve prepared with known dilutions of MCA in 5% DMSO in 100 mM Tris-HCl buffer, pH 8.0. 2.6. Determination of laccase activity (EC 1.10.3.2) Activity of laccase (benzenediol: oxygen oxidoreductase) was determined in desalted samples of culture filtrates and mycelial extracts according to [19] using syringaldazine as a substrate. 0.1 M citrate-NaOH buffer, pH 5.0 was used for determinations. 2.7. Determination of peroxidase (HRP-like) activity (EC 1.11.1.7) Peroxidase activity was assayed in desalted samples of culture filtrates and mycelial extracts according to [20]. 0.003% H2O2, 0.01% o-dianisidine, and 0.1 M citrateNaOH buffer, pH 5.0, were used for determinations. 2.8. Determination of protein Protein concentration was measured according to the method described by Lowry and modified by [21]. Bovine serum albumin was used as standard.
2.3. Preparation of extracellular culture fluid
2.9. Reagents
Extracellular samples were collected as described previously [14] by separating culture fluid from mycelium by filtration. The filtrates were desalted through a Sephadex G-25 column. The elution was performed with 0.001 M Tris, pH 7.0.
SucLLVY-MCA, 7-amino-4-methylcoumarin, CbzLLLal (MG132), DMSO, syringaldazine were obtained from SIGMA, St. Louis.
2.4. Preparation of mycelial extracts
3. Results and discussion
Mycelia (dry weight about 50 – 60 mg per flask) were harvested and homogenized in an ice-chilled motor-driven
White-rot fungi are being studied mainly because of their ligninolytic activity which appears during the nutritional
M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
539
shift from trophophase to idiophase triggered by carbon- or nitrogen starvation [22]. The wood-degrading fungi Trametes versicolor and Phlebia radiata are, similarly to the intensively studied fungus Phanerochaete chrysosporium, very efficient white-rotters in nature [23,24]. Our previous studies have demonstrated numerous intraand extracellular proteolytic enzymes, present both in nutrient-rich and carbon- or nitrogen-starved cultures of Trametes versicolor and Phlebia radiata [13]. Our experiments with PMSF (irreversible inhibitor of serine proteinases) and chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) have indicated that both intracellular and extracellular proteases can be involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation [14]. 3.1. Proteasome assays Experiments with mycelial extracts of T. versicolor and P. radiata showed the ability of these extracts to hydrolyze Suc-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (SucLLLVYMCA), a well known substrate used to detect proteasome activity. Proteasome activity was found in mycelial extracts from both trophophasic (non-starved) and starved cultures of these fungi (Fig. 1). In Eukarya, the 20S proteasome contains two chymotrypsin-like, two trypsin-like, and two active sites shown to have caspase-like specificity [25]. These sites cleave preferentially after large hydrophobic residues, basic residues, and acidic residues, respectively. Comparatively small differences in the levels of chymotrypsin-like (SucLLLVY-MCA-hydrolyzing) activity were found between starved and non-starved mycelia. The crude proteasome activity detected in mycelial extracts of T. versicolor and P. radiata was sensitive in vitro to MG132 (CbzLLLal), one of the more potent peptide aldehyde inhibitors of the proteasome. This agent inhibited the crude activity by about 20%. 3.2. Effect of proteasome inactivation Peptidyl aldehyde analogues are reversible proteasome inhibitors. The aldehyde moiety of these inhibitors forms a hemi-acetyl adduct with the nucleophilic hydroxyl group of the N-terminal threonine in the proteasome [26]. Most of the reported peptide aldehyde inhibitors of the proteasome have been directed against the chymotrypsin-like activity of the 20S proteasome. The specific agent, MG 132 (CbzLLLal) has been previously shown to inhibit purified yeast and mammalian 26S proteasome [15]. To assess its effect on the metabolism of white-rot fungi, MG132 was added to cultures of T. versicolor and P. radiata at the time of transfer of mycelia to the nutrient-deprived or trophophasic media. Figs. 2A–2C show the effect of the proteasome inhibitor MG132 (CbzLLLal) on activities of laccase and peroxidase in carbon-deprived, nitrogen-deprived, and trophophasic
Fig. 1. Activity of mycelial extracts from non-starved (square symbol), carbon-starved (diamond-symbol), and nitrogen-starved (triangle symbol) cultures of (A) Trametes versicolor and (B) Phlebia radiata, against the fluorogenic peptide substrate SucLLVY-MCA. Average of at least three independent experiments performed in triplicate.
