Research Paper
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Acta Biochim Biophys Sin
2005,37: 797–806
doi:10.1111/j.1745-7270.2005.00118.x
Two Human ACAT2 mRNA Variants
Produced by Alternative Splicing and Coding for Novel Isoenzymes
Xiao-Min YAO1,2#, Can-Hua WANG1,2#, Bao-Liang SONG1, Xin-Ying YANG1, Zhen-Zhen WANG1, Wei QI1, Zhi-Xin LIN2, Catherine C. Y. CHANG3, Ta-Yuan CHANG3, and Bo-Liang LI1*
1 State
Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China;
2 Department
of Biochemistry and Technology, Shanghai Jiaotong University, Shanghai 200030,
China;
3 Department
of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756, USA
Received: September
12, 2005
Accepted: October
10, 2005
This work was
supported by the grants from the Major State Basic Research Development Program
of China (2002CB513000), Shanghai Science and Technology Committee, Chinese
Academy of Sciences, and National Institutes of Health (HL60306)
# These authors
contributed equally to this work
*Corresponding
author: Tel, 86-21-54921278; Fax, 86-21-54921011; E-mail, [email protected]
Abstract Acyl coenzyme A:cholesterol acyltransferase
2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is
highly expressed in small intestine and fetal liver, but its expression is
greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a
protein, designated ACAT2a, with 522 amino acids. We have previously reported
the organization of the human ACAT2 gene and the differentiation-dependent
promoter activity in intestinal Caco-2 cells. In the current work, two human
ACAT2 mRNA variants produced by alternative splicing are cloned and predicted
to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379
amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack
of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA).
Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA
variants were detected by RT-PCR, and Western blot analysis confirmed the
presence of their corresponding proteins in human liver and intestine cells.
Furthermore, phosphorylation and enzymatic activity analyses demonstrated that
the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD,
and their enzymatic activities reduced to 25%–35%
of that of ACAT2a. These evidences indicate that alternative splicing produces
two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our
findings might help to understand the regulation of the ACAT2 gene expression
under certain physiological and pathological conditions.
Key words ACAT2 gene; alternative splicing; ACAT2
mRNA variant; isoenzyme
Acyl coenzyme A:cholesterol acyltransferase (ACAT) is a
membrane-bound enzyme present in eukaryotic cells. It catalyzes the formation of
cholesterol ester from long-chain fatty acyl coenzyme A and cholesterol [1],
and participates in various physiological processes including cellular
cholesterol homeostasis in tissue cells, dietary cholesterol absorption in
small intestine, apoB-containing lipoprotein assembly in small intestine and
liver, and steroidgenesis in the adrenal glands [2–4]. ACAT is also involved
in the pathophysiological processes that lead to various human diseases, such
as atherosclerosis [5–7] and Alzheimer’s disease [8,9]. The ACAT inhibitor can decrease
foam cell formation and amyloid b-peptide production [8,10]. For these reasons,
ACAT has been one of the major pharmaceutical targets for developing
cholesterol-lowering and anti-atherosclerosis agents [5–9].So far, two ACAT (ACAT1 and ACAT2) genes have been characterized in
eukaryotes. Chang et al. first reported human ACAT1 cDNA in 1993 [11].
According to the knockout experiments and the conserved sequences of the ACAT1
gene, ACAT2 cDNA was identified in monkey, mouse or human by different
laboratory [12–14]. The organizations of their genomic DNAs were then investigated
[15–17].
In humans, ACAT1 is ubiquitously expressed in various tissues and cells,
whereas ACAT2 is highly expressed in small intestine and fetal liver but is
greatly diminished in adult liver [18–21]. In differentiating intestinal Caco-2
cells, ACAT2 protein content increases by 5- to 10-fold, but ACAT1 protein
content remains relatively constant [20]. So far, it has been reported that the
expression of human ACAT1 gene is regulated at different levels [22–24]. We have
reported that human ACAT2 gene promoter displays cell-type specific and
differentiation-dependent activity in intestinal Caco-2 cells [16]. To provide
the molecular basis for regulation and function of the ACAT2 gene expression,
here we report that alternative splicing produces the two human ACAT2 mRNA
variants that encode two novel ACAT2 isoenzymes without phosphorylatable site
SLLD and with lower enzymatic activity.
Materials and Methods
Reagents
All the restriction endonucleases, pGEM-T easy vector and moloney
murine leukemia virus (MMLV) reverse transcriptase were ordered from Promega
(Madison, USA). The expression vector pcDNA3, phenylmethylsulfonyl fluoride (PMSF),
Trizol total RNA extraction kit and cell culture reagents were purchased from
Invitrogen (Carlsbad, USA). Anti-ACAT2 antibodies DM54, described previously,
and A21, newly prepared, were used for Western blot analysis [25]. Goat
antirabbit IgG conjugated with horseradish peroxidase (HRP) was from Pierce
(Rockford, USA). Anti-b-actin antibody and enhanced chemiluminescence (ECL) detection reagent
were from Santa Cruz Biotechnology (Santa Cruz, USA). Protease inhibitor
cocktail was from Sigma (St. Louis, USA). Taq DNA polymerase and dNTPs
were from Sino-American Biotech (Shanghai, China). Dithiothreitol (DTT) was
from Amersham Biosciences (Piscataway, USA). GSH-Sepharose 4B and [g–32P]ATP
(3000 Ci/mmol) were from Amersham Pharmacia Biotech (Uppsala, Sweden). All the
oligonucleotides were synthesized with an automated DNA synthesizer in the
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences. [3H]oleoyl-coenzyme
A was synthesized as described previously [26].
