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Regulation of Ceramide Signaling by Plasma Membrane Coenzyme Q Reductases
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[16] Regulation of Ceramide Signaling by Plasma Membrane Coenzyme Q Reductases By Pla´cido Navas and Jose´ Manuel Villalba Introduction
Lipid signaling in mammalian cells involves sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and GD3 ganglioside. Ceramide has received much attention, because an increase in ceramide levels initiates cellular responses for cell growth, differentiation, stress, and apoptosis.1 Ceramide accumulation in the cell can occur either from de novo synthesis or from the hydrolysis of sphingomyelin by different sphingomyelinases (SMase, sphingomyelin phosphodiesterase; E.C. 3.1.4.12). A lysosomal acidic pH-optimum SMase (aSMase), and a Mg2þ-dependent neutral SMase (nSMase), which is an integral plasma membrane protein, have been related to apoptosis triggered by stress-inducing agents. Although a role for the aSMase in cell signaling has been questioned, the nSMase is apparently the most important in ceramide signaling according to the number of agents stimulating this enzyme and its favorable position at the plasma membrane, where sphingomyelin is highly concentrated.2,3 Because of its important role in signal transduction and the continuous availability of its substrate sphingomyelin, the activity of signaling SMase must be strictly controlled in cells.4 Much is known about factors that activate nSMase such as TNF- , arachinodate, and phosphatidylserine, but little is known about inhibitors of this enzyme. These inhibitory factors would be valuable tools to better understand the role of the enzyme and its product ceramide in signal transduction.5 Plasma membrane contains an electron transport system that reduces coenzyme Q (CoQ) in this membrane, which guarantees its antioxidant properties but also mediates the regeneration of other primary antioxidants such as ascorbate and -tocopherol. Coenzyme Q seems to be the 1
Y. A. Hannun, Science 274, 1855 (1996). B. Se´gui, C. Bezombes, E. Uro-Coste, J. A. Medin, N. Andrieu-Abadie, N. Auge´, A. Brouchet, G. Laurent, R. Salvayre, J. P. Jaffre´zou, and T. Levade, FASEB J. 14, 36 (2000). 3 C. Bezombes, B. Se´gui, O. Cuvillier, A. P. Bruno, E. Uro-Coste, V. Gouaze´, N. AndrieuAbadie, S. Carpentier, G. Laurent, R. Salvayre, J. P. Jaffre´zou, and T. Levade, FASEB J. 15, 297 (2001). 4 K. Hofmann and V. M. Dixit, Trends Biochem. Sci. 23, 374 (1998). 5 C. C. Lindsey, C. Go´mez-Dı´az, J. M. Villalba, and T. R. R. Pettus, Tetrahedron 58, 4559 (2000). 2
METHODS IN ENZYMOLOGY, VOL. 378
Copyright 2004, Elsevier Inc. All rights reserved. 0076-6879/04 $35.00
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central component of this plasma membrane redox system6 but also the key factor in preventing apoptosis triggered by externally induced oxidative stress.7 Antagonists of CoQ such as short-chain ubiquinone analogues and capsaicin trigger the apoptotic program.8,9 Furthermore, the activation of trans-plasma membrane redox system by the depletion of mitochondrial DNA is due to the increase of CoQ content in the plasma membrane,10 which prevents both ceramide accumulation and apoptosis induced by serum removal.7 Ceramide acts as a mediator of stress responses modulating enzymes of the eicosanoid pathway, protein kinases, nuclear factors, and gene expression.11 Ceramide is also able to induce cell death after its intracellular accumulation1,12 by activating proteases of the caspase family such as caspase 3.13 We have recently documented that CoQ is an efficient noncompetitive inhibitor of the Mgþþ-dependent nSMase.14 CoQ prevents both ceramide accumulation and activation of caspase-3, protecting cells from apoptosis induced by serum withdrawal.15 Mitochondria have been related to a ceramide effect on caspase 3 activation16 and could be responsible for the activation observed in ceramide-dependent apoptosis induced by serum withdrawal.15 However, caspase 3 can be activated in mitochondria-free cytosol after its incubation with lipid extracts obtained from cells grown in serum-free media. When cells used to make the lipid extracts were grown either in the presence of 10% fetal calf serum or in a serum-free medium supplemented with CoQ, the activation in vitro of caspase 3 was not observed.17 6