cultures of T. versicolor, respectively. Changes in both extra- and intracellular activities in nutrient-starved cultures occurred as a result of MG132 addition. A significant decrease in laccase and peroxidase activities was observed in carbon-deprived cultures (Fig. 2A). The inhibitor addition under nitrogen limitation resulted in an approximately 7-fold and 5-fold increase in extracellular laccase and peroxiase activities respectively (Fig. 2B). In contrast to starved cultures, no significant effect could be detected for trophophasic cultures of T. versicolor after exposure to the proteasome inhibitor (Fig. 2C). The effect of MG132 addition to cultures of P. radiata was similar to that observed in the case of T. versicolor, however the changes in laccase and peroxidase activities found in starved cultures of P. radiata were not so strong (Fig. 3). The difference between the effect of MG132 in the carbon starvation response and in the nitrogen starvation response can be explained on the basis of the previously observed difference between the onset of ligninolytic activity triggered by carbohydrate or nitrogen starvation [7].
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M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
Fig. 2. Effect of proteasome inhibitor, MG 132 (CbzLLLal), on activities of extracellular laccase (shaded bars), extracellular peroxidase (black bars), intracellular laccase (empty bars), and intracellular peroxidase (hatched bars) in cultures of Trametes versicolor: (A) carbon-starved, (B) nitrogenstarved, and (C) trophophasic (non-starved). Data are means ⫾ SD from at least three independent experiments performed in duplicate.
Fig. 3. Effect of proteasome inhibitor, MG 132 (CbzLLLal), on activities of extracellular laccase (shaded bars), extracellular peroxidase (black bars), intracellular laccase (empty bars), and intracellular peroxidase (hatched bars) in cultures of Phlebia radiata: (A) carbon-starved, (B) nitrogenstarved, and (C) trophophasic (non-starved). Data are means ⫾ SD from at least three independent experiments performed in duplicate.
4. Conclusions
found to be very useful as inhibitors in studies clarifying the role of the proteasome in different intracellular degradative processes. The present study showed that in carbon-starved cultures of white-rot fungi, addition of the proteasome inhibitor (MG132) decreased laccase and peroxidase activities
The lack of specific inhibitors of the proteasome pathway has long been a major factor limiting an understanding of its function in vivo. Recently several peptide aldehydes were
M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
whereas in nitrogen-starved cultures, addition of MG132 increased activities of these enzymes. Although the present work constitutes only a preliminary step in studying proteasome-mediated proteolysis in lignin-degrading Basidiomycetes, the results obtained here strongly suggest that proteasomes exist in the white-rot fungi and play a role in the metabolism of these microorganisms.
Acknowledgments This work was partly supported by the State Committee for Scientific Research (KBN Grant-No 6 P04A 027 17, to M.S.), EC Contract ICA2-CT-2000 –10050, and BS/ BiNoZ/4.
References [1] Fenn P, Kirk TK. Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium. Arch Microbiol 1981;130:59 – 65. [2] Goldberg AL, Akopian TN, Kisselev AF, Lee DH, Rohrwild M. New insights into the mechanisms and importance of the proteasome in intracellular protein degradation. Biol Chem 1997;378:131– 40. [3] Voges D, Zwickl P, Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu Rev Biochem 1999;68:1015– 68. [4] Coux O, Tanaka K, Goldberg AL. Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 1996;65:801– 47. [5] Hilt W, Wolf DH. Stress-induced proteolysis in yeast. Mol Microbiol 1992;6:2437– 42. [6] Keyser P, Kirk TK, Zeikus JG. Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in absence of lignin in response to nitrogen starvation. J Bacteriol 1978;135:790 –7. [7] Jeffries TW, Choi S, Kirk TK. Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl Environ Microbiol 1981;42:290 – 6. [8] Cuervo AM, Dice JF. Lysosomes, a meeting point of proteins, chaperones, and proteases. J Mol Med 1998;76:6 –12. [9] North MJ. Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiol Rev 1982;46:308 – 40. [10] Eriksson K-E, Pettersson B. Acid proteases from Sporotrichum pulverulentum. Methods Enzymol 1988;160:501– 8.
