Cholesterol Is Superior to 7-Ketocholesterol or
7
-Hydroxycholesterol as an Allosteric Activator for Acyl-coenzyme
A:Cholesterol Acyltransferase 1*
Yi
Zhang
,
Chunjiang
Yu
,
Jay
Liu
,
Thomas A.
Spencer§,
Catherine C. Y.
Chang
, and
Ta-Yuan
Chang
¶
From the
Department of Biochemistry, Dartmouth
Medical School, Hanover, New Hampshire 03755 and the
§ Department of Chemistry, Dartmouth College,
Hanover, New Hampshire 03755
Received for publication, November 13, 2002, and in revised form, January 15, 2003
 |
ABSTRACT |
We compared the abilities of cholesterol
versus various oxysterols as substrate and/or as activator
for the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), by
monitoring the activity of purified human ACAT1 in response to sterols
solubilized in mixed micelles or in reconstituted vesicles. The results
showed that 5
,6
-epoxycholesterol and 7
-hydroxycholesterol are
comparable with cholesterol as the favored substrates, whereas
7-ketocholesterol, 7
-hydroxycholesterol, 5
,6
-epoxycholesterol,
and 24(S),25-epoxycholesterol are very poor substrates for
the enzyme. We then tested the ability of 7-ketocholesterol as an
activator when cholesterol was measured as the substrate, and
vice versa. When cholesterol was measured as the substrate,
the addition of 7-ketocholesterol could not activate the enzyme. In
contrast, when 7-ketocholesterol was measured as the substrate, the
addition of cholesterol significantly activated the enzyme and changed
the shape of the substrate saturation curve from sigmoidal to
essentially hyperbolic. Additional results show that, as an activator,
cholesterol is much better than all the oxysterols tested. These
results suggest that ACAT1 contains two types of sterol binding sites;
the structural requirement for the ACAT activator site is more
stringent than it is for the ACAT substrate site. Upon activation by
cholesterol, ACAT1 becomes promiscuous toward various sterols as its substrate.
 |
INTRODUCTION |
Acyl-coenzyme A:cholesterol acyltransferase
(ACAT)1 is a membrane-bound
enzyme located in the endoplasmic reticulum (ER). It is present in a
variety of cell types and tissues and utilizes two lipophilic
substrates, cholesterol and long-chain fatty acyl-coenzyme A, to
catalyze the formation of neutral lipid cholesteryl esters. In mammals,
two ACAT isoforms exist (ACAT1 and ACAT2) (reviewed in Ref. 1). The
tissue distribution of ACAT1 is essentially ubiquitous, whereas that of
ACAT2 is more restricted. The physiological roles of these isoforms in
various tissues are under active investigation. At the single cell
level, ACAT participates in controlling the cellular membrane
cholesterol level. Unlike many other enzymes involved in cholesterol
metabolism, regulation of ACAT by sterol occurs at the
post-translational level. In mixed micelles or in reconstituted
vesicles, both ACAT1 and ACAT2 display a sigmoidal response to
cholesterol as their substrates (2). These results are consistent with
the concept that the ACAT activity is allosterically regulated by
membrane cholesterol content in the ER (reviewed in Ref. 3).
Oxysterols are sterols containing a second oxygen atom, present as a
carbonyl, hydroxyl, or epoxide group in rings A or B or in the side
chain, in addition to the C3 hydroxyl group. A large number of
oxysterols are found in various locations, including food products,
plasma, or inside the cells (reviewed in Refs. 4 and 5). They are
produced by various enzymes in vivo, and/or by chemical
oxidation in vitro. 24-Hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and
7
-hydroxycholesterol are the four main oxysterols enzymatically
derived (5, 6) and can be found in the plasma and inside certain cell
types. 7-Ketocholesterol, 7
-hydroxycholesterol,
7
-hydroxycholesterol, and 27-hydroxycholesterol are the four major
oxysterols found in human atherosclerotic lesions (5, 7). In
addition, 7-ketocholesterol, 7
-hydroxycholesterol,
7
-hydroxycholesterol, 5
,6
-epoxycholesterol, and
5
,6
-epoxycholesterol are the major oxysterols present in oxidized
low density lipoprotein preparations in vitro (8, 9).
