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 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
Department of
Chemistry and Biochemistry, UCLA, Los Angeles, California 90024
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ABSTRACT |
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Endoplasmic reticulum-associated amyloid
Recent studies of mutations underlying familial Alzheimer's
disease have strengthened links between amyloid 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
To investigate whether the enzymatic activity of ERAB/HADH II was
correlated with its interaction with A 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- Assays of ERAB Enzymatic Activity and A
Binding of ERAB/HADH II to A 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/ 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 A
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
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.
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
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 ERAB/HADH II Activity Is Essential for Promotion of A
Recent studies have demonstrated that processing of
COS cells cotransfected to overexpress wtERAB/HADH II and
In order to further validate these data, it was necessary to correlate
cells expressing the transfected genes (by immunocytochemistry for
ERAB/HADH II or
Using this experimental system, the effect of mutERAB
(Y168G/K172G), in place of wtERAB/HADH II, was assessed in COS cells overexpressing ERAB/HADH II Oxidation of Linear Alcohol Substrates and the Effect
of A
Because enzymatic activity of ERAB/HADH II was essential for
potentiation of A Change in Subcellular Distribution of ERAB/HADH II and Generation
of Reactive Aldehydes in an A
First, subcellular distribution of ERAB/HADH II in neuroblastoma
cells transfected to overexpress ERAB/HADH II alone or ERAB/HADH II +
Having established that ERAB/HADH II changed its distribution in cells
cotransfected to overexpress both ERAB/HADH II and 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 From another vantage point, ERAB/HADH II takes on a quite different
role in cells subjected to an environment rich in A Data from the cotransfection systems with pcDNA3/wtERAB and
pAdlox/ We hypothesize that when lower levels of A-peptide (A
)-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 A
, and contributes to A
-induced
cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the
NADH-dependent reduction of S-acetoacetyl-CoA
with a Km of
68 µM and a
Vmax of
430 µmol/min/mg. The contribution
of ERAB/HADH II enzymatic activity to A
-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
-amyloid precursor protein
(
APP(V717G)) showed DNA fragmentation, cotransfection with
Y168G/K172G-altered ERAB and
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 17
-estradiol. Addition of
micromolar levels of synthetic A
(1-40) to purified ERAB/HADH II
inhibited, in parallel, reduction of S-acetoacetyl-CoA
(Ki
1.6 µM), as well as oxidation
of 17
-estradiol (Ki
3.2 µM) and
(
)-2-octanol (Ki
2.6 µM).
Because micromolar levels of A
were required to inhibit ERAB/HADH II
activity, whereas A
binding to ERAB/HADH II occurred at much lower
concentrations (Km
40-70 nM), the
latter more closely simulating A
levels within cells, A
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
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
APP(V717G) alone. Generation of such toxic
aldehydes was not observed in cells contransfected to overexpress
Y168G/K172G-altered ERAB and
APP(V717G). We conclude that the
generalized alcohol dehydrogenase activity of ERAB/HADH II is central
to the cytotoxicity observed in an A
-rich environment.
INTRODUCTION
Top
Abstract
Introduction
References
-peptide
(A
)1 and the pathogenesis
of this devastating neurodegenerative disorder (1-6). Most work
analyzing toxic effects of A
on cellular properties has employed
high concentrations of A
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 A
are much lower, the amyloidogenic peptide might interact
with particular cellular target molecules, thereby magnifying the
capacity of A
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 A
(10-14). Another potential cellular interaction site is the
endoplasmic reticulum-associated A
-binding protein, initially
designated ERAB and first identified using the yeast two-hybrid system
to screen for species that bound A
(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 A
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 A
-induced cytotoxicity.
-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
-oxidation have been identified (19, 20).
These patients have hepatomegaly, cardiomegaly, encephalopathies,
peripheral neuropathy, rhabdomyolysis, and myoglobinuria, suggesting a
role for fatty acid
-oxidation enzymes in the metabolic balance of a
range of organs.
