Role of ERAB/L-3-Hydroxyacyl-coenzyme A Dehydrogenase Type II Activity in Abeta -induced Cytotoxicity*

Shi Du YanDagger , Yigong Shi§, Aiping Zhu, Jin Fu, Huaijie Zhu, Yucui Zhu, Lenneen Gibson, Eric Stern, Kate Collison, Futwan Al-Mohanna, Satoshi Ogawaparallel , Alex Roher**, Steven G. ClarkeDagger Dagger , and David M. Stern

From the Departments of Pathology, Physiology and Surgery, College of Physicians and Surgeons of Columbia University, New York, New York 10032, the  King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, the parallel  Department of Anatomy and Neuroscience, Osaka University School of Medicine, Osaka, Japan, the § Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, the ** Haldeman Laboratory for Alzheimer's Disease Research, Sun Health Research Institute, Sun City, Arizona 85372, and the Dagger Dagger  Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90024

    ABSTRACT
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Abstract
Introduction
References

Endoplasmic reticulum-associated amyloid beta -peptide (Abeta )-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 Abeta , and contributes to Abeta -induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approx 68 µM and a Vmax of approx 430 µmol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta -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 beta -amyloid precursor protein (beta APP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and beta APP(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 17beta -estradiol. Addition of micromolar levels of synthetic Abeta (1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approx  1.6 µM), as well as oxidation of 17beta -estradiol (Ki approx 3.2 µM) and (-)-2-octanol (Ki approx  2.6 µM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approx  40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta 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 beta APP(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 beta APP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and beta APP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta -rich environment.

    INTRODUCTION
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Abstract
Introduction
References

Recent studies of mutations underlying familial Alzheimer's disease have strengthened links between amyloid beta -peptide (Abeta )1 and the pathogenesis of this devastating neurodegenerative disorder (1-6). Most work analyzing toxic effects of Abeta on cellular properties has employed high concentrations of Abeta fibrils that are thought to nonspecifically injure cells by destabilizing cell membranes (1, 4, 6-9). We have hypothesized, however, that early in Alzheimer's disease (AD), when levels of Abeta are much lower, the amyloidogenic peptide might interact with particular cellular target molecules, thereby magnifying the capacity of Abeta to perturb cellular functions (10-14). At the cell surface, receptors, for example, receptor for advanced glycation end products (RAGE), the p75 component of the neurotrophin receptor, and the macrophage scavenger receptor, are such molecular targets of Abeta (10-14). Another potential cellular interaction site is the endoplasmic reticulum-associated Abeta -binding protein, initially designated ERAB and first identified using the yeast two-hybrid system to screen for species that bound Abeta (15). ERAB was localized to the endoplasmic reticulum and mitochondria in cultured cells (15). Its expression was found to be increased in AD brain, and, in vitro, ERAB facilitated Abeta 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 (16). These results suggested a primary role for ERAB in cell metabolism, in addition to having the potential to contribute to Abeta -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) (17, 18), a participant in the third reaction of the fatty acid beta -oxidation spiral (19, 20). This identification is consistent with a recent report concerning properties of a human L-3-hydroxyacyl-CoA dehydrogenase (21), the cDNA sequence of which is identical to that of human ERAB.2 Although consequences of ERAB/HADH II deficiency are not known, heritable disorders with defects in fatty acid beta -oxidation have been identified (19, 20). These patients have hepatomegaly, cardiomegaly, encephalopathies, peripheral neuropathy, rhabdomyolysis, and myoglobinuria, suggesting a role for fatty acid beta -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 Abeta 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 of S-acetoacetyl-CoA. To analyze the role of ERAB/HADH II enzymatic activity in potentiation of Abeta 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 (16). Cells overexpressing mutant ERAB were relatively protected from Abeta cytotoxicity consequent to overexpression of mutant beta APP(V717G), as demonstrated by suppression of apoptosis. In contrast, cells overexpressing both wild-type ERAB/HADH II and beta APP (V717G) suffered increased cytotoxicity. Thus, it appears that activity of the enzyme is necessary for full toxicity. In cells overexpressing ERAB/HADH II and beta APP(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 (22-25) and have been observed in the brain of AD patients (26-32). These data lead us to propose that enzymatically active ERAB/HADH II, an alcohol dehydrogenase with broad substrate specificity, may have a role in metabolic homeostasis but can switch to a pathologic role in an Abeta -rich environment, as in AD, potentiating cell stress and cytotoxicity.

    EXPERIMENTAL PROCEDURES

Preparation of Wild-type and Mutant Forms of ERAB-- 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 mM isopropyl-1-thio-beta -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 M NaCl. These fractions were pooled, diluted 6-fold, and applied to Source 15S resin in 0.1 M MES (pH 6.0)/0.1 M NaCl (5 mg of protein per 1 ml of resin). The column was eluted with an ascending salt gradient, and ERAB/HADH II emerged at approx 0.15 M NaCl. ERAB/HADH II-rich fractions were concentrated by ultrafiltration to approx 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 (15) or antibody prepared in rabbits, according to standard methods (33), 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 approx 27 kDa, consistent with previous observations (15, 17, 18, 21). 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).

Assays of ERAB Enzymatic Activity and Abeta Binding-- ERAB/HADH II was studied for its activity to reduce S-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 (34), 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, and n-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 17beta -estradiol employed ERAB/HADH II (30 µg/ml), a range of 17beta -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 Abeta (1-40) or Abeta (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. Abeta (1-40) was freshly prepared and dissolved in distilled water. Abeta (1-42) was also freshly prepared and dissolved in Me2SO. The final concentration of Me2SO was <1%, and control experiments using this concentration of Me2SO alone (i.e. without Abeta (1-42)) had no effect on the reactions under study. Kinetic data were analyzed by PRISM (Scitech, San Diego, CA) to determine Km, Vmax, and Ki, and lines shown in the figures represent theoretical curves according to kinetic parameters calculated by the program. One unit of enzyme activity was defined as that converting 1.0 µmol of substrate to product per min. For Ki, a one-site competitive inhibition model was used (34).