Oxysterols possess a wide range of biological properties and may play
regulatory roles in cholesterol metabolism. For example, when added to
the culture medium of intact cells, most oxysterols, including
7
-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol, greatly suppressed the cholesterol biosynthesis rate (10, 11). Several
oxysterols are high affinity ligands for the nuclear receptor LXR
1
(13, 14), and one of them, 24(S),25-epoxycholesterol, has
been proposed as a participant in cholesterol regulation in the liver
(12). On the other hand, the roles of oxysterols in controlling
cholesterol homeostasis in vivo are still under debate, partly because in various mammalian systems examined, various oxysterols are present in very low concentrations, with much shorter half-lives relative to cholesterol (15).
Oxysterols have also been shown to have effects on ACAT activity. For
example, when added to medium of tissue culture cells, oxysterols such
as 7-ketocholesterol or 25-hydroxycholesterol, in addition to their
suppressive effect on cholesterol biosynthesis rate, stimulated
cholesterol esterification rate and increased ACAT activity (16). When
cholesterol was added in the same manner, it failed to provide the same
response. Despite numerous studies, the mechanism of oxysterol-mediated
activation of cholesteryl ester biosynthesis had not been clarified. It
could be due to the presence of a putative oxysterol binding site
present in ACAT1 or could be due to the ability of oxysterol to
mobilize cellular cholesterol to the ER; other mechanism(s) could not
be ruled out. We and other investigators used various crude cell
extract systems and had demonstrated the apparent activation of ACAT by
25-hydroxycholesterol in vitro (17-19). However, in these
studies, the enzyme ACAT and the sterols (cholesterol and
25-hydroxycholesterol) serving as substrate and/or activator were
present in different membranes. Thus, one could not rule out the
possibility that the apparent activation by 25-hydroxycholesterol was
due to its ability to translocate cholesterol from a cholesterol-rich
membrane to a cholesterol-poor membrane where ACAT is located
(discussed in Ref. 19). Studies were also performed attempting to
determine the sterol substrate specificity of ACAT. When individual
sterols were delivered in vesicle form to the enzyme, Cases et
al. (20) have shown that ACAT seemed to utilize various oxysterols
much more efficiently than cholesterol. Again, these
studies involved the use of crude enzyme extracts, with ACAT and
sterols present in different membranes. Among various sterols, the
ability to move from the donor membrane to the ACAT-containing membrane
differs greatly (21). The transfer rates for oxysterols were much
faster (more than 10 times) than those for cholesterol; these
differences could greatly mask the true sterol specificity for the
enzyme. Thus, the intrinsic substrate specificity of ACAT could not be determined from these studies. In oxidized low density
lipoprotein-loaded macrophages, large amounts of esterified oxysterols
and esterified cholesterol are present in the cytosolic fraction of the
cells; these cytosolic steryl esters are the products of ACAT reaction (7). How ACAT can utilize oxysterols in the presence of large amounts
of cellular cholesterol is not clear, partly because the relative
specificity of ACAT toward oxysterols and cholesterol as substrate or
as activator has not been clarified.
In the current work, we compared the abilities of several selected
oxysterols versus cholesterol as ACAT substrates or as ACAT
activators. The oxysterols evaluated included, as representatives of
ring A or B oxysterols, 7
-, and 7
-hydroxycholesterol,
7-ketocholesterol, and 5
,6
- and 5
,6
-epoxycholesterol and,
as a representative of side-chain oxysterols,
24(S),25-epoxycholesterol. We first placed sterols and the
enzyme in mixed micelles (22), using human ACAT1 purified to
homogeneity as the enzyme source and analyzed the enzyme activity in
response to varying sterol concentrations. In mixed micelles, the
sterol is in direct contact with the enzyme in solution form (23). This
assay system also avoids the formation of sterol microdomain(s). On the
other hand, the environment provided by the mixed micelles is not close
to that of ACAT1 under physiological conditions, because ACAT1 is an
integral membrane protein residing in the ER. We therefore tested the
validity of information learned from using the mixed micelles system by
using the reconstituted vesicle system (24, 25). The latter system
provides an environment close to that of ACAT1 under physiological
conditions. In addition, the sterol and the enzyme ACAT1 reside in the
same vesicles, thus eliminating the sterol transfer step between two
different vesicles prior to enzyme catalysis. Our results show that
ACAT1 can accommodate cholesterol, 5
,6
-epoxycholesterol, and
7
-hydroxycholesterol as its three preferred substrates. In contrast,
for activation of ACAT1, cholesterol is superior to all of the
oxysterols tested, including 7-ketocholesterol,
7
-hydroxycholesterol, 7
-hydroxycholesterol, 5
,6
-epoxycholesterol, 5
,6
-epoxycholesterol, or
7
-hydroxycholesterol. Thus, the structural requirement for sterol as
an ACAT activator is more stringent than it is for sterol as an ACAT substrate.