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[11] Dass SB, Dosoretz CG, Reddy CA, Grethlein HE. Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions. Arch Microbiol 1995;163:254 – 8. [12] Datta A. Purification and characterization of a novel protease from solid substrate cultures of Phanerochaete chrysosporium. J Biol Chem 1992;267:728 –36. [13] Staszczak M, Nowak G, Grzywnowicz K, Leonowicz A. Proteolytic activities in cultures of selected white-rot fungi. J Basic Microbiol 1996;36:193–203. [14] Staszczak M, Zdunek E, Leonowicz A. Studies on the role of proteases in the white-rot fungus Trametes versicolor: effect of PMSF and chloroquine on ligninolytic enzymes activity. J Basic Microbiol 2000;40:51– 63. [15] Lee DH, Goldberg AL. Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae. J Biol Chem 1996;271:27280 – 4. [16] Fahraeus G, Reinhammar B. Large scale production and purification of laccase of the fungus Polyporus versicolor and some properties of laccase A. Acta Chem Scand 1967;21:2367–78. [17] Tanaka K, Yoshimura T, Ichihara A. Role of substrate in reversible activation of proteasomes (multi-protease complexes} by sodium dodecyl sulfate. J Biochem 1989;106:495–500. [18] Skoda B, Malek L. Dry pea seed proteasome. Plant Physiol 1992;99: 1515–9. [19] Leonowicz A, Grzywnowicz K. Quantitative estimation of laccase forms in some white-rot fungi using syringaldazine as a substrate. Enzyme Microb Technol 1981;3:55– 8. [20] Claiborne A, Fridovich I. Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase 1. Spectral properties of the products of dianisidine oxidation. Biochemistry 1979;18:2324 –9. [21] Schacterle GR, Pollack RL. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem 1973;51:654 –5. [22] Kirk TK, Farrell RL. Enzymatic combustion: the microbial degradation of lignin. Ann Rev Microbiol 1987;41:465–505. [23] Hatakka A. Lignin-modifying enzymes from selected white-rot fungi: production and role in lignin degradation. FEMS Microbiol Rev 1994;13:125–35. [24] Tuomela M, Vikman M, Hatakka A, Itavaara M. Biodegradation of lignin in compost environment: a review. Bioresource Technology 2000;72:169 – 83. [25] Kisselev A, Akopian TN, Castillo V, Goldberg AL. Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown. Mol Cell 1999;4:395– 402. [26] Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 1998;8:397– 403.
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Proteasomal degradation pathways in Trametes versicolor and Phlebia radiata Magdalena Staszczak Department of Biochemistry, Maria Curie-Sklodowska University, Plac Marii Curie-Sklodowskiej 3, 20 – 031 Lublin, Poland
Abstract Possible involvement of the non-lysosomal (proteasome-mediated) pathway in the regulation of ligninolytic activities was studied. Proteasome activity was detected in mycelial extracts of the efficient lignin-degrading white rot fungi, Trametes versicolor and Phlebia radiata, by monitoring cleavage of a fluorogenic (7-amido-4-methyl-coumarin-linked) peptide substrate (SucLLVY-MCA). One of the more potent peptide aldehyde inhibitors of the proteasome, MG132 (CbzLLLal) was used to define a role for the proteasome-mediated protein degradation in their metabolism. Proteasome activity was assayed in cultures of T. versicolor and P. radiata following a nutritional shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. In carbon-starved cultures, addition of MG132 decreased intracellular and extracellular laccase and peroxidase activities whereas in nitrogen-starved cultures, addition of this proteasome inhibitor increased intracellular activities of these enzymes by 1.5 to 2 fold and extracellular activities by 4 to 7 fold in T. versicolor. Similar but less dramatic changes were observed with P. radiata. The results indicate that proteasomes play a role in the metabolism of these organisms. © 2002 Elsevier Science Inc. All rights reserved.