F. Navarro, P. Navas, J. R. Burgess, R. I. Bello, R. de Cabo, A. Arroyo, and J. M. Villalba, FASEB J. 12, 1665 (1998). 7 M. P. Barroso, C. Go´ mez-Dı´az, J. M. Villalba, M. I. Buro´ n, G. Lo´ pez-Lluch, and P. Navas, J. Bioenerg. Biomembr. 29, 259 (1997). 8 E. J. Wolvetang, J. A. Larm, P. Moutsoulas, and A. Lawen, Cell Growth Differ. 7, 1315 (1996). 9 A. Macho, M. A. Calzado, J. Mun˜ oz-Blanco, C. Go´ mez-Dı´az, C. Gajate, F. Mollinedo, P. Navas, and E. Mun˜ oz, Cell Death Differ. 6, 155 (1999). 10 C. Go´ mez-Dı´az, J. M. Villalba, R. Pe´ rez-Vicente, F. L. Crane, and P. Navas, Biochem. Biophys. Res. Commun. 234, 79 (1997). 11 Y. A. Hannun, J. Biol. Chem. 269, 3125 (1994). 12 W. D. Jarvis, R. N. Kolesnick, F. A. Fornari, R. S. Traylor, D. A. Gewirtz, and S. Grant, Proc. Natl. Acad. Sci. USA 91, 73 (1994). 13 N. Mizushima, R. Koike, H. Kohsaka, Y. Kushi, S. Handa, H. Yagita, and N. Miyasaka, FEBS Lett. 395, 267 (1996). 14 S. F. Martı´n, F. Navarro, N. Forthoffer, P. Navas, and J. M. Villalba, J. Bioenerg. Biomembr. 33, 143 (2001). 15 P. Navas, D. M. Fernandez-Ayala, S. F. Martı´n, G. Lo´ pez-Lluch, R. de Cabo, J. C. Rodrı´guez-Aguilera, and J. M. Villalba, Free Radic. Res. 36, 369 (2002). 16 S. A. Susı´n, N. Zamzami, M. Castedo, E. Daugas, H.-G. Wang, S. Gelei, F. Fassy, J. C. Reed, and G. Kroemer, J. Exp. Med. 186, 25 (1997).
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These results suggest that CoQ may play a role in the regulation of the nSMase in vivo. The inhibition of nSMase in the plasma membrane is carried out more efficiently by the reduced form of CoQ (CoQH2, ubiquinol) and also depends on the length of the isoprenoid side chain.18 If inhibition of plasma membrane nSMase by ubiquinol has physiological significance, then endogenous levels of ubiquinol should also exert this regulatory action. Moreover, because endogenous CoQ can be reduced in plasma membranes by the intrinsic trans-membrane redox system, the activity of plasma membrane NAD(P)H-dependent dehydrogenases should also modulate the activity of nSMase. This function of CoQ occurs at the initiation phase of apoptosis by preventing the activation of the nSMase in the plasma membrane through the direct inhibition of this enzyme. The analysis of the CoQ-dependent regulation of nSMase in the plasma membrane requires a highly purified membrane fraction free of endomembrane contamination and particularly of mitochondrial membranes. The procedure of two-phase partition has been shown to be the best to obtain plasma membranes without mitochondrial contamination from epithelia and cultured cells, and these membranes can be then used to purify nSMase and to analyze the relationship of the activity of this enzyme and the intrinsic content of CoQ. We review here the methods that we have found more useful to study the role of CoQ and its reductases in the regulation of liver plasma membrane nSMase. Methods
Plasma Membrane Purification by Two-Phase Partition Liver tissue is homogenized in a blender or similar equipment in a homogenization medium (2 ml/g fresh weight) containing 37 mM Tris-maleate, pH 6.4; 0.5 M sucrose; 5 mM MgCl2; 5 mM -mercaptoethanol; 1 mM polymethylsulfonyl fluoride (PMSF); and 20 g/l each of chymostatin, leupeptin, antipain, and pepstatin A (CLAP). The homogenate is then centrifuged for 15 min at 5000g. The supernatant is discarded, and the light brown top portion of the pellet is resuspended in 5 ml of 1 mM sodium bicarbonate, a basic solution that has been found to be very effective to protect the functions of plasma membrane.19 After homogenization in a 17
D. J. M. Ferna´ ndez-Ayala, S. A. Martı´n, M. P. Barroso, C. Go´ mez-Dı´az, J. M. Villalba, J. C. Rodrı´guez-Aguilera, G. Lo´ pez-Lluch, and P. Navas, Antiox. Redox Signal 2, 263 (2000). 18 S. F. Martı´n, C. Go´ mez-Dı´az, P. Navas, and J. M. Villalba, Biochem. Biophys. Res. Commun. 297, 581 (2002). 19 P. Navas, D. D. Nowack, and D. J. Morre´ , Cancer Res. 49, 2147 (1989).