 |
EXPERIMENTAL PROCEDURES |
Materials
Cholesterol, 7
-hydroxycholesterol,
5
,6
-epoxycholesterol, 5
,6
-epoxycholesterol,
7-ketocholesterol,
-sitosterol, CHAPS, taurocholate, oleoyl-coenzyme
A, egg phosphatidylcholine (PC), cholesteryl oleate, fatty acid-free
bovine serum albumin, 4-dimethylaminopyridine, imidazole, oleic
anhydride, triethylamine, and primulin dye were all from Sigma.
7
-Hydroxycholesterol, 25-hydroxycholesterol, and
27-hydroxycholesterol were from Steraloids.
24(S),25-epoxycholesterol was synthesized as previously
described (26, 27). All of the sterols showed single spots in TLC
analysis and were used without further purification.
2,6-Di-tert-butyl-p-cresol was from Eastman Kodak
Co. The Centrisart ultrafiltration device (cut-off molecular mass, 300 kDa) was from Sartorius. Organic solvents used were reagent grade and were from Fisher. Grace's insect cell medium was
from Invitrogen. The software program Prism (GraphPad Software, Inc.)
was from Sigma.
Methods
Enzyme Purification--
The source of enzyme was recombinant
human ACAT1 expressed in insect Hi5 cells and purified to
electrophoretic homogeneity. The purification procedure was as
described previously (22, 28), using nickel column chromatography and
ACAT1 monoclonal column chromatography. For some of the experiments
reported in this work (see Fig. 4), the enzyme source used was
HisACAT1
1-65 (29). The ACAT1 monoclonal antibody only
recognizes the N terminus of the enzyme. The
HisACAT1
1-65 enzyme lacked the N-terminal and thus
could only be partially purified, using nickel column chromatography
(29).
ACAT Enzyme Activity Assay--
The enzyme was assayed in
sterol/PC/taurocholate mixed micelles or in reconstituted vesicles
conducted as described previously (22), using the radioactive substrate
[3H]oleoyl coenzyme A at 4 × 104
dpm/nmol. The mixed micelles were prepared as described previously (22), containing varying sterol concentrations, reported as mol % sterol/sterol + PC as indicated in various figures, in 11.2 mM PC and 18.6 mM taurocholate. For assays that
contained two different sterols, each sterol/PC/taurocholate mixed
micelles sample was made separately and then mixed together and used
within 6 h. The reconstituted vesicles were prepared essentially
as described previously (30, 31). In the current work, we used
taurocholate instead of cholate as the detergent to prepare the bile
salt/PC/sterol micelles, with taurocholate at 18.6 mM and
the PC at 11.2 mM. We then used cholestyramine to remove
bile salt from mixed micelles, which rapidly led to vesicle formation.
Control experiments using radioactive taurocholate showed that after
two treatments of cholestyramine, more than 99% of the taurocholate
was removed from the resultant vesicles.
Examining the Sterol Solubility in Mixed Micelles--
The
sterol solubility in micellar solutions was monitored by subjecting the
solutions to ultracentrifugation (at 50,0000 × g for
40 min) followed by ultrafiltration through a Sartorius Centrisart
ultrafiltration device that contains a membrane with a molecular mass
cut-off of 300 kDa as described (32). After the ultracentrifugation and
the ultrafiltration steps, the sterols were quantitated according to
the method described previously (33). Briefly, the lipids present in
the untreated samples or in the supernatants after treatment were
extracted with chloroform/methanol (2:1), spotted onto the TLC plate,
and separated using the solvent system hexane/acetone/acetic acid = 80:20:1 (v/v/v). The plate was sprayed with a 0.05% solution of
primulin dye and then scanned by using the STORM 860 imaging system to
detect the laser-excited fluorescent signals. Spots of a given sterol
were quantitated by integration of variable pixel intensities using the
ImageQuant software. Standard curves were produced by quantitating
increasing amounts (10-200 µg) of a given sterol sample spotted in
parallel lanes.