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 A
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 A
cytotoxicity consequent
to overexpression of mutant
APP(V717G), as demonstrated by
suppression of apoptosis. In contrast, cells overexpressing both
wild-type ERAB/HADH II and
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
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
A
-rich environment, as in AD, potentiating cell stress and cytotoxicity.
EXPERIMENTAL PROCEDURES
-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
0.15
M 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 (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
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).
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 17
-estradiol employed ERAB/HADH II
(30 µg/ml), a range of 17
-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 A
(1-40) or A
(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. A
(1-40) was freshly prepared and
dissolved in distilled water. A
(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
A
(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).
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
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 A
(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).
APP(V717G). The
latter was made by inserting a construct encoding
APP(V717G) (36)
into the HindIII cloning sites in the pAdlox vector (37).
Alternatively, a construct encoding wild-type (wt)
APP(1-695) was
inserted into the SalI cloning sites of the pMT vector (38)
to make pMT/wt
APP.
APP was detected with rabbit anti-C-terminal
APP IgG (369W) generously provided by Dr. Sam Gandy (New York
University, New York, NY) (39).
-rich
environment (the latter provided by transfection with
pAdlox/
APP(V717G)). COS and neuroblastoma cells transiently
transfected with pcDNA3/ERAB with or without pAdlox/
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/wt
APP in place of
APP(V717G). In order to determine the relative apoptosis index (RAI)
(15), cells were also evaluated for expression of ERAB/HADH II or
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
APP were detected separately in duplicate cultures;
the TUNEL assay was performed on the same cultures in which ERAB/HADH
II or
APP was visualized.
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).
RESULTS
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.
68 µM and a
Vmax of
430 units/mg (Fig.
2A and Table
I). These values are similar to bovine
HADH type II (Km of
20 µM and
Vmax of
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 A
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 A
-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 A
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 17 -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 17
-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.
ERAB/HADH II activity
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 17
-estradiol (Fig. 2,
B-C; see below). However, mutERAB bound A
in a manner
comparable to wild-type ERAB (Fig. 3B); apparent Kd values for ERAB/HADH II binding to immobilized
A
(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
-amyloid was observed with A
(1-42) (data not
shown). Thus, mutERAB(Y168G/K172G) was an inactive form of the enzyme
that retained the ability to interact with A
, allowing us to probe
the role of ERAB/HADH II enzymatic activity in potentiation of A
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 A
.
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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 A (1-40). Wells were incubated with A
(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.
APP, with
generation of A
, 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
APP(V717G) forms increased levels of
A
(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 A
-induced
cytotoxicity by cotransfection with plasmids causing overexpression of
wild-type or mutant ERAB/HADH II and
APP(V717G). First, expression
of ERAB/HADH II and
APP was evaluated under our experimental
conditions by immunoblotting using antibody to ERAB/HADH II and
C-terminal
APP antibody (396W) (39). COS cells transiently
transfected with pAdlox/
APP(V717G) overexpressed
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
APP-immunoreactive bands
with molecular masses of
110-140 kDa (often coalescing into one
broad band) (39) were seen, although more rapidly migrating
immunoreactive forms of
APP were also observed (Fig. 4A, lane
6). The latter forms were more evident in cells expressing
APP(V717G) alone (Fig. 4A, lane 6) compared with those
co-expressing
APP(V717G) and wild-type or mutant ERAB/HADH II
(although levels of the latter forms were comparable in cells overexpressing wtERAB/HADH II +
APP(V717G) or mutERAB/HADH II +
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/
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,
APP and/or ERAB/HADH II, was somewhat
less in neuroblastoma than in COS cells. Further experiments were
performed with COS cells overexpressing wt
APP following transfection
with pMT/wt
APP alone or cotransfection with pMT/wt
APP + wtERAB/HADH II. Immunoblotting showed wt
APP (Fig. 4A, upper panel, lanes 9 and 10) to be expressed at levels
comparable to mutant
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/wt
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 APP(V717G). A, immunoblotting for
APP (top panel) and ERAB/HADH II (bottom
panel). Lysates of transiently transfected COS cells were
subjected to SDS-PAGE (10% for
APP, and 12% for ERAB/HADH II)
followed by immunoblotting using antibody to
APP (369W) (top
panel) or antibody to ERAB/HADH II (bottom panel).