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Teflon-glass potter, the mixture is centrifuged again for 15 min at 5000g, and the top half of the resulting pellet is recovered to obtain a crude membrane fraction. Plasma membranes were isolated from crude fractions by the two-phase partition method,19 using a phase system composed of 6.0% (w/w) dextran T-500 (20% in water) (Pharmacia, Barcelona, Spain), 6.0% polyethylene glycol 3350 (40% water) (Fisher, Fair Lawn, NJ), in 0.1 M sucrose and 5 mM potassium phosphate, pH 7.2. The mixture must be inverted vigorously 40 times at 4 and centrifuged at 350g for 5 min to separate the phases. The upper phase, containing the plasma membrane, is then diluted in 25 ml of 1 mM sodium bicarbonate and recovered by centrifugation at 20,000g for 30 min. After this last centrifugation, plasma membranes are resuspended in 50 mM TRIS-HCl, pH 7.5, containing 10% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol (DTT), 1 mM PMSF, and 20 g/l CLAP, and stored at 80 . Purification of SMase from Plasma Membrane The entire purification process must be performed at 4 . Plasma membranes (about 23 mg in 11.5 ml) are extracted with 0.5 M KCl and 5 mM ethylenediaminetetraacetic acid (EDTA) for 30 min to remove peripheral proteins, and then the membrane residue is separated by ultracentrifugation at 100,000g for 90 min. The resulting pellet is then resuspended in 12 ml of 50 mM TRIS-HCl, pH 7.4, containing 2 mM EDTA, 0.25% 3[3-chloraminopropyl diethylammonio]-1-propane sulfonate (CHAPS), 5 mM dithiothreitol (DTT), 1 mM PMSF, and 20 g/ml CLAP. The detergent/plasma membrane mixture is rocked overnight and then centrifuged for 60 min at 100,000g to separate solubilized integral proteins. The proteins contained in the supernatant are used in a series of four-column chromatographies following the activity of nSMase in the elution profile. These chromatographies are as follows. Gel Filtration Chromatography. Solubilized proteins are fractionated in a column (2.6 60 cm) of Sephacryl S-300 HR. The gel is pre-equilibrated with 900 ml buffer A (5 mM TRIS-HCl, pH 7.4, 2 mM EDTA, 0.25% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP). A volume of 13 ml of the sample is loaded, and the column is eluted with 1 volume buffer A at 20 ml/h. Fractions of 6 ml are collected. The most active fractions containing nSMase activity that eluted into the included volume are pooled and used directly for the following chromatography step. Heparin-Sepharose Chromatography. Fractions from Sephacryl S-300 HR are loaded onto a 5-ml column of heparin sepharose-high performance, which has been pre-equilibrated with 50 ml buffer A. The column is washed with 50 ml of buffer A after sample loading, and then bound proteins are
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eluted with a 20-ml linear gradient of 0–100% buffer B (50 mM TRIS-HCl, pH 7.4, 2 M NaCl, 2 mM EDTA, 0.25% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP) at a flow rate of 30 ml/h. Fractions of 4 ml (2 ml during gradient elution) are now collected during sample loading and column washing. Anion-Exchange Chromatography. Pooled fractions from the heparinsepharose chromatography are previously desalted when needed by gel filtration in Sephadex G-25 (PD-10 columns) or directly added at 4 ml/h to a 1-ml HiTrap Q column pre-equilibrated with 20 ml buffer A. After washing the column with at least 10 column volumes of buffer A, bound proteins are eluted by a 20-ml linear gradient of 0–100% buffer C (50 mM TRIS-HCl, pH 7.4, 1 M NaCl, 2 mM EDTA, 0.25% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP). Fractions of 0.7 ml are collected during gradient elution. Hydrophobic Interaction Chromatography. Active fractions collected from anion exchange chromatography are finally applied to a 1-ml phenyl-sepharose high-performance column, which has been pre-equilibrated with 30 ml buffer C. After sample loading, the column is washed with buffer C, and then bound proteins are eluted with a 20-ml linear gradient of 0–100% buffer A. After this step, the column is washed again with 4 ml of buffer A, and bound proteins are then eluted with buffer D (50 mM TRIS-HCl pH 7.4, 2 M NaCl, 2 mM EDTA, 0.5% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP) at a flow rate of 30 ml/h. Fractions of 3 ml are collected during sample loading and column washing, of 0.6 ml during gradient elution, and of 1 ml during the last elution step. Purification must be monitored by both the determination of the nSMase activity in each chromatographical separation, following the protocol described in the next section, and by discontinuous sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% polyacrylamide) as described elsewhere.20 The analysis of the protein fraction with nSMase activity purified by this procedure has shown that it is mostly composed by the magnesium-dependent isoform,14 which is the only inhibited one by CoQ.18 Assay for nSMase Activity and Coenzyme Q Inhibition Mg2þ-dependent neutral SMase activity is assayed in a 50 mM TRISHCl buffer, pH 7.4, containing 0.05% Triton X-100 and 10 mM MgCl2. To exclude putative inhibitions caused by a shift from the optimal pH of the enzyme, the pH of assay buffer must be carefully controlled in each 20