Chemical Synthesis of Sterol Oleate Esters--
Various
nonradioactive sterol oleate esters were used as internal markers and
visualized by iodine staining after TLC analysis. Other than
cholesteryl oleate and 25-hydroxycholesterol (from Steraloids), the
sterol esters described in the current work were not commercially
available. They were chemically synthesized based on the general
instruction provided by Molecular Probes, Inc. (Eugene, OR). Typically,
5 µmol of a given sterol was weighed in an amber vial, and 0.166 ml
of methylene chloride was added and swirled briefly. 0.075 ml of
triethylamine, 3 mg of oleic anhydride, and 0.042 mg of
4-dimethylaminopyridine as catalyst and 2 µl of 0.1%
2,6-di-tert-butyl-p-cresol in methylene chloride as antioxidant were then added. The vial was sealed under nitrogen and
stirred with a magnetic stir bar overnight in the dark. 12 h
later, the same amounts of oleic anhydride, 4-dimethylaminopyridine, and 2,6-di-tert-butyl-p-cresol described before
were added, and the reaction was continued for an additional 10 h.
The sterol oleates synthesized were used as the internal markers for
TLC without purification. When TLC plates were run in a petroleum ether/ether/acetic acid (90:10:1) solvent system, the
Rf values for oleates of the following sterols were
as follows: cholesterol, 0.87; 5
,6
-epoxycholesterol, 0.51;
5
,6
-epoxycholesterol, 0.58; 7-ketocholesterol, 0.36;
7
-hydroxycholesterol, 0.21; 7
-hydroxycholesterol, 0.28;
-sitosterol, 0.89; 25-hydroxycholesterol, 0.30;
24(S),25-epoxycholesterol, 0.46.
 |
RESULTS |
In order to quantitatively compare the abilities of various
sterols that serve as ACAT substrate and/or activator, a single phase
system composed of the enzyme, phospholipid, and sterol is needed. We
had previously developed a mixed micelles system for measuring ACAT
activity. This system consisted of mixing egg PC, taurocholate, and
cholesterol at appropriate ratios, followed by bath-sonication at cold
temperature until optical clearance. The enzyme ACAT solubilized in low
concentration of the detergent CHAPS was then added to the mixed
micelles before the reaction began. It is possible that the micelles
prepared by sonication may still contain a minor portion of sterol in
the form of supersaturated microcrystals and/or multilamellar or
unilamellar vesicles. To remove microcrystals and/or vesicles from the
micelles, Moschetta et al. (32) developed a simple and
effective procedure that involved ultracentrifugation and
ultrafiltration. We used this procedure to monitor the quality of the
mixed micelles that contained either cholesterol, or 7-ketocholesterol,
or 5
,6
-epoxycholestanol. The results showed that essentially
100% of these three sterols existed in the micellar phase at
concentrations from 0.25 to 2 mM (which is equivalent to
0.022-0.15 mol % sterol/sterol + PC) (data not shown). Other sterols,
including 5
,6
-epoxycholesterol,
-sitosterol,
7
-hydroxycholesterol, and 7
-hydroxycholesterol, behaved in the
same manner (results not shown). In contrast, oxysterols that contain
the hydroxy groups at the side chain, including 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, could only produce clear micellar solutions at no more than 0.5 mM (0.043 mol
% sterol/sterol + PC). Therefore, in our current study, we have
focused our effort on comparing various sterols that readily form mixed
micelles. We compared the ability of seven sterols to serve as
substrates of ACAT by varying their concentrations from 0.047 to 2.0 mM, which amount to 0.004-0.15 mol % sterol/sterol + PC.
