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INTRODUCTION |
The human short chain L-3-hydroxyacyl-CoA
dehydrogenase (SCHAD)1 gene,
mapped at chromosome Xp11.2, encodes a single-domain dehydrogenase composed of four identical subunits (1). The primary structure of this
L-3-hydroxyacyl-CoA dehydrogenase is identical to an
amyloid
-peptide-binding protein called endoplasmic
reticulum-associated-binding protein (ERAB) (1-3). Human short chain
L-3-hydroxyacyl-CoA dehydrogenase has a molecular mass of
108 kDa, and its structure is quite distinct from other
L-3-hydroxyacyl-CoA dehydrogenases that bear the signature pattern of the L-3-hydroxyacyl-CoA dehydrogenase family (4, 5). Although this enzyme is not homologous to the classic
L-3-hydroxyacyl-CoA dehydrogenase (L-3-HAD),
which is encoded by the human HADHSC gene located at
chromosome 4q22-26 (6), functional convergence during evolution has
conferred on them the capability of catalyzing the same reaction:
L-3-hydroxyacyl-CoA + NAD+
3-ketoacyl-CoA + NADH + H+ (1).
Transiently expressed ERAB was reportedly localized at the endoplasmic
reticulum of the cultured cells (2). However, this finding does not
agree with the presumed function of human SCHAD/ERAB in fatty acid
-oxidation (1). Therefore, the intracellular distribution of this
dehydrogenase needed to be re-examined. Determination of the role(s)
that human SCHAD plays in normal cells is of paramount importance for
elucidating its role(s) in the pathogenesis of Alzheimer's disease.
Because some members of the short chain dehydrogenase family display
activities toward several structurally distinct substrates (7), we set
out to determine whether human SCHAD harbors more than one enzymatic
activity. Human SCHAD is clearly homologous to the amino-terminal
domain of the peroxisomal multifunctional protein-2 (MFP-2), which
exhibits D-3-hydroxyacyl-CoA dehydrogenase (D-3-HAD) activity of opposite stereospecificity (8).
Interestingly, this domain also displays some 17
-hydroxysteroid
dehydrogenase (17
-HSD) activity (9). MFP-2 was found to be identical
to the previously described 17
-hydroxysteroid dehydrogenase type 4 (8, 9), although the maximal velocity (Vmax) of
the 17
-HSD was only about 1/8000 of that of D-3-HAD (9).
These clues prompted us to further characterize the catalytic
properties of human brain SCHAD.
In this report, we provide evidence that human brain SCHAD is a new
17
-hydroxysteroid dehydrogenase. Moreover, it also harbors alcohol
dehydrogenase activity. This single domain multifunctional enzyme not
only plays a part in the mitochondrial fatty acid
-oxidation, but
also functions in steroid hormone metabolism.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs and Transient Transfections--
The cDNA
insert (BamHI-EcoRI) containing the whole coding
region of human brain SCHAD was removed from the recombinant plasmid pGEM-T-HBHAD (1) by digestion with proper restriction enzymes and
subcloned into the BamHI-EcoRI site of the vector
pcDNA3.1 (Invitrogen) to yield a mammalian expression plasmid
designated pcDNA3.1/SCHAD. The cDNA of human brain SCHAD was
also amplified from pGEM-T-HBHAD by polymerase chain reaction using a
pair of primers: 5'-AAGCTTCCGCCACCATGGCAGCAGCGTGTCGGA-3' and
5'-CTGCAGAAGGCTGCATACGAATGGCC-3'. The resulting polymerase chain
reaction product was cloned into the pcDNA3.1/CT-GFP-TOPO vector
(Invitrogen) according to the procedures of the manufacturer to
generate a SCHAD-GFP (green fluorescent protein) fusion protein
expression plasmid designated pcDNA3.1/SCHAD-CT-GFP. COS-7 and
PC-12 cells were grown according to the instructions of the supplier
(ATCC). All culture media were purchased from Life Technologies, Inc.
Subconfluent cells were plated in 35-mm plates. The next day, cells
were washed with fresh medium and transfected with 1 µg of
pcDNA3.1/CT-GFP (Invitrogen), pcDNA3.1/SCHAD-CT-GFP,
pcDNA3.1/SCHAD, and pcDNA3.1, respectively, using LipofectAMINE
PLUS reagent (Life Technologies, Inc.) in accordance with the protocol
of the manufacturer. From 24 to 48 h post-transfection, the cells
were examined for GFP expression or were used for immunocytochemical staining.
Antibody Preparation and Immunocytochemistry--
Anti-SCHAD
antibodies were generated by immunizing a rabbit with the peptide
RLDGAIRMQP coupled to keyhole limpet hemocyanin with glutaraldehyde.