Binding of ERAB/HADH II to Abeta was studied using 125I-labeled purified recombinant ERAB/HADH II (15). The protein was labeled by the Iodobead method to a final specific radioactivity of approx 2000 cpm/ng (an average of five labelings) and the tracer was >90% precipitable in trichloroacetic acid (10%). Wells were incubated overnight at 4 °C with Abeta (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 with 125I-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 (35).

Cell Transfection Studies-- 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 (15). Where indicated, cultures were subjected to transient transfection with pAdlox/beta APP(V717G). The latter was made by inserting a construct encoding beta APP(V717G) (36) into the HindIII cloning sites in the pAdlox vector (37). Alternatively, a construct encoding wild-type (wt) beta APP(1-695) was inserted into the SalI cloning sites of the pMT vector (38) to make pMT/wtbeta APP. beta APP was detected with rabbit anti-C-terminal beta APP IgG (369W) generously provided by Dr. Sam Gandy (New York University, New York, NY) (39).

Assays for DNA Fragmentation and Subcellular Localization-- The effect of ERAB/HADH II on cellular properties was determined in the absence and the presence of an Abeta -rich environment (the latter provided by transfection with pAdlox/beta APP(V717G)). COS and neuroblastoma cells transiently transfected with pcDNA3/ERAB with or without pAdlox/beta APP(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/wtbeta APP in place of beta APP(V717G). In order to determine the relative apoptosis index (RAI) (15), cells were also evaluated for expression of ERAB/HADH II or beta APP 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 beta APP were detected separately in duplicate cultures; the TUNEL assay was performed on the same cultures in which ERAB/HADH II or beta APP 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 (40). 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 mM 1-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 mM NaCl; 1 mM EDTA; 10 µg/ml aprotinin; 1 mM phenylmethylsulfonyl 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 mM dithiothreitol) 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 (10) in order to identify the plasma membrane-rich fraction), GRP78/Bip (endoplasmic reticulum; StressGen; Victoria, Canada) (40), and cytochrome c (mitochondria; StressGen). The mitochondria-rich fraction was also prepared using the method of Du et al. (41).

Confocal microscopy (Zeiss) employed rabbit anti-human ERAB/HADH II IgG, monoclonal antibody to protein disulfide isomerase (PDI) (StressGen) and a specific marker for mitochondria (Mito-TrackerTM Red CMXRos, Molecular Probes, Eugene, OR). For detection of malondialdehyde (MDA) and 4-hydroxynonenal (HNE) epitopes, murine monoclonal anti-MDA-lysine IgG (1 µg/ml; MDA2) and murine monoclonal anti-HNE-lysine IgG (1 µg/ml; NA59) were used, respectively (22-25). Antibodies were generated in the Immunology Core of the La Jolla SCOR in Molecular Medicine and Atherosclerosis and were kindly supplied by Dr. Joe Witztum (University of California, San Diego, CA). Cultures were fixed using 4% paraformaldehyde plus 5% sucrose (pH 7.4) containing 50 µM butylated hydroxytoluene and 1 mM EDTA (22-23). Sites of primary antibody binding were visualized by addition of rhodamine-conjugated goat anti-mouse IgG (Sigma). Confocal microscopy images were quantitated using the National Institutes of Health Image program.

    RESULTS

Characterization of Recombinant Wild-type and Mutant Forms of ERAB/HADH II-- E. coli was transformed with a plasmid encoding wt human ERAB/HADH II, and lysates were prepared. The recombinant protein was purified by sequential SP Sepharose and Source 15S chromatography, followed by gel filtration on Superdex 200. The final material was homogeneous on SDS-PAGE, displaying a single band migrating at an apparent molecular mass of approx 27 kDa (Fig. 1A), and, on amino acid sequencing, only the N-terminal sequence of ERAB/HADH II was obtained: Ala-Ala-Ala-Cys-Arg-Ser-Val-Lys-Gly Leu-Val-Ala-Val-Ile-Thr-Gly-Gly-Ala-Ser-Gly-Leu. Immunoblotting with anti-human ERAB IgG (Fig. 1B, lane 1) confirmed the presence of ERAB/HADH II epitopes in the recombinant ERAB/HADH II preparations. Addition of excess free ERAB/HADH II during incubation of blots with the primary antibody prevented appearance of the ERAB/HADH II-immunoreactive band (Fig. 1B, lane 2). Preimmune antibody did not visualize the ERAB/HADH II band on blots (Fig. 1B, lane 3).


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Fig. 1.   Characterization of E. coli-derived ERAB/HADH II. A, nonreduced (lane 1) and reduced (lane 2) SDS-PAGE (12%) of purified recombinant ERAB/HADH II (1 µg/lane). Protein was visualized by Coomassie Blue staining. Standard proteins were run simultaneously, and their migration is indicated by the bars on the far left. Each bar is labeled with the corresponding molecular mass in kDa. B. Immunoblotting. Purified recombinant ERAB/HADH II was subjected to reduced SDS-PAGE (12%) followed by immunoblotting. ERAB antigen was visualized using rabbit anti-full-length recombinant ERAB/HADH II IgG (3.5 µg/ml) (lane 1), followed by application of secondary antibody and the detection system. In lane 2, excess soluble ERAB/HADH II (50-fold molar excess over the amount of primary antibody) was added during incubation of blots with the primary antibody. In lane 3, ERAB/HADH II was present on the membrane (as in lane 1), but the same amount preimmune IgG was used in place of immune IgG. Details of procedure are described in the text.