In some experiments, the highest concentrations were extended to 2.3 mM (0.17 mol % sterol/sterol + PC). The solubility of
24(S),25-epoxycholesterol was higher than that of oxysterols with
hydroxy groups at the side chain; this property allowed us to study
24(S),25-epoxycholesterol at concentrations between 0.0047 and 0.1 mol % sterol/sterol + PC. The results (Fig.
1) show that 5
,6
-epoxycholestanol,
cholesterol, and 7
-hydroxycholesterol are the three best substrates.
All three sterols exhibit sigmoidal substrate saturation curves; the
K0.5 values (the concentration at which
half-maximal velocity is achieved) for these three sterols were
similar, varying between 0.7 and 0.9 mol % sterol/sterol + PC. At
sterol concentrations above K0.5, 5
,6
-epoxycholestanol is a slightly better substrate than
cholesterol, whereas 7
-hydroxycholesterol is ~70% as efficient as
cholesterol as the substrate (results of three independent
experiments). The other three sterols tested, 7-ketocholesterol,
7
-hydroxycholesterol, and
-sitosterol (a major plant sterol) were
all vastly inferior to cholesterol as the substrate. In addition,
24(S),25-epoxycholesterol tested at concentrations up to 0.1 mol % sterol/sterol + PC is also a poor substrate. The
inset showed the same data using a smaller scale to report
the ACAT activity. It demonstrates that ACAT1 can definitely use any of
the latter four sterols as substrates although in a far less efficient
manner. We next tested the effect of cholesterol when 7-ketocholesterol
serves as the substrate, and vice versa. The results (Fig.
2A) show that cholesterol
added at low concentrations (from 0.01 to 0.1 mol % sterol/sterol + PC) significantly activated the enzyme when 7-ketocholesterol was
measured as the substrate. It also changed the substrate saturation curve from being sigmoidal to essentially hyperbolic. Calculations (using the software program Prism) showed that the Hill coefficient decreased from 3.0 without cholesterol to 1.1 with cholesterol added at
0.1 mol % sterol/sterol + PC. The K0.5 value
decreased from 0.13 mol % sterol/sterol + PC without cholesterol to
0.04 mol % sterol/sterol + PC with cholesterol. The
Vmax value increased from 150 nmol/min without
cholesterol to 200 nmol/min with cholesterol. The activation effect by
cholesterol shown in Fig. 2A could not be explained, because
cholesterol is a better substrate and binds to the catalytic site more
efficiently. If this were the case, cholesterol should have caused
severe inhibition when 7-ketocholesterol was measured as the substrate.
When cholesterol was measured as the substrate, 7-ketocholesterol added
at low concentrations (from 0.005 to 0.025 mol % sterol/sterol + PC)
caused slight inhibition of the enzyme (Fig. 2B). The
inhibition was more prominent at higher cholesterol concentrations.
Thus, the inhibitory effect of 7-ketocholesterol is presumably through
substrate competition. 7-Ketocholesterol did not alter the sigmoidicity
of the cholesterol substrate saturation curve. Calculations showed that
the K0.5 value for cholesterol stayed at 0.07 mol % sterol/sterol + PC, and the Hill coefficient stayed around 2.2, with or without 7-ketocholesterol added.

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Fig. 1.
Sterol substrate saturation curves of
HisACAT1 in mixed micelles. The mixed micelles were prepared as
described under "Experimental Procedures." The ACAT activity
assays were performed in duplicate. Points shown were averages of
duplicate assays; error bars indicated variations
from the mean. The results shown are representative of two separate
experiments. The inset shows replot of the same data with
smaller scale in the ACAT activity.
5 ,6 -epoxy,
5 ,6 -epoxycholesterol;
5 ,6 -epoxy,
5 ,6 -epoxycholesterol; 7-keto, 7-ketocholesterol;
7 OH, 7 -hydroxycholesterol;
7 OH, 7 -hydroxycholesterol;
24(S),25-epoxy, 24(S),25-epoxycholesterol;
-sito, -sitosterol.
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Fig. 2.
Sterol substrate saturation curves of
HisACAT1 with both cholesterol and 7-ketocholesterol present in mixed
micelles. The final concentrations of cholesterol or
7-ketocholesterol were as indicated. A, 7-ketocholesterol
substrate saturation curve in the presence of the indicated
concentrations of cholesterol. B, cholesterol substrate
saturation curve in the presence of the indicated concentrations of
7-ketocholesterol. The assays were done in duplicate. The results shown
are representative of two separate experiments.