Cells transfected with pcDNA3.1/SCHAD or pcDNA3.1 and growing
on coverslips were incubated with primary anti-SCHAD antibody. The
bound primary antibody was then detected with fluorescein
isothiocyanate-labeled goat anti-rabbit IgG (Sigma) according to a
published protocol (10). Cells were imaged using a laser scanning
confocal microscope as described below.
Mitochondria Staining and Confocal Microscopy--
Living cells
were incubated in culture medium containing 1 µM
MitoTrackerTM Red CMXRos (Molecular Probes) for 15 min at
37 °C. The live cells were then either mounted in fresh culture
medium and examined directly or were immunostained as described above.
Fluorescense-labeled cells were examined using a Nikon Eclipse E800
microscope coupled to a Nikon PCM 2000 dual laser scanning confocal
microscopy system. Images were analyzed with a
C-Imaging-SIMPLE32TM image analysis system (Compix
Inc.).
Overexpression and Purification of Human Brain
L-3-Hydroxyacyl-CoA Dehydrogenase--
IPTG
induction of Escherichia coli BL21
(DE3)pLysS/pSBET-HBHAD transformants, and the purification of human
brain SCHAD from the transformants containing the overexpressed enzyme,
were performed as described previously (1).
Protein Analysis and Enzyme Assays--
Protein concentrations
were determined by the method of Bradford (11). Proteins were separated
on a 4-20% gradient gel at pH 8.3, as described previously (12). The
activity of L-3-hydroxyacyl-CoA dehydrogenase was measured
with acetoacetyl-CoA as substrate according to the published method
(13). Alcohol dehydrogenase and hydroxysteroid dehydrogenase activities
were assayed at 25 °C by spectrophotometrically measuring the
absorbance change at 340 nm as a function of time (14, 15). The molar
extinction coefficient used for calculating rates is 6220 M
1 cm
1. A standard assay
mixture for dehydrogenation reactions contained 0.1 M
potassium phosphate (pH 8.0), fatty acid-free bovine serum albumin (0.1 mg/ml), 100 µM steroid substrate (added in 8 µl of ethanol) or 160 mM alcohol substrate, 1 mM
NAD+ (or NADP+), and appropriate quantities of
enzyme. When the reduction of substrate was measured, the pH of
phosphate buffer was adjusted to 6.6, and 100 µM NADH (or
NADPH), and keto/aldehyde substrate were substituted for
NAD+ (or NADP+) and alcohol substrate,
respectively. Confirmation of steroid products, after extraction with
diethyl ether, was achieved by thin-layer chromatography. Kinetic
parameters for the different substrates were estimated by analysis of
the kinetic data with the computer program Leonora (16). A unit of
activity is defined as the amount of enzyme that catalyzes the
conversion of 1 µmol of substrate to product/min.
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RESULTS |
Subcellular Location of Human Short Chain
L-3-Hydroxyacyl-CoA Dehydrogenase--
The GFP and
GFP-tagged human brain short chain L-3-hydroxyacyl-CoA
dehydrogenase (SCHAD-GFP) were expressed in COS-7 cells transiently
transfected with pcDNA3.1/CT-GFP and pcDNA3.1/SCHAD-CT-GFP, respectively. Because GFP and its fusion protein fluoresce under UV
light, GFP is a convenient molecular reporter to monitor patterns of
protein localization in living cells (17). As shown in Fig. 1a, GFP was distributed
uniformly throughout the cytoplasm and nucleus. In contrast, the
resulting SCHAD-GFP fusion protein displayed a granulated pattern of
localization (see Fig. 1d). Once mitochondria were
specifically stained by a mitochondrion-selective dye with red
fluorescence (Fig. 1, b and e), the punctate
organelles where the GFP-tagged SCHAD localized were unequivocally
identified as mitochondria (see Fig. 1f). Because GFP itself
cannot enter mitochondria (Fig. 1c), these results indicate
that mitochondria are the proper location for this dehydrogenase.

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Fig. 1.
Localization of human short chain
L-3-hydroxyacyl-CoA dehydrogenase in COS-7
cells. Cells were transfected with pcDNA3.1/CT-GFP
(a-c) or pcDNA3.1/SCHAD-CT-GFP (d-f). Cells
transfected with pcDNA3.1/SCHAD were immunostained with primary
anti-SCHAD and then with the fluorescense-tagged secondary antibody
(g-i). Mitochondria stained with MitoTrackerTM
Red CMXRos are shown in red (b, e, and
h); GFP, SCHAD-GFP, and SCHAD are shown in green
(a, d, and g). c,
f, and i show merged images of a and
b, d and e, and g and
h, respectively. The results shown are representative of
three independent experiments.