Initial comparison with Swiss-Prot and Protein Data Bank using the FASTA algorithm indicated that the ERAB sequence most closely resembled the family of short-chain alcohol dehydrogenases, including hydroxysteroid dehydrogenases and acetoacetyl-CoA reductases (15). Studies of bovine HADH II, the bovine counterpart of ERAB, demonstrated that it possesses 3-hydroxyacyl-CoA dehydrogenase activity, catalyzing both forward and reverse reactions (18). In this work, we show that in the presence of NADH, ERAB reduced S-acetoacetyl-CoA with a Km of approx 68 µM and a Vmax of approx 430 units/mg (Fig. 2A and Table I). These values are similar to bovine HADH type II (Km of approx 20 µM and Vmax of approx 530 units/mg) (18), and to those of human HADH type II (HADH II) described in a report published during preparation of our manuscript (21). Thus, ERAB is an HADH and, based on amino acid sequence, is identical to human HADH type II. However, the interaction of ERAB with Abeta results in quite unexpected properties compared with what might be expected for a metabolic enzyme, leading us to term it ERAB/HADH II (to emphasize Abeta -induced modulation of its properties, see below). These data suggested the importance of determining the contribution of ERAB/HADH II enzymatic activity to its role in potentiation of Abeta cytotoxicity.


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Fig. 2.   Characterization of ERAB/HADH II enzymatic activity: reduction of S-acetoacetyl-CoA (A) and oxidation of octanol (B) and 17beta -estradiol (C). Experiments utilized either wild-type ERAB/HADH II (filled squares) or mutant ERAB(Y168G/K172G) (open squares). The same concentration of wild-type and mutant ERAB was used in each case. A, ERAB/HADH II (0.33 µg/ml) was incubated with the indicated concentration of S-acetoacetyl-CoA and NADH (0.1 mM). B, ERAB/HADH II (20 µg/ml) was incubated with the indicated concentration of (-)-octanol and NAD+ (7.5 mM). C, ERAB/HADH II (30 µg/ml) was incubated with the indicated concentration of 17beta -estradiol and NAD+ (0.4 mM). The velocity (V) of the reaction (units/mg of protein) is plotted versus added substrate concentration. Details of the experimental methods are described in the text. The broken line represents the theoretical curve according to the Km and Vmax values (see Table I) calculated by the computer program. Experimental procedures are described in the text.

                              
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Table I
ERAB/HADH II activity

ERAB/HADH II Activity Is Essential for Promotion of Abeta Cytotoxicity-- Tyr168 and Lys172 are part of a highly conserved sequence (residues 168-172) that has been putatively assigned as part of the active site of the enzyme (16). Thus, to prepare an active site-blocked form of human ERAB/HADH II, we mutated these residues simultaneously or separately by site-directed mutagenesis to Gly. Mutant (mut) ERAB(Y168G/K172G) was expressed in E. coli and purified as described above for wtERAB/HADH II. The final material was homogeneous on SDS-PAGE (Fig. 3A, two left lanes), migrated virtually identically with wtERAB/HADH II, had the expected N-terminal sequence (data not shown), and was immunoreactive with anti-ERAB IgG (Fig. 3A, two right lanes). Purified mutERAB(Y168G/K172G) was devoid of activity toward S-acetoacetyl-CoA (Fig. 2A), as well as octanol and 17beta -estradiol (Fig. 2, B-C; see below). However, mutERAB bound Abeta in a manner comparable to wild-type ERAB (Fig. 3B); apparent Kd values for ERAB/HADH II binding to immobilized Abeta (1-40) were 64.5 ± 9.0 and 38.9 ± 9.3 nM for wild-type and mutant ERAB/HADH II, respectively. Similar binding of ERAB/HADH II to beta -amyloid was observed with Abeta (1-42) (data not shown). Thus, mutERAB(Y168G/K172G) was an inactive form of the enzyme that retained the ability to interact with Abeta , allowing us to probe the role of ERAB/HADH II enzymatic activity in potentiation of Abeta cytotoxicity. Similar results were independently obtained with the two other ERAB/HADH II mutants, Y168G and K172G; in each case, the mutant form was devoid of enzymatic activity but retained the capacity to bind Abeta .


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Fig. 3.   Characterization of mutant ERAB/HADH II (Y168G/K172G). A, left two lanes, reduced SDS-PAGE (12%) of wild-type ERAB (ERAB/HADH II) and mutERAB/HADH II (5 µg/lane). Protein was visualized by Coomassie Blue staining. A, right two lanes, SDS-PAGE of ERAB/mutERAB (as above) followed by immunoblotting with anti-ERAB/HADH II IgG. Migration of simultaneously run molecular mass markers is indicated on the left in kilodaltons (kD). B, binding of 125I-ERAB/HADH II to Abeta (1-40). Wells were incubated with Abeta (5 µg/ml), followed by blocking excess sites in the well with albumin-containing buffer and then addition of 125I-ERAB/HADH II (either wild-type or mutERAB) alone or in the presence of 100-fold molar excess of unlabeled ERAB/HADH II (either wild-type or mutERAB). Specific binding (total minus nonspecific binding) is plotted versus added ERAB/HADH II (nM). The broken line shows the best-fit line by nonlinear least squares analysis.