1. Introduction The growth of wood decaying fungi, especially under natural conditions, requires control of their nitrogen economy. This involves regulation of proteolytic activities for the intracellular protein turnover, extracellular digestion of protein sources, and modification of proteins through limited proteolysis. Continuous protein turnover is involved in basic cellular functions such as the modulation of the levels of structural, catalytic and regulatory proteins, adjustment to stress, as well as preferential removal of defective proteins. Rapid protein turnover was shown in the white-rot fungus Phanerochaete chrysosporium during the transition between depletion of medium nitrogen and the onset of ligninolytic activity [1]. Eukaryotic cells contain two major systems for protein degradation: lysosomal (vacuolar) and non-lysosomal (nonvacuolar). The lysosome was long believed to be the only site for protein breakdown in cells, but it is now clear that intracellular proteolysis is largely accomplished by a highly selective non-lysosomal pathway that requires ATP and a proteasome particle [2– 4]. The 26S proteasome consists of
Tel.: ⫹48-81-5375-775; fax: ⫹48-81-5375-102. E-mail address: [email protected].
a barrel-shaped proteolytically active core (20S proteasome) and 19S regulatory complexes. During the past decade, this multisubunit, multicatalytic complex of the cytosol and nucleus, was shown to have vital regulatory functions. It degrades many important proteins involved in signaling pathway, in cell cycle control, and in general metabolism, including transcription factors and key metabolic enzymes. In recent years, it has become clear that intracellular proteolysis plays an essential role in response to stress conditions such as high temperatures or nutrient deprivation [5]. Lignin-modifying enzymes of white-rot fungi are mainly expressed during the secondary phase of growth (i.e. idiophase), when the limitation of carbon, nitrogen and sulphur occurs [6,7]. It has been demonstrated for many eukaryotic organisms, including yeasts, that both the lysosomal (vacuolar) and non-lysosomal (non-vacuolar) proteolytic systems are activated by nutrient starvation [8]. Thus intracellular proteolytic enzymes are likely to be involved in the metabolic transition from primary growth (trophophase) to idiophase triggered in the white-rot fungi by nutrient deprivation. Even though the synthesis of intracellular and extracellular proteases is a common feature among fungi [9], comparatively little study has been devoted to proteases of wood-degrading Basidiomycetes [10 –13]. Our previous study showed that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 0 1 0 - 8
538
M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
laccase and peroxidase activity in cultures of Trametes versicolor under nutrient limitation [14]. A major goal of the present study was to detect proteasome activity in the efficient lignin-degraders and to examine whether the specific agent (CbzLLLal, MG 132) that blocks the function of yeast and mammalian 26S proteasome [15], can affect the levels of ligninolytic enzymes of white-rot fungi. 2. Materials and methods 2.1. Culture conditions Mycelia of white-rot fungi Trametes versicolor (ATCC 44308, strain FCL 7) and Phlebia radiata (ATCC 64658, strain FCL 99a) were maintained, through periodic (every 7 days) inoculation with floating discs of mycelium (5 mm of the diameter) as surface cultures at 26°C, in scintillation flasks containing 10 ml of nutrient-rich growth medium [16] with glucose as a carbon source and L-asparagine as a nitrogen source. After the 7-day cultivation period, mycelia were transferred to fresh growth media deprived of glucose (carbon starvation) or L-asparagine (nitrogen starvation), and to trophophasic media. The trophophasic media contained glucose and asparagine of the same concentrations as those measured after seven days of fungal growth. Other conditions of starvation experiments were essentially those described previously [14]. Three independent experiments were performed in duplicate. 2.2. Effect of proteasome inactivation The proteasome inhibitor, MG132 (CbzLLLal) was added (to a final concentration of 40 M) at the time of transfer of mycelia to the nutrient-deprived or trophophasic media. An equivalent amount of solvent used (DMSO) was added to parallel sets of cultures (nutrient-deprived and trophophasic). At 6 h after exposure to the proteasome inhibitor, enzymatic activities were assayed by the methods described below. The results are expressed as a percentage of remaining activity. For each determination, the specific activity of samples from the parallel culture (carbon-deprived, nitrogen-deprived and trophophasic) without the inhibitor was taken to be 100%.