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|
Human ACAT1 is a homotetrameric enzyme (34). We had previously shown
that by deleting the first 65 amino acid residues from the N-terminal,
the enzyme could be converted to a dimeric form. This form was
designated as hACAT1
1-65. The dimeric enzyme is 5-10
times more active than the native ACAT1 in terms of catalytic
efficiency (29). We compared the effect of cholesterol and
7-ketocholesterol using hACAT1
1-65 as the enzyme source. The results show (Fig.
3A) that cholesterol
significantly activated the enzyme activity when 7-ketocholesterol was
measured as the substrate, whereas 7-ketocholesterol added had minimal effect on the enzyme activity when cholesterol was measured as the
substrate (Fig. 3B). Thus, the ability of cholesterol to
activate the enzyme does not require the enzyme to exist at the
tetrameric form.

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Fig. 3.
Sterol substrate saturation curves of
HisACAT1 1-65, with both
cholesterol and 7-ketocholesterol present in mixed micelles.
HisACAT1 1-65 was partially purified as described under
"Experimental Procedures." A, 7-ketocholesterol
substrate saturation curve in the presence of indicated concentration
of cholesterol (at 0 or 0.04 mol %). B, cholesterol
substrate saturation curve in the presence of indicated concentration
of 7-ketocholesterol (at 0 or 0.04 mol %). The assays were done in
duplicate.
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We next tested the effect of cholesterol when
5
,6
-epoxycholestanol was measured as the substrate, and
vice versa. The results showed that cholesterol added at low
concentrations significantly activated the enzyme when
5
,6
-epoxycholesterol was used as the substrate, whereas
5
,6
-epoxycholesterol added inhibited the enzyme when cholesterol
was measured as the substrate (data not shown). This result suggested
that cholesterol may be a better activator than
5
,6
-epoxycholesterol, although the latter sterol is a slightly
better substrate than cholesterol (Fig. 1). To test this
interpretation, we next used 7-ketocholesterol as the substrate at two
different concentrations (at 0.08 or 0.12 mol % sterol/sterol + PC)
and compared cholesterol versus various other sterols as indicated, including 5
,6
-epoxycholesterol, for their abilities to
activate the enzyme. The results (Fig. 4)
showed that among all the sterols tested, cholesterol is the only
sterol that caused significant activation of the enzyme.

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Fig. 4.
Effects of various sterols as indicated (at
0.04 mol %) on HisACAT1 when 7-ketocholesterol was measured as the
substrate. The concentration of 7-ketocholesterol was fixed at
0.08 or 0.12 mol %. The results shown were averages of duplicate
assays.
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In intact cells, ACAT resides mainly in the ER and uses sterol present
in the ER as the enzymatic substrate (3). To measure ACAT activity in a
system that is similar to the ER, we next tested the effects of various
sterols present in vesicle form. To avoid the step of sterol transfer
between donor vesicles to the vesicles where ACAT resides, we had
previously developed a reconstituted vesicle system by diluting the
ACAT solubilized in detergent into a large excess of preformed vesicles
with defined sterol and PC composition (24). We now used the
reconstituted vesicle system to compare the abilities of cholesterol,
5
,6
-epoxycholesterol, and 7-ketocholesterol to serve as ACAT
substrates. The results show (Fig. 5)
that 5
,6
-epoxycholesterol and cholesterol are much better
substrates than 7-ketocholesterol. For each of the sterols tested, the
enzyme responded to the sterol content present in the vesicles in a
sigmoidal-like manner. We next tested the effect of cholesterol on ACAT
activity when 7-ketocholesterol was measured as the substrate, and
vice versa. The results show that cholesterol added at low
concentrations significantly activated the enzyme when
7-ketocholesterol (Fig. 6A)
was measured as the substrate, whereas 7-ketocholesterol (Fig.