U. K. Laemmli, Nature 277, 680 (1970).
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experiment in which the compounds used can modify the pH. Samples (50– 100 g plasma membrane fraction in 5–10 l, or 15–30 g purified nSMase, in 25–50 l of column eluates) are mixed with assay buffer plus 10 nmol of a mixture of cold sphingomyelin and [methyl-14C]-sphingomyelin (specific radioactivity 10,000 cpm/nmol). After incubation for 30–60 min at 37 , the reaction is stopped by adding 900 l chloroform/methanol (2:1) and 200 l distilled water. Tubes are vortexed and then centrifuged at 1500g for 5 min to achieve separation of phases. [14C]-phosphorylcholine present in the aqueous phase is quantified using a liquid scintillation counter. nSMase activity was expressed as cpm g protein1 min1. Studies of nSMase inhibition by CoQ/CoQH2 in plasma membrane were carried out by modifying the concentrations of these compounds in membrane samples.7,14,15,18 To obtain plasma membranes free of quinones, membrane samples (about 3 mg protein) are extracted after lyo philization with 1 ml of heptane for 6 h at 20 in the dark. Heptane is then decanted and evaporated, and membranes are resuspended in the nSMase assay buffer. The analysis with HPLC will be used to demonstrate that quinones have been removed. Supplementation with quinones is carried out by the incubation of membranes with ethanolic solutions of the corresponding quinone. The oxidized form is dissolved directly from a commercial source in pure ethanol, and a yellow solution is obtained. This is also used to produce the reduced form ubiquinol. To prepare CoQ10H2, oxidized CoQ10 is dissolved in ethanol at a 1 mM concentration, and then microliter amounts of a solution of sodium borohydride (10 mg/ml in water) are added until the yellow color of the CoQ10 solution is lost and a colorless mixture is obtained. A volume of 1 M NaCl and hexane are added, and the mixture is then vortexed for some seconds. Phases are separated by a brief centrifugation in a microfuge, and the upper hexane phase (containing the hydroquinone) is withdrawn and dried under a nitrogen stream. The resulting dried samples are resuspended in ethanol and have to be used immediately to prevent reoxidation. To test the effect of CoQ(H2) addition on nSMase activity, the required amount of oxidized or reduced CoQ (in ethanol) is added to a 1.5-ml tube, and the solvent is evaporated under a N2 stream. The dried antioxidant is dissolved in 50 l of assay buffer (which contains Triton X-100, see preceding), and the sample is then added. A stronger inhibition is achieved if membranes or purified nSMase are preincubated (up to 60 min at 37 ) in the presence of the antioxidant. Afterwards, another 50 l of assay buffer containing the substrate sphingomyelin is incorporated into the reaction mixture, and the assay is carried out as described previously.
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Conclusion
Ceramide is an important lipid-signaling molecule, because its intracellular accumulation triggers cell growth arrest and cell death.1,12 Ceramide can be accumulated by synthesis by ceramide synthase, but most is produced from the hydrolysis of sphingomyelin by a number of SMases.21 This sphingomyelin is an abundant component of the outer leaflet of the plasma membrane bilayer, and it is the most important candidate to be the source of ceramide as a consequence of an externally induced oxidative stress.15,22 It has been important to develop a procedure to purify the Mgþþdependent nSMase from plasma membrane with the guarantee that enzyme isoforms from other cellular membranes are not present.14,18 The use of fractions highly enriched in plasma membrane to study the properties of this enzyme has facilitated the understanding of its properties and also the tentative redox mechanism of its regulation, mainly its inhibition by CoQH2,18 which is recycled by NAD(P)H-dependent reductases at this membrane.6 Also, the purification of this enzyme has further clarified its inhibition by ubiquinol and opened new perspectives for the study of enzyme regulation mechanisms. Recently, inhibitors of the plasma membrane nSMase have attracted increasing interest because of their potential clinical relevance as therapeutic agents in the treatment of several diseases, such as immune and cardiovascular diseases. These inhibitors include scyphostatin,23 manumycin A,24 and F11334s,25 which interestingly display structural similarities with CoQ, increasing the importance of this quinone as a natural and specific regulator of the plasma membrane nSMase. Acknowledgment This work has been partially supported by the Spanish MCyT grants numbers BMC2002–01602 and BMC2002–01078.
21
G. S. Dbaibo, W. El-Assad, A. Krikorian, B. Liu, K. Diab, N. Z. Idriss, M. El-Sabban, T. A. Driscoll, D. K. Perry, and Y. A. Hannun, FEBS Lett. 503, 7 (2001). 22 N. Andrieu-Abadie, V. Gouaze´ , R. Salvayre, and T. Levade, Free Radic. Biol. Med. 31, 717 (2001). 23 F. Nara, M. Tanaka, S. Masuda-Inoue, Y. Yamasato, H. Doi-Yoshioka, K. Sukuki-Konagai, S. Kumakura, and T. Ogita, J. Antibiot. (Tokyo) 52, 531 (1999). 24 C. Arenz, M. Thutewohl, O. Block, H. Waldmann, H. J. Altenbach, and A. Giannis, Chembiochem. 2, 141 (2001). 25 M. Tanaka, F. Nara, Y. Yamasato, Y. Ono, and T. Ogita, J. Antibiot. (Tokyo) 52, 827 (1999).