Recent studies have demonstrated that processing of beta APP, with generation of Abeta , can occur within multiple intracellular compartments (36, 42-46), including endoplasmic reticulum, a site where ERAB/HADH II has been localized. In particular, a construct encoding the London mutant of beta APP(V717G) forms increased levels of Abeta (1-42) in endoplasmic reticulum following transient transfection (36). This led us to develop a model system using COS and neuroblastoma cells for testing the effects of ERAB/HADH II on Abeta -induced cytotoxicity by cotransfection with plasmids causing overexpression of wild-type or mutant ERAB/HADH II and beta APP(V717G). First, expression of ERAB/HADH II and beta APP was evaluated under our experimental conditions by immunoblotting using antibody to ERAB/HADH II and C-terminal beta APP antibody (396W) (39). COS cells transiently transfected with pAdlox/beta APP(V717G) overexpressed beta APP antigen (Fig. 4A, upper panel, lanes 3, 5, and 6), compared with controls (Fig. 4A, upper panel, lanes 1, 2, 4, and 7), whether cells were cotransfected to overexpress wtERAB or mutERAB (Fig. 4A, upper panel, lanes 3 and 5) or not (Fig. 4A, upper panel, lane 6). Several closely spaced beta APP-immunoreactive bands with molecular masses of approx 110-140 kDa (often coalescing into one broad band) (39) were seen, although more rapidly migrating immunoreactive forms of beta APP were also observed (Fig. 4A, lane 6). The latter forms were more evident in cells expressing beta APP(V717G) alone (Fig. 4A, lane 6) compared with those co-expressing beta APP(V717G) and wild-type or mutant ERAB/HADH II (although levels of the latter forms were comparable in cells overexpressing wtERAB/HADH II + beta APP(V717G) or mutERAB/HADH II beta APP(V717G)) (Fig. 4A, lanes 3 and 5). Similarly, human ERAB/HADH II antigen was overexpressed by COS cells transfected with pcDNA3/wtERAB (Fig. 4A, lower panel, lanes 2 and 3) or pcDNA3/mutERAB (Fig. 4A, lower panel, lanes 4 and 5), whether cotransfected with pAdlox/beta APP(V717G) (Fig. 4A, lower panel, lanes 3 and 5) or not (Fig. 4A, lower panel, lanes 2 and 4). Comparable results were observed in cotransfection studies with neuroblastoma cells, although the absolute amount of the transfected gene products, beta APP and/or ERAB/HADH II, was somewhat less in neuroblastoma than in COS cells. Further experiments were performed with COS cells overexpressing wtbeta APP following transfection with pMT/wtbeta APP alone or cotransfection with pMT/wtbeta APP + wtERAB/HADH II. Immunoblotting showed wtbeta APP (Fig. 4A, upper panel, lanes 9 and 10) to be expressed at levels comparable to mutant beta APP (lanes 3, 5, and 6), whether wtERAB/HADH II was present (lane 9) or not (lane 10). Similarly, cotransfection of COS cells with pMT/wtbeta APP did not alter expression of wild-type ERAB/HADH II (Fig. 4A, lower panel, lanes 8 and 9).


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Fig. 4.   Cotransfection of COS cells to overexpress ERAB/HADH II and beta APP(V717G). A, immunoblotting for beta APP (top panel) and ERAB/HADH II (bottom panel). Lysates of transiently transfected COS cells were subjected to SDS-PAGE (10% for beta APP, and 12% for ERAB/HADH II) followed by immunoblotting using antibody to beta APP (369W) (top panel) or antibody to ERAB/HADH II (bottom panel). Either the mutant form of beta APP(V717G) or wtbeta APP was overexpressed. Lanes correspond to samples from COS cells transfected with the following constructs in the left panels: lane 1, pcDNA3; lane 2, pcDNA3/wtERAB; lane 3, pAdlox/beta APP(V717G) + pcDNA3/wtERAB; lane 4, pcDNA3/mutERAB; lane 5, pAdlox/beta APP(V717G) + pcDNA3/mutERAB; lane 6, pAdlox/beta APP(V717G); lane 7, pAdlox; lane 8, pcDNA3/wtERAB; lane 9, pMT/wtbeta APP + pcDNA3/wtERAB; lane 10, pMT/wtbeta APP. B, ELISA for cytoplasmic histone-associated DNA fragments. COS cells were transfected/cotransfected with plasmids resulting in overexpression of the indicated protein (wtERAB/HADH II, mutERAB, beta APP(V717G), or wtbeta APP) or with LipofectAMINE alone. At the far left, the transfected gene product(s) are indicated. After 48 h, samples were harvested for the ELISA. * denotes p < 0.01; # denotes p < 0.05. C, TUNEL assay. COS cells were transfected with pcDNA3/wtERAB alone (I), pcDNA3/wtERAB + pAdlox/beta APP(V717G) (II), or pAdlox/beta APP(V717G) alone (III). Forty-eight hours later, the TUNEL assay was performed. D, RAI. Cells were transfected/cotransfected with the indicated constructs, and 48 h later, cultures were subjected to the TUNEL assay and immunostained to detect ERAB/HADH II or beta APP antigen (ERAB/HADH II and beta APP antigens were determined in duplicate cultures; ERAB/HADH II immunostaining and TUNEL assay, or beta APP immunostaining and TUNEL assay, were performed on the same slide). The RAI is a percentage denoting the ratio of the number of TUNEL positive nuclei in cells with the indicated antigen divided by the total number of cells with the indicated antigen in the same cell population. RAI0 indicates the fraction of TUNEL positive nuclei in cells not staining for the transfected antigen divided by the total number of cells not expressing the indicated antigen in the same cell population. The constructs used for transfection/cotransfection are indicated on the far left.

COS cells cotransfected to overexpress wtERAB/HADH II and beta APP(V717G) displayed increased DNA fragmentation, as shown by ELISA for cytosolic histone-associated DNA fragments (Fig. 4B) and TUNEL assay (Fig. 4C, I-III, depicting representative fields of cells overexpressing wtERAB/HADH II alone (I), beta APP(V717G) alone (III), or wtERAB+beta APP(V717G) (II)). When COS cells were cotransfected to overexpress wtERAB with wtbeta APP (the latter in place of mutant beta APP), evidence of increased DNA fragmentation was also observed compared with cultures overexpressing either wtERAB or wtbeta APP alone (Fig. 4B). However, somewhat lower levels of DNA fragmentation were observed following overexpression of wtbeta APP compared with beta APP(V717G), consistent with the previously observed higher levels of intracellular Abeta (especially Abeta (1-42) in endoplasmic reticulum) with this mutant form of beta APP (36).