6B) added inhibited the enzyme, presumably through substrate
competition, when cholesterol was measured as the substrate. In data
not shown, we also tested the effect of cholesterol on ACAT activity
when 5
,6
-epoxycholestanol was measured as the substrate, and
vice versa, and have obtained essentially the same results
as described in Fig. 6, A and B. To further test
this finding using the reconstituted vesicle system, we used
7-ketocholesterol as the variable substrate and compared cholesterol
and 5
,6
-epoxycholesterol, added at 0.4 mol % sterol/sterol + PC,
for their ability to activate the enzyme. The results (Fig. 7) show that cholesterol is superior to
5
,6
-epoxycholesterol as the activator. Thus, the results using
the reconstituted vesicles system fully corroborated the results using
the mixed micelles system.

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Fig. 5.
Sterol substrate saturation curves of
HisACAT1 in reconstituted vesicles. Reconstituted vesicles
containing cholesterol, 7-ketocholesterol, or
5 ,6 -epoxycholesterol at the indicated concentrations were
prepared as described under "Experimental Procedures." Results were
averages of duplicate assays; error bars indicate
variations from the mean.
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Fig. 6.
Sterol substrate saturation curves of
HisACAT1 with both cholesterol and 7-ketocholesterol present in
reconstituted vesicles. The cholesterol/PC/taurocholate and
7-ketocholesterol/PC/taurocholate mixed micelles were made separately
and then mixed and treated with cholestyramine to produce the
reconstituted vesicles. A, 7-ketocholesterol substrate
saturation curves with cholesterol at the indicated concentration (0 or
0.04 mol %). B, cholesterol substrate saturation curves
with 7-ketocholesterol at the indicated concentration (0 or 0.04 mol
%).
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Fig. 7.
7-Ketocholesterol substrate saturation curves
of HisACAT1 with cholesterol or 5 ,6 -epoxycholesterol present in
reconstituted vesicles. The final concentration of cholesterol or
5 ,6 -epoxycholesterol added was at 0.04 mol %. The assays were
done in duplicate.
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 |
DISCUSSION |
In the current work, we employed the mixed micelles assay system
to compare the abilities of various sterols to serve as a substrate for
ACAT1. The results show that cholesterol and 5
,6
-epoxycholesterol are the two best substrates tested. 7
-Hydroxycholesterol is the third best substrate, being 70% as efficient, whereas other sterols such as 7-ketocholesterol were 10% or less as efficient. Modification of cholesterol by including the 7
-hydroxy moiety in steroid ring B
is moderately tolerated as the enzymatic substrate. We then developed a
method to compare the abilities of various sterols serving as ACAT
activators. The results show that cholesterol is far superior as an
activator to all other sterols tested, including 5
,6
-epoxycholesterol or 7
-hydroxycholesterol. Thus, the
structural specificity of the activator site is much more stringent
than its sterol substrate site. The results obtained by using the mixed micelles have essentially been confirmed by using the reconstituted vesicles system.
In bile salt-based mixed micelles, a sterol-specific microdomain(s) has
not been reported. In PC-based vesicles, the physical properties of
cholesterol, 5
,6
-epoxycholestanol, 7
-hydroxycholesterol, 7
-hydroxycholesterol, and 7-ketocholesterol have previously been shown to be similar to those of cholesterol (19, 35, 36). Therefore, we
believe that the sterol specificity demonstrated in our current work is
mainly due to the intrinsic specificity of the enzyme, not due to
subtle difference in biophysical properties of sterols in micelles or
in vesicles. We also show that the ability of cholesterol to serve as
an activator is preserved when the oligomeric structure of ACAT changes
from the tetrameric form to a dimeric form. To explain these results,
we propose the following model: ACAT1 contains a sterol substrate site
and an allosteric sterol activator site. The activator site is
restricted to cholesterol only, whereas the substrate site is more
promiscuous. When 7-ketocholesterol is the substrate, activation by
cholesterol at low concentration decreases the
K0.5 value as well as increasing the
Vmax value toward 7-ketocholesterol. When
5
,6
-epoxycholesterol is the substrate, activation by cholesterol
at low concentration decreases the K0.5 value
without affecting the Vmax value of the enzyme
toward 5
,6
-epoxycholesterol. In the future, using various
biochemical approaches that include photoaffinity labeling and
site-specific mutagenesis, studies can test the validity of this model.