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plasma membrane quinone reductases
[16] Regulation of Ceramide Signaling by Plasma Membrane Coenzyme Q Reductases By Pla´cido Navas and Jose´ Manuel Villalba Introduction
Lipid signaling in mammalian cells involves sphingolipids such as sphingomyelin, sphingosine-1-phosphate, and GD3 ganglioside. Ceramide has received much attention, because an increase in ceramide levels initiates cellular responses for cell growth, differentiation, stress, and apoptosis.1 Ceramide accumulation in the cell can occur either from de novo synthesis or from the hydrolysis of sphingomyelin by different sphingomyelinases (SMase, sphingomyelin phosphodiesterase; E.C. 3.1.4.12). A lysosomal acidic pH-optimum SMase (aSMase), and a Mg2þ-dependent neutral SMase (nSMase), which is an integral plasma membrane protein, have been related to apoptosis triggered by stress-inducing agents. Although a role for the aSMase in cell signaling has been questioned, the nSMase is apparently the most important in ceramide signaling according to the number of agents stimulating this enzyme and its favorable position at the plasma membrane, where sphingomyelin is highly concentrated.2,3 Because of its important role in signal transduction and the continuous availability of its substrate sphingomyelin, the activity of signaling SMase must be strictly controlled in cells.4 Much is known about factors that activate nSMase such as TNF- , arachinodate, and phosphatidylserine, but little is known about inhibitors of this enzyme. These inhibitory factors would be valuable tools to better understand the role of the enzyme and its product ceramide in signal transduction.5 Plasma membrane contains an electron transport system that reduces coenzyme Q (CoQ) in this membrane, which guarantees its antioxidant properties but also mediates the regeneration of other primary antioxidants such as ascorbate and -tocopherol. Coenzyme Q seems to be the 1
Y. A. Hannun, Science 274, 1855 (1996). B. Se´gui, C. Bezombes, E. Uro-Coste, J. A. Medin, N. Andrieu-Abadie, N. Auge´, A. Brouchet, G. Laurent, R. Salvayre, J. P. Jaffre´zou, and T. Levade, FASEB J. 14, 36 (2000). 3 C. Bezombes, B. Se´gui, O. Cuvillier, A. P. Bruno, E. Uro-Coste, V. Gouaze´, N. AndrieuAbadie, S. Carpentier, G. Laurent, R. Salvayre, J. P. Jaffre´zou, and T. Levade, FASEB J. 15, 297 (2001). 4 K. Hofmann and V. M. Dixit, Trends Biochem. Sci. 23, 374 (1998). 5 C. C. Lindsey, C. Go´mez-Dı´az, J. M. Villalba, and T. R. R. Pettus, Tetrahedron 58, 4559 (2000). 2
METHODS IN ENZYMOLOGY, VOL. 378
Copyright 2004, Elsevier Inc. All rights reserved. 0076-6879/04 $35.00
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central component of this plasma membrane redox system6 but also the key factor in preventing apoptosis triggered by externally induced oxidative stress.7 Antagonists of CoQ such as short-chain ubiquinone analogues and capsaicin trigger the apoptotic program.8,9 Furthermore, the activation of trans-plasma membrane redox system by the depletion of mitochondrial DNA is due to the increase of CoQ content in the plasma membrane,10 which prevents both ceramide accumulation and apoptosis induced by serum removal.7 Ceramide acts as a mediator of stress responses modulating enzymes of the eicosanoid pathway, protein kinases, nuclear factors, and gene expression.11 Ceramide is also able to induce cell death after its intracellular accumulation1,12 by activating proteases of the caspase family such as caspase 3.13 We have recently documented that CoQ is an efficient noncompetitive inhibitor of the Mgþþ-dependent nSMase.14 CoQ prevents both ceramide accumulation and activation of caspase-3, protecting cells from apoptosis induced by serum withdrawal.15 Mitochondria have been related to a ceramide effect on caspase 3 activation16 and could be responsible for the activation observed in ceramide-dependent apoptosis induced by serum withdrawal.15 However, caspase 3 can be activated in mitochondria-free cytosol after its incubation with lipid extracts obtained from cells grown in serum-free media. When cells used to make the lipid extracts were grown either in the presence of 10% fetal calf serum or in a serum-free medium supplemented with CoQ, the activation in vitro of caspase 3 was not observed.17 6
