Role of ERAB/l-3-Hydroxyacyl-coenzyme A Dehydrogenase Type II Activity in Aβ-induced Cytotoxicity
1999; Elsevier BV; Volume: 274; Issue: 4 Linguagem: Inglês
10.1074/jbc.274.4.2145
ISSN1083-351X
AutoresShi Du Yan, Yigong Shi, Aiping Zhu, Jin Fu, Huaijie Zhu, Yucui Zhu, Lenneen Gibson, Eric Stern, Kate S. Collison, Futwan Al‐Mohanna, Satoshi Ogawa, Alex E. Roher, Steven Clarke, David M. Stern,
Tópico(s)Ginkgo biloba and Cashew Applications
ResumoEndoplasmic reticulum-associated amyloid β-peptide (Aβ)-binding protein (ERAB)/l-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Aβ, and contributes to Aβ-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a K m of ≈68 μm and aV max of ≈430 μmol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Aβ-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant β-amyloid precursor protein (βAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and βAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2–C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17β-estradiol. Addition of micromolar levels of synthetic Aβ(1–40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (K i ≈ 1.6 μm), as well as oxidation of 17β-estradiol (K i ≈3.2 μm) and (−)-2-octanol (K i ≈ 2.6 μm). Because micromolar levels of Aβ were required to inhibit ERAB/HADH II activity, whereas Aβ binding to ERAB/HADH II occurred at much lower concentrations (K m ≈ 40–70 nm), the latter more closely simulating Aβ levels within cells, Aβ perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and βAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or βAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and βAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Aβ-rich environment. Endoplasmic reticulum-associated amyloid β-peptide (Aβ)-binding protein (ERAB)/l-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Aβ, and contributes to Aβ-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a K m of ≈68 μm and aV max of ≈430 μmol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Aβ-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant β-amyloid precursor protein (βAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and βAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2–C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17β-estradiol. Addition of micromolar levels of synthetic Aβ(1–40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (K i ≈ 1.6 μm), as well as oxidation of 17β-estradiol (K i ≈3.2 μm) and (−)-2-octanol (K i ≈ 2.6 μm). Because micromolar levels of Aβ were required to inhibit ERAB/HADH II activity, whereas Aβ binding to ERAB/HADH II occurred at much lower concentrations (K m ≈ 40–70 nm), the latter more closely simulating Aβ levels within cells, Aβ perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and βAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or βAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and βAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Aβ-rich environment. amyloid β-peptide Alzheimer's disease endoplasmic reticulum-associated Aβ-binding protein l-3-hydroxyacyl-CoA dehydrogenase type II 4-hydroxynonenal malondialdehyde mutant protein disulfide isomerase wild-type relative apoptosis index polyacrylamide gel electrophoresis enzyme-linked immunosorbent assay terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling 4-morpholineethanesulfonic acid. Recent studies of mutations underlying familial Alzheimer's disease have strengthened links between amyloid β-peptide (Aβ)1 and the pathogenesis of this devastating neurodegenerative disorder (1Sisodia S.S. Price D.L. FASEB J. 1995; 9: 366-370Crossref PubMed Scopus (231) Google Scholar, 2Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (838) Google Scholar, 3Morrison J. Hof P. 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ERAB was localized to the endoplasmic reticulum and mitochondria in cultured cells (15Yan S.-D. Soto C. Chen X. Zhu H. Al-Mohanna F. Collison K. Zhu A. Stern E. Saido T. Tohyama M. Ogawa S. Roher A. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (350) Google Scholar). Its expression was found to be increased in AD brain, and, in vitro, ERAB facilitated Aβ cytotoxicity in neuroblastoma and transfected COS cells. Our initial analysis of the ERAB amino acid sequence showed resemblance to the family of short-chain alcohol dehydrogenases, including hydroxysteroid dehydrogenases, Ke6, and acetoacetyl coenzyme A (CoA) reductases (16Schembri M. Bayly R. Davies J. J. Bacteriol. 1995; 177: 4501-4507Crossref PubMed Google Scholar). These results suggested a primary role for ERAB in cell metabolism, in addition to having the potential to contribute to Aβ-induced cytotoxicity. The bovine counterpart of ERAB was recently isolated from liver mitochondrial preparations, and characterized as the mitochondrial 3-hydroxyacyl-CoA dehydrogenase type II (HADH II) (E.C. 1.1.1.35) (17Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar,18Kobayashi A. Jiang L. Hashimoto T. J. Biochem. 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar), a participant in the third reaction of the fatty acid β-oxidation spiral (19Roe C. Coates P. Scriver C. Beaudet L. Sly W. Vaye D. The Metabolic Basis of Inherited Disease. McGraw-Hill, Inc., New York1995: 1501-1533Google Scholar, 20Eaton S. Barlett K. Pourfarzam M. Biochem. J. 1996; 320: 345-357Crossref PubMed Scopus (353) Google Scholar). This identification is consistent with a recent report concerning properties of a humanl-3-hydroxyacyl-CoA dehydrogenase (21He X.-Y. Schulz H. Yang S.-Y. J. Biol. Chem. 1998; 273: 10741-10746Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), the cDNA sequence of which is identical to that of human ERAB. 2ERAB was the name applied to HADH II before their identity was ascertained. The designation ERAB/HADH II is used to emphasize the properties of ERAB with respect to binding of Aβ and potentiation of Aβ toxicity, as well as its metabolic function as a 3-hydroxyacyl-CoA dehydrogenase. We propose to rename the enzyme Aβ binding alcohol dehydrogenase as this reflects both its capacity to bind Aβ and its activity as a generalized alcohol dehydrogenase (see text). Although consequences of ERAB/HADH II deficiency are not known, heritable disorders with defects in fatty acid β-oxidation have been identified (19Roe C. Coates P. Scriver C. Beaudet L. Sly W. Vaye D. The Metabolic Basis of Inherited Disease. McGraw-Hill, Inc., New York1995: 1501-1533Google Scholar, 20Eaton S. Barlett K. Pourfarzam M. Biochem. J. 1996; 320: 345-357Crossref PubMed Scopus (353) Google Scholar). These patients have hepatomegaly, cardiomegaly, encephalopathies, peripheral neuropathy, rhabdomyolysis, and myoglobinuria, suggesting a role for fatty acid β-oxidation enzymes in the metabolic balance of a range of organs. To investigate whether the enzymatic activity of ERAB/HADH II was correlated with its interaction with Aβ and cytotoxicity, we have further characterized this protein. We find that it has the ability to catalyze the oxidation of alcohol groups in a range of substrates, including linear alcohols and estradiol, as well as the reduction ofS-acetoacetyl-CoA. To analyze the role of ERAB/HADH II enzymatic activity in potentiation of Aβ toxicity, a catalytically crippled form was prepared containing two substitutions in the highly conserved sequence of residues 168–172 (YSASK), putatively assigned as part of the active center of the enzyme (16Schembri M. Bayly R. Davies J. J. Bacteriol. 1995; 177: 4501-4507Crossref PubMed Google Scholar). Cells overexpressing mutant ERAB were relatively protected from Aβ cytotoxicity consequent to overexpression of mutant βAPP(V717G), as demonstrated by suppression of apoptosis. In contrast, cells overexpressing both wild-type ERAB/HADH II and βAPP (V717G) suffered increased cytotoxicity. Thus, it appears that activity of the enzyme is necessary for full toxicity. In cells overexpressing ERAB/HADH II and βAPP(V717G), the intracellular distribution of ERAB/HADH II was altered, and generation of reactive aldehydes, including malondialdehyde and 4-hydroxynonenal, occurred. Such reactive aldehydes provide a barometer of oxidant cell stress (22Rosenfeld M. Palinski W. Herttula S. Butler S. Witztum J. Arteriosclerosis. 