In order to further validate these data, it was necessary to correlate cells expressing the transfected genes (by immunocytochemistry for ERAB/HADH II or beta APP) with those undergoing nuclear DNA fragmentation (by TUNEL assay). This was especially important, as the transient transfection system employed for these studies has an efficiency of approx 20-25%. For this reason, we utilized a RAI (15) to compare, in the same population, cells successfully transfected to overexpress wild-type/mutant ERAB/HADH II or beta APP and those undergoing apoptosis (Fig. 4D). The RAI denotes a ratio of cells with TUNEL-positive nuclei overexpressing ERAB/HADH II (RAIERAB) or beta APP (RAIbeta APP) divided by the total number of cells expressing either ERAB/HADH II or beta APP. RAI0 is the percentage of TUNEL-positive nuclei in cells in the same wells that did not express the transfected gene products, divided by the total number of cells that had negligible levels of these protein products. Cells transfected with pcDNA3/wtERAB alone displayed a low level of DNA fragmentation (RAIERAB approx  13%), similar to that observed in nontransfected cells in the same wells (RAI0 approx  8%). Comparable results were observed when cells were transfected to overexpress beta APP(V717G) alone; RAIbeta APP was low, at approx 9%, and RAIbeta APP approx  RAI0 (11%). In contrast, COS cells cotransfected with pcDNA3/wtERAB + pAdlox/beta APP(V717G) had high levels of DNA fragmentation (RAIERAB approx  64% and RAIbeta APP approx  60%, the latter in separate experiments) compared with unsuccessfully transfected cells in the same well (RAI0 approx  7%). These data indicate that overexpression of wtERAB along with mutant beta APP markedly enhanced cellular toxicity compared with either alone. Similar results were observed with neuroblastoma cells.

Using this experimental system, the effect of mutERAB (Y168G/K172G), in place of wtERAB/HADH II, was assessed in COS cells overexpressing beta APP(V717G). As noted above, levels of ERAB/HADH II in COS cells transfected with pcDNA3/mutERAB were comparable to those observed in COS cells transfected with pcDNA3/wtERAB (Fig. 4A, lower panel). Also, cotransfection of pcDNA3/mutERAB + pAdlox/beta APP(V717G) resulted in expression of beta APP antigen at levels comparable to those seen in cells transfected with the construct encoding wtERAB/HADH II (Fig. 4A, upper panel). However, cotransfection of COS cells with pcDNA3/mutERAB + pAdlox/beta APP(V717G) did not show an increase in cytoplasmic histone-associated DNA fragments, compared with cells transfected with pAdlox/beta APP(V717G) alone or other controls (Fig. 4B). Furthermore, using the RAI, cells cotransfected to overexpress beta APP(V717G) and mutERAB did not display an increase in RAIERAB (approx 12%) or RAIbeta APP (approx 9%), compared with RAI0 (approx 5%) (Fig. 4D). Thus, although mutERAB (Y168G/K172G) was expressed similarly to wtERAB/HADH II, the enzymatically inactive form was ineffective for potentiating apoptosis in COS cells overexpressing beta APP(V717G). Comparable results were observed when mutERAB(Y168G/K172G) was replaced with the two other mutant forms of ERAB/HADH II, Y168G and K172G (data not shown).

ERAB/HADH II Oxidation of Linear Alcohol Substrates and the Effect of Abeta -- To probe mechanisms through which ERAB/HADH II induced its toxic effects in an Abeta -rich environment, possible dehydrogenation of a range alcohol substrates was studied, based on the hypothesis that such activity might give rise to aldehydes with associated toxic properties. Using a series of alcohols with carbon chains of differing lengths, ERAB/HADH II-dependent oxidation was evaluated; the most favorable catalytic efficiencies were seen for the stereoisomers of 2-octanol, with kcat/Km of approx 540-1400 M-1 s-1 and Km values of approx 40-80 mM (Fig. 2B and Table I). ERAB/HADH II activity toward steroid substrates was studied using 17beta -estradiol (Fig. 2C), NAD+-dependent dehydrogenation occurred with a catalytic efficiency (kcat/Km) of approx 7.4 × 105 M-1 s-1 had a Km value of approx 14 µM (see Table I). It is significant that this latter catalytic efficiency is only about 4-fold less than that seen in the reduction of S-acetoacetyl-CoA (Table I). These data were consistent with the concept that ERAB/HADH II could effectively metabolize a range of alcohols, although the activity toward linear alcohol substrates was less than that as an hydroxysteroid dehydrogenase.

Because enzymatic activity of ERAB/HADH II was essential for potentiation of Abeta cytotoxicity, it was possible that the amyloidogenic peptide enhanced the enzymatic activity of ERAB/HADH II or modulated substrate specificity, possibly favoring linear alcohols with resultant augmented formation of aldehydes. Addition of increasing concentrations of synthetic Abeta (1-40) to ERAB/HADH II diminished NADH-dependent reduction of S-acetoacetyl-CoA (Fig. 5A). Inhibition fit to a one-site competitive model with Ki approx  1.6 ± 0.5 µM (34). Similarly, Abeta suppressed ERAB/HADH II-mediated, NAD+-dependent oxidation of octanol (Ki approx  2.6 ± 0.3 µM; Fig. 5B) and 17beta -estradiol (Ki approx  3.2 ± 0.2 µM; Fig. 5C). Comparable results were obtained when Abeta (1-42) was used in place of Abeta (1-40) (data not shown). Such levels of Abeta that attenuated ERAB/HADH II activity toward each of these substrates were considerably higher than those required to occupy Abeta binding sites on ERAB/HADH II; the Kd for Abeta binding to ERAB/HADH II was approx 40-70 nM (see Fig. 3B). This was an important consideration, because intracellular Abeta would more likely be present at nanomolar levels than in the micromolar range. Thus, there is probably a secondary effect of binding of additional molecules of Abeta (or perhaps aggregated/fibrillar Abeta ) to ERAB/HADH II, but these effects are distinct from the lower amounts of Abeta that occupy ERAB/HADH II binding sites.