Either the mutant form of
APP(V717G) or wt
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/
APP(V717G) + pcDNA3/wtERAB; lane 4, pcDNA3/mutERAB; lane 5, pAdlox/
APP(V717G) + pcDNA3/mutERAB; lane 6, pAdlox/
APP(V717G); lane
7, pAdlox; lane 8, pcDNA3/wtERAB; lane
9, pMT/wt
APP + pcDNA3/wtERAB; lane 10, pMT/wt
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,
APP(V717G), or wt
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/
APP(V717G) (II), or pAdlox/
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
APP
antigen (ERAB/HADH II and
APP antigens were determined in duplicate
cultures; ERAB/HADH II immunostaining and TUNEL assay, or
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.
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),
APP(V717G) alone
(III), or wtERAB+
APP(V717G) (II)). When COS
cells were cotransfected to overexpress wtERAB with wt
APP (the
latter in place of mutant
APP), evidence of increased DNA
fragmentation was also observed compared with cultures overexpressing
either wtERAB or wt
APP alone (Fig. 4B). However, somewhat
lower levels of DNA fragmentation were observed following
overexpression of wt
APP compared with
APP(V717G), consistent with
the previously observed higher levels of intracellular A
(especially
A
(1-42) in endoplasmic reticulum) with this mutant form of
APP
(36).
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
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
APP and those undergoing
apoptosis (Fig. 4D). The RAI denotes a ratio of cells
with TUNEL-positive nuclei overexpressing ERAB/HADH II
(RAIERAB) or
APP (RAI
APP) divided by the
total number of cells expressing either ERAB/HADH II or
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
13%), similar to that observed in nontransfected cells in the same
wells (RAI0
8%). Comparable results were observed when
cells were transfected to overexpress
APP(V717G) alone;
RAI
APP was low, at
9%, and RAI
APP
RAI0 (11%). In contrast, COS cells cotransfected with
pcDNA3/wtERAB + pAdlox/
APP(V717G) had high levels of DNA
fragmentation (RAIERAB
64% and RAI
APP
60%, the latter in separate experiments) compared with
unsuccessfully transfected cells in the same well (RAI0
7%). These data indicate that overexpression of wtERAB along with
mutant
APP markedly enhanced cellular toxicity compared with either
alone. Similar results were observed with neuroblastoma cells.
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/
APP(V717G) resulted in expression of
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/
APP(V717G) did not show an increase in cytoplasmic
histone-associated DNA fragments, compared with cells transfected with
pAdlox/
APP(V717G) alone or other controls (Fig. 4B).
Furthermore, using the RAI, cells cotransfected to overexpress
APP(V717G) and mutERAB did not display an increase in
RAIERAB (
12%) or RAI
APP (
9%), compared with RAI0 (
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
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).
--
To probe mechanisms through which ERAB/HADH II induced
its toxic effects in an A
-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
540-1400 M
1 s
1 and
Km values of
40-80 mM (Fig.
2B and Table I). ERAB/HADH II activity toward steroid
substrates was studied using 17
-estradiol (Fig. 2C),
NAD+-dependent dehydrogenation occurred with a
catalytic efficiency (kcat/Km) of
7.4 × 105 M
1 s
1 had a
Km value of
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.
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 A
(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
1.6 ± 0.5 µM (34). Similarly, A
suppressed ERAB/HADH
II-mediated, NAD+-dependent oxidation of
octanol (Ki
2.6 ± 0.3 µM;
Fig. 5B) and 17
-estradiol (Ki
3.2 ± 0.2 µM; Fig. 5C). Comparable results were obtained when A
(1-42) was used in place of A
(1-40) (data not shown). Such levels of A
that attenuated ERAB/HADH II
activity toward each of these substrates were considerably higher than
those required to occupy A
binding sites on ERAB/HADH II; the
Kd for A
binding to ERAB/HADH II was
40-70 nM (see Fig. 3B). This was an important
consideration, because intracellular A
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 A
(or perhaps aggregated/fibrillar A
) to ERAB/HADH II, but these
effects are distinct from the lower amounts of A
that occupy
ERAB/HADH II binding sites.