F. Navarro, P. Navas, J. R. Burgess, R. I. Bello, R. de Cabo, A. Arroyo, and J. M. Villalba, FASEB J. 12, 1665 (1998). 7 M. P. Barroso, C. Go´ mez-Dı´az, J. M. Villalba, M. I. Buro´ n, G. Lo´ pez-Lluch, and P. Navas, J. Bioenerg. Biomembr. 29, 259 (1997). 8 E. J. Wolvetang, J. A. Larm, P. Moutsoulas, and A. Lawen, Cell Growth Differ. 7, 1315 (1996). 9 A. Macho, M. A. Calzado, J. Mun˜ oz-Blanco, C. Go´ mez-Dı´az, C. Gajate, F. Mollinedo, P. Navas, and E. Mun˜ oz, Cell Death Differ. 6, 155 (1999). 10 C. Go´ mez-Dı´az, J. M. Villalba, R. Pe´ rez-Vicente, F. L. Crane, and P. Navas, Biochem. Biophys. Res. Commun. 234, 79 (1997). 11 Y. A. Hannun, J. Biol. Chem. 269, 3125 (1994). 12 W. D. Jarvis, R. N. Kolesnick, F. A. Fornari, R. S. Traylor, D. A. Gewirtz, and S. Grant, Proc. Natl. Acad. Sci. USA 91, 73 (1994). 13 N. Mizushima, R. Koike, H. Kohsaka, Y. Kushi, S. Handa, H. Yagita, and N. Miyasaka, FEBS Lett. 395, 267 (1996). 14 S. F. Martı´n, F. Navarro, N. Forthoffer, P. Navas, and J. M. Villalba, J. Bioenerg. Biomembr. 33, 143 (2001). 15 P. Navas, D. M. Fernandez-Ayala, S. F. Martı´n, G. Lo´ pez-Lluch, R. de Cabo, J. C. Rodrı´guez-Aguilera, and J. M. Villalba, Free Radic. Res. 36, 369 (2002). 16 S. A. Susı´n, N. Zamzami, M. Castedo, E. Daugas, H.-G. Wang, S. Gelei, F. Fassy, J. C. Reed, and G. Kroemer, J. Exp. Med. 186, 25 (1997).
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These results suggest that CoQ may play a role in the regulation of the nSMase in vivo. The inhibition of nSMase in the plasma membrane is carried out more efficiently by the reduced form of CoQ (CoQH2, ubiquinol) and also depends on the length of the isoprenoid side chain.18 If inhibition of plasma membrane nSMase by ubiquinol has physiological significance, then endogenous levels of ubiquinol should also exert this regulatory action. Moreover, because endogenous CoQ can be reduced in plasma membranes by the intrinsic trans-membrane redox system, the activity of plasma membrane NAD(P)H-dependent dehydrogenases should also modulate the activity of nSMase. This function of CoQ occurs at the initiation phase of apoptosis by preventing the activation of the nSMase in the plasma membrane through the direct inhibition of this enzyme. The analysis of the CoQ-dependent regulation of nSMase in the plasma membrane requires a highly purified membrane fraction free of endomembrane contamination and particularly of mitochondrial membranes. The procedure of two-phase partition has been shown to be the best to obtain plasma membranes without mitochondrial contamination from epithelia and cultured cells, and these membranes can be then used to purify nSMase and to analyze the relationship of the activity of this enzyme and the intrinsic content of CoQ. We review here the methods that we have found more useful to study the role of CoQ and its reductases in the regulation of liver plasma membrane nSMase. Methods
Plasma Membrane Purification by Two-Phase Partition Liver tissue is homogenized in a blender or similar equipment in a homogenization medium (2 ml/g fresh weight) containing 37 mM Tris-maleate, pH 6.4; 0.5 M sucrose; 5 mM MgCl2; 5 mM -mercaptoethanol; 1 mM polymethylsulfonyl fluoride (PMSF); and 20 g/l each of chymostatin, leupeptin, antipain, and pepstatin A (CLAP). The homogenate is then centrifuged for 15 min at 5000g. The supernatant is discarded, and the light brown top portion of the pellet is resuspended in 5 ml of 1 mM sodium bicarbonate, a basic solution that has been found to be very effective to protect the functions of plasma membrane.19 After homogenization in a 17
D. J. M. Ferna´ ndez-Ayala, S. A. Martı´n, M. P. Barroso, C. Go´ mez-Dı´az, J. M. Villalba, J. C. Rodrı´guez-Aguilera, G. Lo´ pez-Lluch, and P. Navas, Antiox. Redox Signal 2, 263 (2000). 18 S. F. Martı´n, C. Go´ mez-Dı´az, P. Navas, and J. M. Villalba, Biochem. Biophys. Res. Commun. 297, 581 (2002). 19 P. Navas, D. D. Nowack, and D. J. Morre´ , Cancer Res. 49, 2147 (1989).