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Fig. 5.   Effect of Abeta (1-40) on ERAB/HADH II enzymatic activity. A, reduction of S-acetoacetyl-CoA. ERAB/HADH II (0.67 µg/ml) was incubated with S-acetoacetyl-CoA (0.18 mM), NADH (0.1 mM), and the indicated concentrations of Abeta . B, oxidation of octanol. ERAB/HADH II (10 µg/ml) was incubated with (-)-octanol (210 mM), NAD+ (7.5 mM), and the indicated concentrations of Abeta . C, oxidation of 17beta -estradiol. ERAB/HADH II (25 µg/ml) was incubated with 17beta -estradiol (61 µM), NAD+ (0.4 mM), and the indicated concentrations of Abeta . Velocity (V) of the reaction (units/mg of protein) is plotted versus log [Abeta ] (nM). Data were fit to a one-site model for competitive inhibition.

Change in Subcellular Distribution of ERAB/HADH II and Generation of Reactive Aldehydes in an Abeta -rich Environment-- In a previous study, we observed that exogenous Abeta induced a change in the subcellular distribution of ERAB/HADH II in neuroblastoma cells (15). This suggested the possibility that ERAB/HADH II might find different substrates and/or activate enzyme systems following its transposition to new sites within the cell. Because of the association of oxidant stress with AD (9, 25-31), we considered the possibility that following translocation within the cell, ERAB/HADH II participated in events generating reactive aldehydes, MDA and/or HNE, both of which have been observed in the brain of AD patients (26-32).

First, subcellular distribution of ERAB/HADH II in neuroblastoma cells transfected to overexpress ERAB/HADH II alone or ERAB/HADH II + beta APP(V717G) was studied. Confocal microscopy of neuroblastoma cells transfected with pcDNA3/ERAB, either wtERAB or mutERAB(Y168G/K172G), displayed ERAB/HADH II principally in endoplasmic reticulum and mitochondria (Fig. 6, A-F, shows results with wtERAB). The distribution of ERAB/HADH II (Fig. 6, C and F) overlapped (Fig. 6, A and D), in part, with an endoplasmic reticulum marker, PDI (Fig. 6B) and a mitochondrial marker (Fig. 6E). Consistent with these results, subcellular fractionation of neuroblastoma cells transfected with pcDNA3/wtERAB showed ERAB/HADH II antigen to be principally in fractions enriched for mitochondrial and endoplasmic reticulum markers (Fig. 7A). The distribution of mutERAB in neuroblastoma cells similarly transfected with pcDNA3/mutERAB was comparable to that observed with wtERAB/HADH II, as seen in subcellular fractionation studies (Fig. 7C) and by confocal microscopy (data not shown). Cotransfection of neuroblastoma cells with pcDNA3/wtERAB and pAdlox/beta APP(V717G) displayed a different pattern of ERAB/HADH II antigen (Fig. 6, G--L). By confocal microscopy the altered distribution of ERAB/HADH II in cotransfectants (Fig. 6, I and L) was evident, with lesser amounts in endoplasmic reticulum (Fig. 6, G and H), although it was still in mitochondria (Fig. 6, J and K). Strikingly, in cotransfectants with pcDNA3/wtERAB and pAdlox/beta APP(V717G), ERAB/HADH II antigen seemed to be present in multiple sites within the cell, including the plasma membrane. In general, ERAB/HADH II antigen appeared to be more diffusely distributed in cotransfectants with some antigen in cell membrane, cytosol, and nuclear membrane and, in certain instances, associated with the nucleus. Subcellular fractionation studies showed a shift in ERAB/HADH II antigen to fractions rich in plasma membrane markers in cotransfectants (Fig. 7B). Similar results were obtained when mutERAB(Y168G/K172G) was overexpressed in place of wtERAB/HADH II (Fig. 7D). COS cells subjected to the cotransfection procedure and the same analysis as described above showed similar, though less striking results. Experiments in which wtbeta APP was overexpressed along with wild-type ERAB/HADH II also showed translocation of ERAB/HADH II from endoplasmic reticulum-associated to plasma membrane-associated fractions (Fig. 7E), although to a somewhat lesser extent than was seen with mutant beta APP(V717G).


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Fig. 6.   Subcellular distribution of ERAB/HADH II in neuroblastoma cells following transient transfection with or without beta APP(V717G). A-F, neuroblastoma cells were transfected with pcDNA3/wtERAB/HADH II alone, and cultures were stained to visualize ERAB/HADH II + endoplasmic reticulum marker PDI (A), PDI alone (B), ERAB/HADH II alone (C), ERAB/HADH II + mitochondrial marker (D), mitochondrial marker alone (E), or ERAB/HADH II alone (F). G-L, neuroblastoma cells were cotransfected with pcDNA3/wtERAB + pAdlox/beta APP(V717G), and cultures were stained to visualize ERAB/HADH II + PDI (G), PDI alone (H), ERAB/HADH II alone (I), ERAB/HADH II + mitochondrial marker (J), mitochondrial marker alone (K), or ERAB/HADH II alone (L). On the left of the figure, the transfected gene products and the antigens visualized by immunostaining are indicated in the corresponding set of three panels.


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Fig. 7.   Subcellular fractionation of neuroblastoma cells either transfected with pcDNA3/wtERAB/HADH II alone (A), cotransfected with pcDNA3/wtERAB + pAdlox/beta APP(V717G) (B), transfected with pcDNA3/mutERAB alone (C), cotransfected with pcDNA3/mutERAB + pAdlox/beta APP(V717G) (D), or cotransfected with pcDNA3/ wtERAB/HADH II + pMT/wtbeta APP (E). Neuroblastoma cells were transfected/cotransfected with the indicated constructs and processed as described in the text. Each fraction prepared from nontransfected neuroblastoma cells was also subjected to Western blotting using antibodies to ERAB/HADH II and/or to proteins associated with specific subcellular structures: GRP78/Bip for endoplasmic reticulum (F, lanes 1 and 2), RAGE for plasma membrane (G, lane 4), and cytochrome c for mitochondria (H, lane 6).