Potter’s homogenizer, in 2 ml of 0.05 M Tris-HCl buffer, pH 7.3. The homogenates were then centrifuged for 10 min at 10000⫻ g, at 4°C. The supernatants were desalted through a Sephadex G-25 column as described above. 2.5. Proteasome assays Proteasome activity was detected in mycelial extracts by cleavage of a fluorogenic peptide substrate (Suc-LLVYMCA) by the modified stopped assay procedure [17,18]. Briefly, assay mixtures containing 5–10 l of the mycelial extract, 100 M Suc-LLVY-MCA (in DMSO), and 100 mM Tris-HCl buffer, pH 8.0, were made up in a total volume of 100 l and then incubated at 37°C for 15 and 30 min. The fluorescent proteolysis product was quantified in a spectrofluorometer (FluoroMax-2, Instruments S.A., Inc., JOBIN YVON/SPEX Division, USA), at an emission wavelength of 440 nm, with excitation at 360 nm. Fluorescence units measured were converted to picomoles of MCA released by using a standard curve prepared with known dilutions of MCA in 5% DMSO in 100 mM Tris-HCl buffer, pH 8.0. 2.6. Determination of laccase activity (EC 1.10.3.2) Activity of laccase (benzenediol: oxygen oxidoreductase) was determined in desalted samples of culture filtrates and mycelial extracts according to [19] using syringaldazine as a substrate. 0.1 M citrate-NaOH buffer, pH 5.0 was used for determinations. 2.7. Determination of peroxidase (HRP-like) activity (EC 1.11.1.7) Peroxidase activity was assayed in desalted samples of culture filtrates and mycelial extracts according to [20]. 0.003% H2O2, 0.01% o-dianisidine, and 0.1 M citrateNaOH buffer, pH 5.0, were used for determinations. 2.8. Determination of protein Protein concentration was measured according to the method described by Lowry and modified by [21]. Bovine serum albumin was used as standard.
2.3. Preparation of extracellular culture fluid
2.9. Reagents
Extracellular samples were collected as described previously [14] by separating culture fluid from mycelium by filtration. The filtrates were desalted through a Sephadex G-25 column. The elution was performed with 0.001 M Tris, pH 7.0.
SucLLVY-MCA, 7-amino-4-methylcoumarin, CbzLLLal (MG132), DMSO, syringaldazine were obtained from SIGMA, St. Louis.
2.4. Preparation of mycelial extracts
3. Results and discussion
Mycelia (dry weight about 50 – 60 mg per flask) were harvested and homogenized in an ice-chilled motor-driven
White-rot fungi are being studied mainly because of their ligninolytic activity which appears during the nutritional
M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
539
shift from trophophase to idiophase triggered by carbon- or nitrogen starvation [22]. The wood-degrading fungi Trametes versicolor and Phlebia radiata are, similarly to the intensively studied fungus Phanerochaete chrysosporium, very efficient white-rotters in nature [23,24]. Our previous studies have demonstrated numerous intraand extracellular proteolytic enzymes, present both in nutrient-rich and carbon- or nitrogen-starved cultures of Trametes versicolor and Phlebia radiata [13]. Our experiments with PMSF (irreversible inhibitor of serine proteinases) and chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) have indicated that both intracellular and extracellular proteases can be involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation [14]. 3.1. Proteasome assays Experiments with mycelial extracts of T. versicolor and P. radiata showed the ability of these extracts to hydrolyze Suc-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (SucLLLVYMCA), a well known substrate used to detect proteasome activity. Proteasome activity was found in mycelial extracts from both trophophasic (non-starved) and starved cultures of these fungi (Fig. 1). In Eukarya, the 20S proteasome contains two chymotrypsin-like, two trypsin-like, and two active sites shown to have caspase-like specificity [25]. These sites cleave preferentially after large hydrophobic residues, basic residues, and acidic residues, respectively. Comparatively small differences in the levels of chymotrypsin-like (SucLLLVY-MCA-hydrolyzing) activity were found between starved and non-starved mycelia. The crude proteasome activity detected in mycelial extracts of T. versicolor and P. radiata was sensitive in vitro to MG132 (CbzLLLal), one of the more potent peptide aldehyde inhibitors of the proteasome. This agent inhibited the crude activity by about 20%. 