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The native SCHAD expressed in COS-7 cells transiently transfected with
pcDNA3.1/SCHAD was located by immunocytochemistry (see Fig.
1g). The green fluorescence of the SCHAD/antibody complex co-localized with the red fluorescence of mitochondria (see Fig. 1,
h and i). When preimmune rabbit serum was used
instead of the anti-SCHAD antibodies, such green fluorescence was not
observed (data not shown). Cells transfected with pcDNA3.1 vector
emitted little green fluorescence after treatment with anti-SCHAD and FITC-labeled secondary antibody (data not shown). We obtained similar
results when using PC-12 instead of COS-7 cells (data not shown).
The human SCHAD gene product was shown to be identical to the
A
-binding-protein, ERAB (1). This finding may cause some confusion
about whether human SCHAD participates in mitochondrial fatty acid
-oxidation, particularly in light of a recent report localizing ERAB
to the endoplasmic reticulum in transiently transfected cells (2). Our
study, employing confocal microscopy and using either the
immunocytochemistry or GFP method, resulted in fluorescence images
differing from those published previously (2). We show that
mitochondria are a major location for human SCHAD in living cells (see
Fig. 1). These results strongly support our proposition (1) that this
human dehydrogenase could act as a mitochondrial fatty acid
-oxidation enzyme.
Capability of Human Short Chain L-3-Hydroxyacyl-CoA
Dehydrogenase to Oxidize Alcohols--
Ethanol is usually a good
substrate for alcohol dehydrogenases (14); however, its rate of
oxidation catalyzed by human brain SCHAD is not detectable (data not
shown). Because we considered SCHAD to be a new member of the short
chain dehydrogenase superfamily (1), we were interested in determining
whether this mitochondrial
-oxidation enzyme is capable of
converting alcohols to aldehydes or ketones. Our experiments revealed
that propanol and longer chain primary alcohols could be slowly
oxidized. For example, the specific activity of the purified enzyme was
estimated to be 2.0 ± 0.3 nmol · min
1 · mg
1 for the dehydrogenation of 1-propanol. However,
secondary alcohols were found to be better substrates. For example,
2-propanol was oxidized approximately 9-fold faster than was 1-propanol
in a standard assay. Nevertheless, the rate of the backward reaction, i.e. the reduction of acetone to produce 2-propanol, was not
detected. These observations suggest that human brain SCHAD, in
contrast to the classic L-3-hydroxyacyl-CoA dehydrogenase,
is a multifunctional enzyme.
Characterization of the Intrinsic Dehydrogenase Activities of Human
Short Chain L-3-Hydroxyacyl-CoA Dehydrogenase--
A
variety of steroid substrates were tested for the ability of human
brain SCHAD to oxidize or to reduce them. We found that SCHAD could
oxidize the most potent estrogen 17
-estradiol to a very weak one,
estrone. This dehydrogenase can also oxidize and thereby inactivate
androsterone to androstanedione. In addition, SCHAD can use NADH as
coenzyme to reduce the potent androgen, 5
-dihydrotestosterone, to a
weak one, dihydroandrosterone. SCHAD also displayed low activities
toward C21 steroid hormones (Table I). Details of this investigation will be
described in a subsequent report. Our results reveal that human brain
SCHAD is a new type of oxidative 17
-hydroxysteroid dehydrogenase,
which possesses some 3
-hydroxysteroid dehydrogenase activity as
well. Its primary catalytic properties were determined by steady-state
kinetic measurements (16, 18) and are summarized in Table
II. To our knowledge, porcine
17
-hydroxysteroid dehydrogenase type 4 was the only known oxidative
17
-HSD that had been purified to near homogeneity (9). It is
noteworthy that the Vmax and the
Km for 17
-estradiol of SCHAD were both 2 orders
of magnitude higher than those reported for porcine 17
-HSD type 4 (9). Therefore, the actual catalytic efficiencies of these two
different oxidative 17
-HSDs are comparable.