Having established that ERAB/HADH II changed its distribution in cells cotransfected to overexpress both ERAB/HADH II and beta APP(V717G), we next sought to determine whether reactive aldehydes (MDA or HNE) were formed under these conditions. COS cells overexpressing wtERAB/HADH II and beta APP(V717G) were studied for MDA and HNE epitopes using murine monoclonal antibodies and confocal microscopy. Cultures expressing either wtERAB/HADH II or beta APP(V717G) alone showed no increase in MDA (Fig. 8A, I-III and IV-VI, respectively) or HNE antigen (Fig. 8B, I-III and IV-VI, respectively). In contrast, cultures expressing both wtERAB/HADH II and beta APP(V717G) showed abundant MDA (Fig. 8A, VII-IX) and HNE antigen (Fig. 8B, VII-IX; quantitative analysis of this immunocytochemical data is shown in Fig. 9, A and C). In cotransfectants, the distribution of MDA and ERAB/HADH II (Fig. 8A, VII), or HNE and ERAB/HADH II (Fig. 8B, VII) antigen appeared to overlap, at least in part. ERAB/HADH II (Fig. 8, A and B, IX) and HNE or MDA (Fig. 8, A and B, VIII) were present in the cell membrane, as well as more diffusely throughout the cell. These experiments were also performed with mutERAB(Y168G/K172G) in place of wtERAB/HADH II (Fig. 8, A and B, X-XV). In contrast to the wild-type enzyme, mutERAB (Fig. 8, A and B, XII and XV) did not generate elevated levels of either MDA or HNE epitopes (Fig. 8, A and B, XI and XIV, and Fig. 9). Thus, the integrity of the active site of ERAB/HADH II is required to support generation of reactive aldehydes, such as malondialdehyde and 4-hydroxynonenal, in cells cotransfected with beta APP(V717G). Further experiments were performed in which cultures were transfected to overexpress wtbeta APP (in place of mutant beta APP) and wtERAB (Fig. 9, B and D). In these studies as well, there was an increase, although to a lesser extent, in generation of reactive aldehyde epitopes in cells expressing wtERAB + wtbeta APP compared with cultures expressing either wtERAB or wtbeta APP alone. For the latter studies, the increase in HNE epitopes (Fig. 9D) was more striking than that for MDA epitopes (Fig. 9B), although over many experiments, it was evident that both reactive aldehydes were produced at higher levels in cells cotransfected to overexpress wtERAB + wtbeta APP than in cultures expressing either wtERAB or wtbeta APP alone.


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Fig. 8.   Expression of MDA and HNE epitopes in COS cells expressing wt or mutERAB/HADH II with or without beta APP(V717G). COS cells were transiently transfected with pcDNA3/wtERAB or pcDNA3/mutERAB(Y168G/K172G) either alone or in the presence of pAdlox/beta APP(V717G). Cultures were fixed and stained with antibodies to MDA and ERAB/HADH II, or HNE and ERAB/HADH II. Panels represent the following. A, I-IX, MDA and ERAB/HADH II staining (wtERAB/HADH II): cells were transfected with pcDNA3/wtERAB alone (I-III), transfected with pAdlox/beta APP(V717G) alone (IV-VI), or cotransfected with pcDNA3/wtERAB + pAdlox/beta APP(V717G) (VII-IX). Cultures were stained to visualize either ERAB/HADH II alone (green) (III, VI, and IX), MDA alone (red) (II, V, and VIII) or ERAB/HADH II + MDA antigens (I, IV, and VII). A, X-XV, MDA and ERAB/HADH II (mutERAB) staining: cells were transfected with pcDNA3/mutERAB alone (XIII-XV) or cotransfected with pcDNA3/mutERAB + pAdlox/beta APP(V717G) (X-XII). Cultures were stained to visualize either ERAB/HADH II alone (green) (XII and XV), MDA alone (red) (XI and IV) or ERAB/HADH II + MDA antigens (X and XIII). B, I-IX, HNE and ERAB/HADH II (wtERAB) staining: staining was identical to panel A, except that antibody to HNE was used in place of antibody to MDA. B, X-XV, HNE and ERAB/HADH II staining (mutERAB): staining was identical to panel A, except that antibody to HNE was used in place of antibody to MDA. In each case, the antigens visualized by immunostaining (labeled above the figure) and the gene products overexpressed by transfection (labeled on the far left) are indicated.


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Fig. 9.   Quantitative analysis of MDA (A and B) and HNE (C and D) epitope expression in COS cells expressing wild-type or mutant ERAB/HADH II with or without beta APP(V717G) (A and B) or wild-type beta APP (wtbeta APP) (C and D). Data from experiments in Fig. 8 (for beta APP(V717G)) or from experiments in which primary data was not shown (for wtbeta APP) were analyzed by the National Institutes of Health Image program. Relative fluorescence intensity (arbitrary units) is shown in COS cell cultures transfected with the indicated constructs. Each bar is labeled with the gene products overexpressed by transfection. * denotes p < 0.01; # denotes p < 0.05.


    DISCUSSION

The properties of ERAB/L-3-hydroxyacyl-CoA dehydrogenase type II reveal an intriguing dichotomy for cellular homeostasis. On the one hand, enzyme activity as an L-3-hydroxyacyl-CoA dehydrogenase is consistent with ERAB/HADH II participation in metabolic homeostasis, namely in the fatty acid beta -oxidation pathway. This concept is in accordance with previous reports demonstrating that bovine (17, 18) and, more recently, human (21) L-3-hydroxyacyl-CoA dehydrogenase reversibly catalyze the third reaction of the fatty acid beta -oxidation cycle in vitro. Although inherited deficiencies of enzymes involved in fatty acid beta -oxidation clearly have serious clinical consequences (19, 20), including a report of short chain 3-hydroxyacyl-CoA dehydrogenase deficiency (47), whether ERAB/HADH II has an important role in normal metabolic balance remains to be determined. ERAB/HADH II also has the capacity to dehydrogenate alcohol functions, including a range of linear alcohols, as well as 17beta -estradiol. Although efficiency of the enzyme is less for the latter reactions than as an HADH, these data emphasize the potentially broad capacity of ERAB/HADH II to dehydrogenate a range of alcohol substrates. Whereas the physiologic significance of this property of ERAB/HADH II is not clear at this time, the potential of ERAB to metabolize 17beta -estradiol, a neuro- and vascular-protective hormone (48-51), deserves further study. The reason for the apparent lack of ERAB/HADH type II activity as a 17beta -hydroxysteroid dehydrogenase in the experiments of He et al. (21) may reflect differences in assay conditions and/or in the nature of the E. coli-derived protein.