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Fig. 5.
Effect of A (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 A
.
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 A
.
C, oxidation of 17
-estradiol. ERAB/HADH II (25 µg/ml)
was incubated with 17
-estradiol (61 µM),
NAD+ (0.4 mM), and the indicated concentrations
of A
. Velocity (V) of the reaction (units/mg of protein)
is plotted versus log [A
] (nM). Data were
fit to a one-site model for competitive inhibition.
-rich Environment--
In a previous
study, we observed that exogenous A
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).
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/
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/
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 wt
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
APP(V717G).
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Fig. 6.
Subcellular distribution of ERAB/HADH II in
neuroblastoma cells following transient transfection with or without
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/
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/ APP(V717G)
(B), transfected with pcDNA3/mutERAB alone
(C), cotransfected with pcDNA3/mutERAB + pAdlox/
APP(V717G) (D), or cotransfected with
pcDNA3/ wtERAB/HADH II + pMT/wt
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).
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
APP(V717G) were studied for MDA and HNE epitopes using murine
monoclonal antibodies and confocal microscopy. Cultures expressing
either wtERAB/HADH II or
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
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
APP(V717G). Further experiments were performed in which cultures were transfected to overexpress wt
APP (in place of mutant
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 + wt
APP compared with cultures
expressing either wtERAB or wt
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 + wt
APP than in cultures expressing either wtERAB or wt
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
APP(V717G). COS cells were transiently transfected with
pcDNA3/wtERAB or pcDNA3/mutERAB(Y168G/K172G) either alone or in
the presence of pAdlox/
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/
APP(V717G) alone (IV-VI), or cotransfected with
pcDNA3/wtERAB + pAdlox/
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/
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 APP(V717G) (A and
B) or wild-type
APP (wt
APP) (C and
D). Data from experiments in Fig. 8 (for
APP(V717G)) or from experiments in which primary data was not shown
(for wt
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
-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
-oxidation cycle in vitro. Although inherited
deficiencies of enzymes involved in fatty acid
-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 17
-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 17
-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 17
-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.
. Previously
(15), we found that cell stress, following exposure of neuroblastoma
cells to synthetic A
, 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
A
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 A
(15). Because these previous
experiments involved nonphysiologic exposure of cultured cells to A
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
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.
APP(V717G) suggest that ERAB/HADH II and, presumably, the
product of cellular
APP(V717G) metabolism, A
, 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 A
would suggest that A
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/wt
APP, the
latter in place of
APP(V717G), suggests that high levels of
cell-associated A
(1-42) generated with the mutant form of
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 A
. However, regardless
of the precise mechanism by which ERAB/HADH II and A
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
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/
APP(V717G), compared with
native ERAB/HADH II. Because A
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 A
. In accordance
with this concept is our finding that ERAB/HADH II binding sites for
A
are half-maximally occupied at a peptide concentration of
40-70 nM, whereas A
suppression of ERAB/HADH II
enzymatic activity occurs at much higher concentrations,
2-3
µM. The latter levels of A
are unlikely to be present
within cells, and, taken together, the data suggest that ERAB/HADH II binding to A
may modulate other properties of the molecule, such as
its localization within the cell.
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 A
-mediated changes in cellular properties) modulating
enzymatically active ERAB/HADH II function/localization, we increased
cellular vulnerability. Taken together, the combination of A
binding
properties and generalized alcohol dehydrogenase activity, in addition
to HADH activity, lead us to propose the new name A
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.
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:
A, amyloid
-peptide; AD, Alzheimer's disease; ERAB, endoplasmic
reticulum-associated A
-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 A and
potentiation of A
toxicity, as well as its metabolic function as a
3-hydroxyacyl-CoA dehydrogenase. We propose to rename the enzyme A
binding alcohol dehydrogenase as this reflects both its capacity to
bind A
and its activity as a generalized alcohol dehydrogenase (see text).
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