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Table II
Kinetic parameters of the hydroxysteroid dehydrogenase inherent in the
short chain L-3-hydroxyacyl-CoA dehydrogenase from human
braina
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The effects of pH on the 17
-HSD and L-3-HAD activities
of human brain short chain L-3-hydroxyacyl-CoA
dehydrogenase were determined. The reduction of acetoacetyl-CoA to
L-3-hydroxybutyryl-CoA exhibited a pH optimum of 6.5-7.0
(Fig. 2), which is quite distinct from
that reported for the bovine liver enzyme (19). In the latter case,
reaction rates continuously increase with decreasing pH (19). For the
oxidation of 17
-estradiol to estrone, human brain SCHAD showed a pH
optimum of 9.5-10.0 (Fig. 2), which is similar to that reported for
human 17
-HSD type 2 (20). SCHAD is different from other 17
-HSDs
because of its inability to catalyze the reduction of either estrone or
androstenedione by NADH or NADPH at an appreciable rate (Table I).
However, its pH-activity profile for the reduction of acetoacetyl-CoA
appears to be similar to the patterns determined for the reduction of
estrone or androstenedione catalyzed by human 17
-HSD type 2 (20).

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Fig. 2.
Effects of pH on the activities of human
short chain L-3-hydroxyacyl-CoA
dehydrogenase. Solid circles and triangles
represent 17 -hydroxysteroid dehydrogenase activity measured with 100 µM 17 -estradiol as substrate and
L-3-hydroxyacyl-CoA dehydrogenase activity assayed with 20 µM acetoacetyl-CoA as substrate, respectively. Initial
rates were measured at 25 °C in the presence of 1 mM
NAD+ ( ) or 100 µM NADH ( ) in potassium
phosphate buffer adjusted to the appropriate pH.
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Human Short Chain L-3-Hydroxyacyl-CoA Dehydrogenase Is
a Novel 17
-Hydroxysteroid Dehydrogenase--
Thus far, five
different types of human 17
-HSD have been identified (20-24).
However, only type 1, which catalyzes the reduction of estrone to
17
-estradiol, has been purified to near homogeneity (25). Recently,
rat 17
-HSD type 6 (26) and mouse 17
-HSD type 7 (27) and Ke 6 (28)
were identified and reported to be new isoenzymes. However, their
corresponding enzymes from human tissues have not yet been identified.
The sequence identity between human 17
-HSD isoenzymes is less than
22% except that human brain SCHAD and the amino-terminal domain of
peroxisomal 17
-HSD type 4 show 29% identity. Nevertheless, all of
them have eight conserved residues that represent "fingerprints"
typical of the short chain dehydrogenase family (29) (see Fig.
3). By comparing the structural and
functional features of SCHAD with the known 17
-HSDs (see Table
III), we conclude that human SCHAD
represents a new type of 17
-HSD.

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Fig. 3.
Alignment of amino acid sequence of human
short chain L-3-hydroxyacyl-CoA dehydrogenase
(SCHAD) (1) with those of human
17 -hydroxysteroid dehydrogenase (HSD) types 4 (23), 3 (22), 2 (20), and 1 (21). Human 17 -HSD type 5 is not
included because its primary structure has not been published (24).
Standard 1-letter amino acid abbreviations are used. Conserved residues
that represent the fingerprint typical of the short chain dehydrogenase
family (29) are marked by asterisks (*), whereas those conserved in
three sequences or more are shown by letters in the
bottom line. Such letters are capitalized where
SCHAD and 17 -HSD type 4 have the same residue.
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Table III
Comparison of human short chain L-3-hydroxyacyl-CoA
dehydrogenase with other human 17 -hydroxysteroid dehydrogenases
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A homolog of the human SCHAD gene was found on Drosophila
chromosome X. A conceptual translation from the open reading frame of
the Drosophila scully gene revealed a 68% identity with the primary structure of human brain SCHAD, even though the
scully gene product has not yet been purified and
characterized (30). The putative Drosophila short chain
L-3-hydroxyacyl-CoA dehydrogenase was thought to function
in fatty acid
-oxidation because such a role agreed with the
presence of aberrant mitochondria and cytoplasmic lipid inclusions in
scully mutants described previously (30). However, other
important scully mutant phenotypes, such as hypogenitalism and germinal cell aplasia, cannot be explained without the knowledge that human brain SCHAD is a multifunctional enzyme with intrinsic 17
-hydroxysteroid dehydrogenase activity. Combining these
observations, it seems likely that the invertebrate counterpart of
human SCHAD, the scully gene product, retains enough
hydroxysteroid dehydrogenase activity to play a significant part in sex
steroid hormone metabolism as well as in fatty acid oxidation.