3.2. Effect of proteasome inactivation Peptidyl aldehyde analogues are reversible proteasome inhibitors. The aldehyde moiety of these inhibitors forms a hemi-acetyl adduct with the nucleophilic hydroxyl group of the N-terminal threonine in the proteasome [26]. Most of the reported peptide aldehyde inhibitors of the proteasome have been directed against the chymotrypsin-like activity of the 20S proteasome. The specific agent, MG 132 (CbzLLLal) has been previously shown to inhibit purified yeast and mammalian 26S proteasome [15]. To assess its effect on the metabolism of white-rot fungi, MG132 was added to cultures of T. versicolor and P. radiata at the time of transfer of mycelia to the nutrient-deprived or trophophasic media. Figs. 2A–2C show the effect of the proteasome inhibitor MG132 (CbzLLLal) on activities of laccase and peroxidase in carbon-deprived, nitrogen-deprived, and trophophasic
Fig. 1. Activity of mycelial extracts from non-starved (square symbol), carbon-starved (diamond-symbol), and nitrogen-starved (triangle symbol) cultures of (A) Trametes versicolor and (B) Phlebia radiata, against the fluorogenic peptide substrate SucLLVY-MCA. Average of at least three independent experiments performed in triplicate.
cultures of T. versicolor, respectively. Changes in both extra- and intracellular activities in nutrient-starved cultures occurred as a result of MG132 addition. A significant decrease in laccase and peroxidase activities was observed in carbon-deprived cultures (Fig. 2A). The inhibitor addition under nitrogen limitation resulted in an approximately 7-fold and 5-fold increase in extracellular laccase and peroxiase activities respectively (Fig. 2B). In contrast to starved cultures, no significant effect could be detected for trophophasic cultures of T. versicolor after exposure to the proteasome inhibitor (Fig. 2C). The effect of MG132 addition to cultures of P. radiata was similar to that observed in the case of T. versicolor, however the changes in laccase and peroxidase activities found in starved cultures of P. radiata were not so strong (Fig. 3). The difference between the effect of MG132 in the carbon starvation response and in the nitrogen starvation response can be explained on the basis of the previously observed difference between the onset of ligninolytic activity triggered by carbohydrate or nitrogen starvation [7].
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M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
Fig. 2. Effect of proteasome inhibitor, MG 132 (CbzLLLal), on activities of extracellular laccase (shaded bars), extracellular peroxidase (black bars), intracellular laccase (empty bars), and intracellular peroxidase (hatched bars) in cultures of Trametes versicolor: (A) carbon-starved, (B) nitrogenstarved, and (C) trophophasic (non-starved). Data are means ⫾ SD from at least three independent experiments performed in duplicate.
Fig. 3. Effect of proteasome inhibitor, MG 132 (CbzLLLal), on activities of extracellular laccase (shaded bars), extracellular peroxidase (black bars), intracellular laccase (empty bars), and intracellular peroxidase (hatched bars) in cultures of Phlebia radiata: (A) carbon-starved, (B) nitrogenstarved, and (C) trophophasic (non-starved). Data are means ⫾ SD from at least three independent experiments performed in duplicate.
4. Conclusions
found to be very useful as inhibitors in studies clarifying the role of the proteasome in different intracellular degradative processes. The present study showed that in carbon-starved cultures of white-rot fungi, addition of the proteasome inhibitor (MG132) decreased laccase and peroxidase activities
The lack of specific inhibitors of the proteasome pathway has long been a major factor limiting an understanding of its function in vivo. Recently several peptide aldehydes were
M. Staszczak / Enzyme and Microbial Technology 30 (2002) 537–541
whereas in nitrogen-starved cultures, addition of MG132 increased activities of these enzymes. Although the present work constitutes only a preliminary step in studying proteasome-mediated proteolysis in lignin-degrading Basidiomycetes, the results obtained here strongly suggest that proteasomes exist in the white-rot fungi and play a role in the metabolism of these microorganisms.
Acknowledgments This work was partly supported by the State Committee for Scientific Research (KBN Grant-No 6 P04A 027 17, to M.S.), EC Contract ICA2-CT-2000 –10050, and BS/ BiNoZ/4.
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