In oxidized low density lipoprotein-loaded macrophages, a large amount
of esterified oxysterols including 7-ketocholesterol are found; those
present in the cytosol were derived from ACAT reaction (7). These
observations could be explained based on our model; ACAT1 present in
macrophages is activated by cholesterol in the cholesterol-rich
environment and becomes more efficient in using 7-ketocholesterol as an
alternative substrate, thus causing an ample amount of
7-ketocholesterol to be esterified. In addition, as shown in our
current work,
-sitosterol (a major plant sterol) is a poor ACAT1
substrate and a poor activator. Our model predicts that
-sitosterol may become a much better substrate of ACAT1 when
the enzyme is under high cholesterol conditions. In the future, this
prediction could be tested under various physiological conditions.
Related to our current work, Brown et al. (37) have recently
shown that when added as cyclodextrin complex, cholesterol, 7-ketocholesterol, 7
-hydroxycholesterol, or other structurally related sterols added to the ER membranes in vitro causes
conformational change of the sterol-sensing protein SCAP. Oxysterols
with hydroxy groups at the side chain, such as 25-hydroxycholesterol or
27-hydroxycholesterol, fail to initiate this change. SCAP is a key
factor involved in sterol-specific transcriptional regulation of
cholesterol biosynthesis. The sterol specificity demonstrated in their
system strongly suggests that it is cholesterol itself that acts on the
sterol regulatory machinery. The authors did not test the abilities of
the sterols to cause conformational change of SCAP in a
dose-dependent manner. Our results reported here show that
cholesterol is superior to either 7-ketocholesterol or 7
-cholesterol
as an ACAT activator in vitro. Both ACAT1 and SCAP are
integral membrane proteins that mainly reside in the ER. Our results
thus reinforce the results of Brown et al. (37), supporting
the concept that the content of cholesterol rather than the content of
an oxysterol such as 7-ketocholesterol or 7
-hydroxycholesterol
located in the ER plays a pivotal role in the regulation of
intracellular cholesterol metabolism. The fact that 7-ketocholesterol
added in intact cells caused significant stimulation in esterification
of cholesterol (16) could be explained by its ability to cause
translocation of cholesterol from the plasma membrane to the ER, where
ACAT resides (3, 38).
Due to their limited solubility in mixed micelles, we could not perform
extensive testing on various oxysterols with hydroxymoiety at the side
chain. We did, however, find that 25-hydroxycholesterol at
concentrations up to 0.05 mol % was also a poor substrate for ACAT1.
When cholesterol was measured as a substrate, 25-hydroxycholesterol added at low concentration failed to increase ACAT activity, whereas cholesterol added at low concentration greatly increased the ACAT activity in utilizing 25-hydroxycholesterol as the substrate (results not shown). In lipid membranes, the oxysterols with hydroxy groups at
the side chain are very different from cholesterol in terms of their
biophysical properties (21, 35, 36). Kauffman et al. (39)
proposed that 25-hydroxycholesterol and 27-hydroxycholesterol might
prefer to interact with PC in "reverse orientation"
(i.e. with its hydrophilic side chain lining up with the
polar moiety of the PC molecules in membranes). Based on earlier
studies and the current results, if oxysterols serve as important
regulators for ACAT activity in intact cells, they may act through
certain novel mechanism(s) rather than by incorporating into the ER
membranes and being recognized by ACAT as a preferred activating molecule.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Keith Suckling and Brian
Jackson of GlaxoSmithKline Pharmaceuticals and members of our
laboratory for stimulating discussions, and we thank Helina Morgan for
careful editing of the manuscript.
 |
FOOTNOTES |
*
This work is supported by National Institutes of Health
Grant HL60306 (to T. Y. C.).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. Tel.:
603-650-1622; Fax: 603-650-1128; E-mail:
Ta.Yuan.Chang@Dartmouth.edu.
Published, JBC Papers in Press, January 17, 2003, DOI 10.1074/jbc.M211559200
 |
ABBREVIATIONS |
The abbreviations used are:
ACAT, acyl-coenzyme
A:cholesterol acyltransferase;
CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid;
ER, endoplasmic reticulum;
PC, phosphatidylcholine.
 |
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