1990; 10: 336-349Crossref PubMed Scopus (426) Google Scholar, 23Palinski W. Herttula S. Rosenfeld M. Butler S. Socher S. Parthasarathy S. Curtiss L. Witztum J. 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Escherichia coli (BL21) was transformed with pGE5-human ERAB/HADH II or mutant forms of ERAB/HADH II, prepared as described below. Transformants were induced with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside for 3 h, and cell extracts were prepared by cell disruption. Extracts were subjected to cation exchange FPLC chromatography on SP Sepharose Fast Flow (Amersham Pharmacia Biotech) and on Source 15S, followed by gel filtration on Superdex 200. The extract from ∼1 liter of bacterial culture was applied to 2 ml of SP Sepharose in 25 mm MES (pH 6.0), 50 mm NaCl, 5 mm dithiothreitol. The resin was washed with equilibration buffer and eluted with an ascending linear salt gradient (0.1–1.0 m NaCl). ERAB/HADH II, detected by its migration on SDS-PAGE and by immunoblotting (see below), eluted in fractions corresponding to 0.15–0.4 mNaCl. These fractions were pooled, diluted 6-fold, and applied to Source 15S resin in 0.1 m MES (pH 6.0)/0.1 mNaCl (5 mg of protein per 1 ml of resin). The column was eluted with an ascending salt gradient, and ERAB/HADH II emerged at ≈0.15m NaCl. ERAB/HADH II-rich fractions were concentrated by ultrafiltration to ≈15 mg/ml and loaded onto a Superdex 200 (30/10) column (1 ml was applied to the column for each run). Peak fractions from Superdex 200 were subjected to SDS-PAGE (12%) and immunoblotting. Immunodetection of ERAB/HADH II employed as primary antibody either anti-ERAB peptide IgG (15Yan S.-D. Soto C. Chen X. Zhu H. Al-Mohanna F. Collison K. Zhu A. Stern E. Saido T. Tohyama M. Ogawa S. Roher A. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (350) Google Scholar) or antibody prepared in rabbits, according to standard methods (33Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York1988Google Scholar), using full-length recombinant human ERAB/HADH II as the immunogen. IgG was purified from rabbit antisera by chromatography on Protein A-Sepharose CL-4B (Amersham Pharmacia Biotech). Sites of primary antibody binding were visualized with peroxidase-conjugated goat anti-rabbit IgG (Sigma). The ERAB/HADH II immunoreactive band migrated with ≈27 kDa, consistent with previous observations (15Yan S.-D. Soto C. Chen X. Zhu H. Al-Mohanna F. Collison K. Zhu A. Stern E. Saido T. Tohyama M. Ogawa S. Roher A. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (350) Google Scholar, 17Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar, 18Kobayashi A. Jiang L. Hashimoto T. J. Biochem. 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar, 53Pahl H. Baeuerle P. Trends Biochem. Sci. 1997; 22: 63-67Abstract Full Text PDF PubMed Scopus (296) Google Scholar). N-terminal sequencing of wild-type and mutant forms of ERAB/HADH II was performed on a Porton 2090 E gas phase protein sequencer (Beckman) equipped with an on-line Hewlett-Packard 1090 HPLC. Site-directed mutagenesis was employed to mutate tyrosine (168) and/or lysine (172) to glycine using a kit from Promega (Madison, WI). ERAB/HADH II was studied for its activity to reduceS-acetoacetyl-CoA, as well as its capacity to dehydrogenate alcohol groups in a range of linear alcohols and in estradiol. The assay for reduction of S-acetoacetyl-CoA employed ERAB/HADH II (333 ng/ml), a range of S-acetoacetyl-CoA concentrations (0.0015–0.36 mm; Sigma), and NADH (0.1 mm; Sigma) in 97 mm potassium phosphate (pH 7.3). The reaction was run for a total of 2 h at 25 °C under steady-state conditions (34Segel I.H. Enzyme