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DISCUSSION |
The results of our present study provide new evidence for a close
relationship between fatty acid metabolism and sterol metabolism. The
recent observation that the sterol regulatory element-binding protein
(SREBP) stimulates the biosynthesis of cholesterol and fatty
acids
particularly unsaturated fatty acids
established interdependent regulatory control at the transcriptional level for these two lipid
pathways (31). Our study, which shows that the human SCHAD gene
product, a mitochondrial
-oxidation enzyme, functions as an
oxidative 17
-hydroxysteroid dehydrogenase (17
-HSD), establishes interdependence of metabolites in these two pathways upon a
single-domain multifunctional enzyme. Together, these observations
suggest that both the biosynthesis and the catabolism of intermediates
in these two lipid pathways are interrelated at both the level of gene expression and enzyme function. These important observations could have
wide ranging implications.
17
-Hydroxysteroid dehydrogenase is essential for sex steroid
metabolism. The reductive 17
-HSDs catalyze the final step in the
synthesis of potent androgens and estrogens, whereas the oxidative 17
-HSDs inactivate these androgens and estrogens. The various 17
-HSD isoenzymes display different tissue and cellular
distributions and substrate specificities (Table III). Expression of
reductive 17
-HSD isoenzymes across tissue types is directly related
to the formation of the respective sex steroid in each tissue type. For
example, 17
-HSD types 3 and 5 catalyze the formation of testosterone in the testis and peripheral tissues, respectively (22, 24). In
contrast, type 1 17
-HSD converts estrone to the potent estrogen 17
-estradiol in both gonadal and nongonadal tissues, including brain
(32). Reportedly, 30-50% of total androgens in males and 75% of
total estrogens in females prior to menopause are synthesized in
peripheral tissues (33). Oxidative 17
-HSD, on the other hand, play a
significant role in maintaining the steady-state level of each sex
steroid within individual cells. 17
-HSD type 2, expressed in
placenta, endometrium, and liver, inactivates androgens as well as
estrogens (20). 17
-HSD type 4, expressed in organs that are rich in
peroxisomes, catalyzes the oxidation of 17
-estradiol (8, 9). We
found that human SCHAD, expressed in a variety of tissues including
brain, catalyzes the conversion of 17
-estradiol and
dihydrotestosterone to inactive steroids (Table I). This is the first
mitochondrial enzyme found to function in the catabolism of steroid
hormones although several reactions essential for the conversion of
cholesterol into sex steroids and bile acids have been previously shown
to occur in mitochondria. Moreover, the oxidation of androsterone
catalyzed by human SCHAD would yield androstanedione, a potent
inhibitor of the aromatase. Therefore, overexpression of this
dehydrogenase may interfere with the generation of 17
-estradiol from
androgens. After menopause, all estrogens in women are synthesized in
peripheral tissues (33). In this situation, human SCHAD and other
oxidative 17
-HSDs would have an exceedingly profound impact on the
control of intracellular estrogen levels.
Recent clinical studies have suggested that estrogen replacement
therapy can delay or prevent Alzheimer's disease (AD) (34). A number
of putative mechanisms whereby estrogens could interfere with the
progression of AD have been proposed, including organizational and
activational effects on the central nervous system (35), increase of
dendritic spines and synapse formation (36), and reduction of neuronal
generation of amyloid
-peptide (37), among others. Brain extracts of
AD patients contain significantly more human brain SCHAD/ERAB than
those of age-matched controls (2).2 A high concentration of
human brain SCHAD/ERAB would likely cause an estrogen-deficient state
in neurons. The identification of human brain SCHAD as a new 17
-HSD
leads us to propose that high concentrations of human brain SCHAD are a
potential risk factor for AD.
Estrogens reportedly attenuate excitotoxicity, oxidative injury, and
amyloid
-peptide (A
) toxicity in hippocampal neurons (38).
Overexpression of human brain SCHAD in cultured cells would deplete
17
-estradiol, thus accounting for the high sensitivity of such cells
to A
-induced stress and apoptosis described previously (2).
Moreover, it was reported that aldehyde reductase confers protection
upon PC-12 cells to glyoxal cytotoxicity (39), so the alcohol
dehydrogenase activity of human brain SCHAD may play the opposite role
of enhancing the toxicity of aldehydes. Because the human brain
SCHAD/A
complex displays considerable enzymatic activities,2 the binding of A
to human brain SCHAD might
exacerbate the adverse effects of this multifunctional enzyme if it
would cause a relocation of SCHAD. On the basis of these observations,
we believe that tuning down SCHAD expression to restore the protective
effects of estrogen may ameliorate the neuronal dysfunction associated with AD. Additionally, because the human SCHAD gene product is responsible for estrogen inactivation, it would be important to assess
if its expression in malignant cells of estrogen-responsive tissues
such as breast cancer and endometrium cancer differs from that in
normal controls. In our effort to expand insight into this important
multifunctional enzyme, we are currently studying the regulatory
mechanism of human SCHAD gene expression.