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Teflon-glass potter, the mixture is centrifuged again for 15 min at 5000g, and the top half of the resulting pellet is recovered to obtain a crude membrane fraction. Plasma membranes were isolated from crude fractions by the two-phase partition method,19 using a phase system composed of 6.0% (w/w) dextran T-500 (20% in water) (Pharmacia, Barcelona, Spain), 6.0% polyethylene glycol 3350 (40% water) (Fisher, Fair Lawn, NJ), in 0.1 M sucrose and 5 mM potassium phosphate, pH 7.2. The mixture must be inverted vigorously 40 times at 4 and centrifuged at 350g for 5 min to separate the phases. The upper phase, containing the plasma membrane, is then diluted in 25 ml of 1 mM sodium bicarbonate and recovered by centrifugation at 20,000g for 30 min. After this last centrifugation, plasma membranes are resuspended in 50 mM TRIS-HCl, pH 7.5, containing 10% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol (DTT), 1 mM PMSF, and 20 g/l CLAP, and stored at 80 . Purification of SMase from Plasma Membrane The entire purification process must be performed at 4 . Plasma membranes (about 23 mg in 11.5 ml) are extracted with 0.5 M KCl and 5 mM ethylenediaminetetraacetic acid (EDTA) for 30 min to remove peripheral proteins, and then the membrane residue is separated by ultracentrifugation at 100,000g for 90 min. The resulting pellet is then resuspended in 12 ml of 50 mM TRIS-HCl, pH 7.4, containing 2 mM EDTA, 0.25% 3[3-chloraminopropyl diethylammonio]-1-propane sulfonate (CHAPS), 5 mM dithiothreitol (DTT), 1 mM PMSF, and 20 g/ml CLAP. The detergent/plasma membrane mixture is rocked overnight and then centrifuged for 60 min at 100,000g to separate solubilized integral proteins. The proteins contained in the supernatant are used in a series of four-column chromatographies following the activity of nSMase in the elution profile. These chromatographies are as follows. Gel Filtration Chromatography. Solubilized proteins are fractionated in a column (2.6 60 cm) of Sephacryl S-300 HR. The gel is pre-equilibrated with 900 ml buffer A (5 mM TRIS-HCl, pH 7.4, 2 mM EDTA, 0.25% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP). A volume of 13 ml of the sample is loaded, and the column is eluted with 1 volume buffer A at 20 ml/h. Fractions of 6 ml are collected. The most active fractions containing nSMase activity that eluted into the included volume are pooled and used directly for the following chromatography step. Heparin-Sepharose Chromatography. Fractions from Sephacryl S-300 HR are loaded onto a 5-ml column of heparin sepharose-high performance, which has been pre-equilibrated with 50 ml buffer A. The column is washed with 50 ml of buffer A after sample loading, and then bound proteins are
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eluted with a 20-ml linear gradient of 0–100% buffer B (50 mM TRIS-HCl, pH 7.4, 2 M NaCl, 2 mM EDTA, 0.25% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP) at a flow rate of 30 ml/h. Fractions of 4 ml (2 ml during gradient elution) are now collected during sample loading and column washing. Anion-Exchange Chromatography. Pooled fractions from the heparinsepharose chromatography are previously desalted when needed by gel filtration in Sephadex G-25 (PD-10 columns) or directly added at 4 ml/h to a 1-ml HiTrap Q column pre-equilibrated with 20 ml buffer A. After washing the column with at least 10 column volumes of buffer A, bound proteins are eluted by a 20-ml linear gradient of 0–100% buffer C (50 mM TRIS-HCl, pH 7.4, 1 M NaCl, 2 mM EDTA, 0.25% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP). Fractions of 0.7 ml are collected during gradient elution. Hydrophobic Interaction Chromatography. Active fractions collected from anion exchange chromatography are finally applied to a 1-ml phenyl-sepharose high-performance column, which has been pre-equilibrated with 30 ml buffer C. After sample loading, the column is washed with buffer C, and then bound proteins are eluted with a 20-ml linear gradient of 0–100% buffer A. After this step, the column is washed again with 4 ml of buffer A, and bound proteins are then eluted with buffer D (50 mM TRIS-HCl pH 7.4, 2 M NaCl, 2 mM EDTA, 0.5% CHAPS, 5 mM DTT, 1 mM PMSF, and 1 g/ml CLAP) at a flow rate of 30 ml/h. Fractions of 3 ml are collected during sample loading and column washing, of 0.6 ml during gradient elution, and of 1 ml during the last elution step. Purification must be monitored by both the determination of the nSMase activity in each chromatographical separation, following the protocol described in the next section, and by discontinuous sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% polyacrylamide) as described elsewhere.20 The analysis of the protein fraction with nSMase activity purified by this procedure has shown that it is mostly composed by the magnesium-dependent isoform,14 which is the only inhibited one by CoQ.18 Assay for nSMase Activity and Coenzyme Q Inhibition Mg2þ-dependent neutral SMase activity is assayed in a 50 mM TRISHCl buffer, pH 7.4, containing 0.05% Triton X-100 and 10 mM MgCl2. To exclude putative inhibitions caused by a shift from the optimal pH of the enzyme, the pH of assay buffer must be carefully controlled in each 20