Kinetics, Behavior, and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience, New York1975Google Scholar), and the change in NADH absorbance at 340 nm was determined every 5 min. Alcohol dehydrogenase assays employed ERAB/HADH II (20 μg/ml), a range of alcohol substrates and concentrations (methanol, ethanol, n-propanol, isopropanol,n-butanol, isobutanol, n-pentanol, (±)-2-octanol, (+)-2-octanol, (−)-2-octanol, andn-decanol; Sigma), and NAD+ (7.5 mm) in 22 mm sodium pyrophosphate, 0.3 mm sodium phosphate (pH 8.8). The reaction was run for 2 h at 25 °C, and the absorbance at 340 nm was monitored every 5 min as described above. Studies to evaluate oxidation of 17β-estradiol employed ERAB/HADH II (30 μg/ml), a range of 17β-estradiol concentrations (3.8–92 μm), and NAD+ (0.4 mm) in 20 mm sodium pyrophosphate (pH 8.9) at 25 °C for 2 h. Where indicated, freshly prepared synthetic Aβ(1–40) or Aβ(1–42), either obtained from California Peptide Inc. (Napa, CA) and purified by reversed-phase and size exclusion high pressure liquid chromatography (purity was confirmed by mass spectrometry, amino acid analysis and peptide mapping) or purchased in purified form from QCB (Hopkinton, MA) was added to the reaction mixture. Aβ(1–40) was freshly prepared and dissolved in distilled water. Aβ(1–42) was also freshly prepared and dissolved in Me2SO. The final concentration of Me2SO was 90% precipitable in trichloroacetic acid (10%). Wells were incubated overnight at 4 °C with Aβ(1–40) diluted into carbonate buffer (0.1 m; pH 9.6). Excess sites in the well were blocked with phosphate-buffered saline containing fatty acid-free bovine serum albumin (10 mg/ml) for 2 h at 37 °C. Wells were aspirated, and binding buffer (minimal essential medium containing fatty acid-free bovine serum albumin, 1 mg/ml; 0.05 ml) was added with125I-ERAB/HADH II alone or in the presence of 100-fold excess unlabeled ERAB/HADH II. Binding was allowed to proceed for 2 h at 37 °C, and each well was washed four times (0.2 ml/wash) over 30 s with ice cold phosphate-buffered saline containing Tween 20 (0.05%). Bound tracer was eluted with Nonidet P-40 (1%) over 5 min at 37 °C. Specific binding was defined as total minus nonspecific binding. Total binding was that observed in the presence of tracer alone. Nonspecific binding was that observed with tracer in the presence of 100-fold excess unlabeled ERAB/HADH II. Binding experiments were performed with four replicates per concentration of tracer. Data were analyzed by nonlinear least squares analysis (Enzfitter) using a one-site model by the method of Klotz and Hunston (35Klotz I. Hunston D. J. Biol. Chem. 1984; 259: 10060-10062Abstract Full Text PDF PubMed Google Scholar). Transient transfection of COS-1 or neuroblastoma (N115) cells (ATCC) employed pcDNA3/human ERAB/HADH II (wild-type or mutant forms) using LipofectAMINE according to previously described methods (15Yan S.-D. Soto C. Chen X. Zhu H. Al-Mohanna F. Collison K. Zhu A. Stern E. Saido T. Tohyama M. Ogawa S. Roher A. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (350) Google Scholar). Where indicated, cultures were subjected to transient transfection with pAdlox/βAPP(V717G). The latter was made by inserting a construct encoding βAPP(V717G) (36Wild-Bode C. Yamazaki T. Capell A. Leimer U. Steiner H. Ihara Y. Haass C. J. Biol. Chem. 1997; 272: 16085-16088Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) into the HindIII cloning sites in the pAdlox vector (37Uyttendaele H. Marazzi G. Wu G. Yan Q. Sassoon D. Kitajewski J. Development. 1996; 122: 2251-2259Crossref PubMed Google Scholar). Alternatively, a construct encoding wild-type (wt) βAPP(1–695) was inserted into the SalI cloning sites of the pMT vector (38Bonthron D. Handin R. Kaufman R. Wasley L. Orr E. Mitsock L. Ewenstein B. Loscalzo J. Ginsburg D. Orkin S. Nature. 1986; 324: 270-273Crossref PubMed Scopus (143) Google Scholar) to make pMT/wtβAPP. βAPP was detected with rabbit anti-C-terminal βAPP IgG (369W) generously provided by Dr. Sam Gandy (New York University, New York, NY) (39Borchelt D. Thinakaran G. Eckman C. Lee M. Davenport F. Ratovitsky T. Prada C.