From another vantage point, ERAB/HADH II takes on a quite different role in cells subjected to an environment rich in Abeta . Previously (15), we found that cell stress, following exposure of neuroblastoma cells to synthetic Abeta , was increased in cultures transfected to overexpress ERAB/HADH II. Cotransfection of the same ERAB/HADH II-expressing cells with a construct specifically generating cytosolic Abeta similarly enhanced cytotoxicity (15). Consistent with these data, blockade of ERAB/HADH II, following intracellular introduction of anti-ERAB F(ab')2 using a liposome-based system, had a protective effect in the presence of Abeta (15). Because these previous experiments involved nonphysiologic exposure of cultured cells to Abeta and intracellular loading of foreign antibody fragments, we have extended our observations using a system in which cultured cells are simultaneously cotransfected with constructs encoding ERAB/HADH II and mutant beta APP(V717G). This system allows endogenous overexpression of both genes without other interventions; such cells suffer intense stress, evidenced by DNA fragmentation and generation of reactive aldehydes (MDA/HNE epitopes). Our results are unlikely to represent a general "endoplasmic reticulum stress response,"(52, 53) as similar cotransfection studies with ERAB/HADH II mutants devoid of enzymatic activity did not induce cell damage, although the level of expression of the transfected constructs was comparable.

Data from the cotransfection systems with pcDNA3/wtERAB and pAdlox/beta APP(V717G) suggest that ERAB/HADH II and, presumably, the product of cellular beta APP(V717G) metabolism, Abeta , interact with cytotoxic consequences for the host cell. The molecular mechanisms underlying such an interaction must be clarified in detail, because direct ERAB/HADH II binding to Abeta would suggest that Abeta generated in the lumen of the endoplasmic reticulum or other membrane-bound compartments could interact with ERAB/HADH II, the latter likely to be present on the cytosolic side of endoplasmic reticulum and in mitochondria. The apparently reduced potentiation of cytotoxicity when cultures were cotransfected with pcDNA3/wtERAB + pMT/wtbeta APP, the latter in place of beta APP(V717G), suggests that high levels of cell-associated Abeta (1-42) generated with the mutant form of beta APP (36) accentuate toxicity in the presence of ERAB/HADH II. These data are also consistent with the possibility of an interaction, either direct or indirect, between ERAB/HADH II and Abeta . However, regardless of the precise mechanism by which ERAB/HADH II and Abeta interact, our data using this cotransfection system provide strong support for the concept that ERAB/HADH II enzymatic activity can exert deleterious effects in cells overexpressing beta APP. Thus, three ERAB/HADH II mutants that were devoid of enzymatic activity and expressed comparably to wtERAB/HADH II showed dramatically reduced DNA fragmentation in cotransfection experiments with pAdlox/beta APP(V717G), compared with native ERAB/HADH II. Because Abeta interaction with these enzymatically inactive ERAB/HADH II mutants was similar to that observed with wtERAB/HADH II, the enzymatic activity of ERAB must be differentiated from those determinants that mediate binding of Abeta . In accordance with this concept is our finding that ERAB/HADH II binding sites for Abeta are half-maximally occupied at a peptide concentration of approx 40-70 nM, whereas Abeta suppression of ERAB/HADH II enzymatic activity occurs at much higher concentrations, approx 2-3 µM. The latter levels of Abeta are unlikely to be present within cells, and, taken together, the data suggest that ERAB/HADH II binding to Abeta may modulate other properties of the molecule, such as its localization within the cell.

We hypothesize that when lower levels of Abeta are present in the brain, cytotoxicity may not be due solely to the amyloidogenic peptide itself, but may be amplified by cellular cofactors such as ERAB/HADH II. By directly forming a complex with ERAB/HADH II and/or indirectly (through undefined Abeta -mediated changes in cellular properties) modulating enzymatically active ERAB/HADH II function/localization, we increased cellular vulnerability. Taken together, the combination of Abeta binding properties and generalized alcohol dehydrogenase activity, in addition to HADH activity, lead us to propose the new name Abeta binding alcohol dehydrogenase or ABAD to better describe the unusual properties of the enzyme previously referred to as ERAB or HADH II.

    ACKNOWLEDGEMENT

We thank Dr. Gabriel Godman (Columbia University) for efforts and advice during the performance of these studies and preparation of the manuscript.

    FOOTNOTES

* This work was supported by Grants AG00690, AG14103, and AG11925 from the United States Public Health Service and by a grant from the Surgical Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pathology, P&S 17-410, College of Physicians & Surgeons of Columbia University, 630 West 168th St., New York, NY 10020. Tel.: 212-305-3958; Fax: 212-305-5337.

The abbreviations used are: Abeta , amyloid beta -peptide; AD, Alzheimer's disease; ERAB, endoplasmic reticulum-associated Abeta -binding protein; HADH II, L-3-hydroxyacyl-CoA dehydrogenase type II; HNE, 4-hydroxynonenal; MDA, malondialdehyde; mut, mutant; PDI, protein disulfide isomerase; wt, wild-type; RAI, relative apoptosis index; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MES, 4-morpholineethanesulfonic acid.

2 ERAB 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 Abeta and potentiation of Abeta toxicity, as well as its metabolic function as a 3-hydroxyacyl-CoA dehydrogenase. We propose to rename the enzyme Abeta binding alcohol dehydrogenase as this reflects both its capacity to bind Abeta and its activity as a generalized alcohol dehydrogenase (see text).

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Abstract
Introduction
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