U. K. Laemmli, Nature 277, 680 (1970).
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experiment in which the compounds used can modify the pH. Samples (50– 100 g plasma membrane fraction in 5–10 l, or 15–30 g purified nSMase, in 25–50 l of column eluates) are mixed with assay buffer plus 10 nmol of a mixture of cold sphingomyelin and [methyl-14C]-sphingomyelin (specific radioactivity 10,000 cpm/nmol). After incubation for 30–60 min at 37 , the reaction is stopped by adding 900 l chloroform/methanol (2:1) and 200 l distilled water. Tubes are vortexed and then centrifuged at 1500g for 5 min to achieve separation of phases. [14C]-phosphorylcholine present in the aqueous phase is quantified using a liquid scintillation counter. nSMase activity was expressed as cpm g protein1 min1. Studies of nSMase inhibition by CoQ/CoQH2 in plasma membrane were carried out by modifying the concentrations of these compounds in membrane samples.7,14,15,18 To obtain plasma membranes free of quinones, membrane samples (about 3 mg protein) are extracted after lyo philization with 1 ml of heptane for 6 h at 20 in the dark. Heptane is then decanted and evaporated, and membranes are resuspended in the nSMase assay buffer. The analysis with HPLC will be used to demonstrate that quinones have been removed. Supplementation with quinones is carried out by the incubation of membranes with ethanolic solutions of the corresponding quinone. The oxidized form is dissolved directly from a commercial source in pure ethanol, and a yellow solution is obtained. This is also used to produce the reduced form ubiquinol. To prepare CoQ10H2, oxidized CoQ10 is dissolved in ethanol at a 1 mM concentration, and then microliter amounts of a solution of sodium borohydride (10 mg/ml in water) are added until the yellow color of the CoQ10 solution is lost and a colorless mixture is obtained. A volume of 1 M NaCl and hexane are added, and the mixture is then vortexed for some seconds. Phases are separated by a brief centrifugation in a microfuge, and the upper hexane phase (containing the hydroquinone) is withdrawn and dried under a nitrogen stream. The resulting dried samples are resuspended in ethanol and have to be used immediately to prevent reoxidation. To test the effect of CoQ(H2) addition on nSMase activity, the required amount of oxidized or reduced CoQ (in ethanol) is added to a 1.5-ml tube, and the solvent is evaporated under a N2 stream. The dried antioxidant is dissolved in 50 l of assay buffer (which contains Triton X-100, see preceding), and the sample is then added. A stronger inhibition is achieved if membranes or purified nSMase are preincubated (up to 60 min at 37 ) in the presence of the antioxidant. Afterwards, another 50 l of assay buffer containing the substrate sphingomyelin is incorporated into the reaction mixture, and the assay is carried out as described previously.
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Conclusion
Ceramide is an important lipid-signaling molecule, because its intracellular accumulation triggers cell growth arrest and cell death.1,12 Ceramide can be accumulated by synthesis by ceramide synthase, but most is produced from the hydrolysis of sphingomyelin by a number of SMases.21 This sphingomyelin is an abundant component of the outer leaflet of the plasma membrane bilayer, and it is the most important candidate to be the source of ceramide as a consequence of an externally induced oxidative stress.15,22 It has been important to develop a procedure to purify the Mgþþdependent nSMase from plasma membrane with the guarantee that enzyme isoforms from other cellular membranes are not present.14,18 The use of fractions highly enriched in plasma membrane to study the properties of this enzyme has facilitated the understanding of its properties and also the tentative redox mechanism of its regulation, mainly its inhibition by CoQH2,18 which is recycled by NAD(P)H-dependent reductases at this membrane.6 Also, the purification of this enzyme has further clarified its inhibition by ubiquinol and opened new perspectives for the study of enzyme regulation mechanisms. Recently, inhibitors of the plasma membrane nSMase have attracted increasing interest because of their potential clinical relevance as therapeutic agents in the treatment of several diseases, such as immune and cardiovascular diseases. These inhibitors include scyphostatin,23 manumycin A,24 and F11334s,25 which interestingly display structural similarities with CoQ, increasing the importance of this quinone as a natural and specific regulator of the plasma membrane nSMase. Acknowledgment This work has been partially supported by the Spanish MCyT grants numbers BMC2002–01602 and BMC2002–01078.
21
G. S. Dbaibo, W. El-Assad, A. Krikorian, B. Liu, K. Diab, N. Z. Idriss, M. El-Sabban, T. A. Driscoll, D. K. Perry, and Y. A. Hannun, FEBS Lett. 503, 7 (2001). 22 N. Andrieu-Abadie, V. Gouaze´ , R. Salvayre, and T. Levade, Free Radic. Biol. Med. 31, 717 (2001). 23 F. Nara, M. Tanaka, S. Masuda-Inoue, Y. Yamasato, H. Doi-Yoshioka, K. Sukuki-Konagai, S. Kumakura, and T. Ogita, J. Antibiot. (Tokyo) 52, 531 (1999). 24 C. Arenz, M. Thutewohl, O. Block, H. Waldmann, H. J. Altenbach, and A. Giannis, Chembiochem. 2, 141 (2001). 25 M. Tanaka, F. Nara, Y. Yamasato, Y. Ono, and T. Ogita, J. Antibiot. (Tokyo) 52, 827 (1999).