-M. Kim G. Seekins S. Yager D. Slunt H. Wang R. Seeger M. Levey A. Gandy S. Copeland N. Jenkins N. Price D. Younkin S. Sisodia S. Neuron. 1996; 17: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (1362) Google Scholar). The effect of ERAB/HADH II on cellular properties was determined in the absence and the presence of an Aβ-rich environment (the latter provided by transfection with pAdlox/βAPP(V717G)). COS and neuroblastoma cells transiently transfected with pcDNA3/ERAB with or without pAdlox/βAPP(V717G) were assayed for evidence of DNA fragmentation using the ELISA for cytoplasmic histone-associated DNA fragments (Cell Death ELISA; Boehringer Mannheim) and the TUNEL assay (Boehringer Mannheim). Similar experiments were performed employing pMT/wtβAPP in place of βAPP(V717G). In order to determine the relative apoptosis index (RAI) (15Yan S.-D. Soto C. Chen X. Zhu H. Al-Mohanna F. Collison K. Zhu A. Stern E. Saido T. Tohyama M. Ogawa S. Roher A. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (350) Google Scholar), cells were also evaluated for expression of ERAB/HADH II or βAPP immunocytochemically (cells were fixed in 2% paraformaldehyde containing 0.1% Nonidet P-40) using primary antibodies to each antigen and peroxidase-conjugated anti-rabbit IgG (Sigma) as the secondary antibody, (because both primary antibodies were prepared in rabbits, ERAB/HADH II and βAPP were detected separately in duplicate cultures; the TUNEL assay was performed on the same cultures in which ERAB/HADH II or βAPP was visualized. Subcellular localization of ERAB/HADH II employed ultracentrifugation of disrupted cells and confocal microscopy. Cells (5 × 108) were transfected as above, and, 12 h later, cells were pelletted and fractionated as described (40Kuwabara K. Matsumoto M. Ikeda J. Hori J. Ogawa S. Maeda Y. Kitagawa K. Imuta N. Kinoshita T. Stern D.M. Yanagi H. Kamada T. J. Biol. Chem. 1996; 271: 5025-5032Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). In brief, cell pellets frozen at −80 °C were thawed, resuspended in 10 ml of Buffer A (0.25 m sucrose; 10 mm HEPES, pH 7.5; 1 mm dithiothreitol; 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin; 0.1 mm1-chloro-3-tosylamido-7-amino-2-heptanone), and cavitated at 400 p.s.i. for 30 min using a nitrogen cavitation bomb apparatus (Kontes Glass Co., Vineland, NJ). Following cell disruption, the lysate was clarified by centrifugation at 10,000 × g for 15 min at 4 °C, and the pellet was resuspended in TNE buffer (10 mm Tris-HCl, pH 8.0; 1% Nonidet P-40; 150 mmNaCl; 1 mm EDTA; 10 μg/ml aprotinin; 1 mmphenylmethylsulfonyl fluoride). The latter material was centrifuged and fractionated through a series of sucrose steps (38, 30, and 20% sucrose prepared in 10 mm HEPES, pH 7.5; 1 mmdithiothreitol) at 100,000 × g for 3 h at 4 °C. Layered fractions (fractions 1–4) were collected by puncturing the tube at the desired depth and gently withdrawing the fluid. The pellet at the bottom of the tube was resuspended in 3 ml of Buffer A (precipitate fraction, termed fraction 5). Following determination of protein content, each fraction (5 μg protein/lane) was subjected to Western blotting using anti-ERAB/HADH II IgG. Enrichment of cellular structures/organelles in subcellular fractions was identified by the presence of marker proteins: RAGE (control cultures were transfected with pcDNA3/RAGE that was detected by Western blotting using specific antibodies to RAGE (10Yan S.-D. Chen X. Fu J. Chen M. Zhu H. Roher A. Slattery T. Zhao L. Nagashima M. Morser J. Migheli A. Nawroth P. Stern D. Schmidt A.-M. Nature. 1996; 382: 685-691Crossref PubMed Scopus (1848) Google Scholar) in order to identify the plasma membrane-rich fraction), GRP78/Bip (endoplasmic reticulum; StressGen; Victoria, Canada) (40Kuwabara K. Matsumoto M. 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