Cell culture and human tissues
All the cell lines used were maintained in basal medium supplemented
with fetal bovine serum (FBS) and antibiotics in a 37 ?C incubator with 5% CO2 and
95% air. The human colon carcinoma cell line Caco-2 was maintained in
Dulbecco’s modified Eagle’s medium containing 20% FBS, 100 U/ml of penicillin
and 100 U/ml of streptomycin; cells seeded for 4, 6, 8 or 12 d were designated
as Caco-2 cell differentiation for 0, 2, 4 or 8 d (0, 2, 4 or 8 d
post-confluency), respectively. Human hepatoblastoma cell line HepG2 and
cervical cancer cell line HeLa were maintained in the same medium as Caco-2,
except that it also contained 10% FBS. Human acute monocytic leukemia cell
line THP-1 was maintained in RPMI 1640 medium containing 10% FBS. Human
embryonal kidney cell line HEK293 and Chinese hamster ovary cell line AC29
were grown in minimal essential medium and Ham’s F12 medium containing 10%
FBS, respectively. The human tissues were obtained from national hospitals
(Shanghai, China). Consents for removal of tissues for research purposes were
obtained from donors or their relatives. The tissues were rapidly frozen and
stored in liquid nitrogen until use.
Reverse
transcription-polymerase chain reaction (RT-PCR)
The 4 mg of total RNA prepared from cultured cells or tissues by the single
step acid guanidinium thiocyanate phenol chloroform method (Invitrogen) was
annealed with 0.5 mg of oligo(dT18) in a total volume of 20 ml containing 50 mM
Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 10 mM DTT.
The single-stranded cDNAs (ss-cDNAs) were synthesized by adding 1 ml (200 U) MMLV
reverse transcriptase and 1 ml of dNTPs (10 mM of each of dATP, dCTP, dGTP and dTTP) to the
annealed products and incubating at 37 ?C for 60 min. The 1 ml of ss-cDNA was
added to a PCR mixture in a final volume of 50 ml containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of
each of dATP, dCTP, dGTP and dTTP, 0.4 mM of each set of the
forward/reverse primers, and 2.5 U of Taq DNA polymerase. The control
experiments were performed to assure that the PCR product was amplified within
the linear amplification range. PCR products (10 ml) were separated and
stained on agarose gel, and quantified using UVP Labwork (Version 4.0)
software. The sequences of forward and reverse primers used in PCR to amplify
the different fragments of human ACAT2 cDNA are listed in Table 1. The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment (291 bp)
was amplified as the control using the forward/reverse primer set GAPDHF (5‘-ACCACAGTCCATGCCATCAC-3‘)/GAPDHR
(5‘-TCCACCACCCTGTTGCTGTA-3‘).
Plasmids
The whole coding sequences of human ACAT2 cDNA fragments were amplified
by RT-PCR using the forward/reverse primer set C41/C42, cloned into pGEM-T easy
vector, and identified by digestion of the restriction enzyme EcoRI and
DNA sequencing. The human ACAT2 cDNA fragments amplified from the identified
plasmids pGEM-ACAT2a, pGEM-ACAT2b and pGEM-ACAT2c were digested with EcoRI
and XbaI, then inserted into the EcoRI/XbaI sites of the
vector pcDNA3 to generate three expression plasmids pACAT2a, pACAT2b and
pACAT2c.
Transfection
The human ACAT2 expression plasmids were individually transfected
into AC29 cells using the calcium phosphate co-precipitation method [27].
Briefly, cells were cultured in 60 mm dishes at a density of 5?105 cells per 5 ml of culture medium containing 10% FBS for 24 h, and
fed with fresh medium for another 2 h before transfection. The 1.2 ml of
transfection mixture (containing 12 mg DNA) for a 60 mm dish was prepared by gently
mixing equal volumes of DNA (125 mM calcium ion at final concentration) and
HeBS (21 mM HEPES, pH 7.1, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4 and
6 mM glucose at final concentration), added into the medium and incubated with
the cells at 37 ?C for 9 h. The transfected cells were rinsed twice with
phosphate buffered saline (PBS) and grown for 48 h in culture medium for Western
blot analysis and ACAT activity assay.
Western blot
The cell lysates from the cultured cells were prepared by harvesting
with a lysis buffer containing 10% SDS, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100
mM DTT, and 1% (V/V) protease inhibitor cocktail, incubating at
37 ?C for 20 min, then shearing with a syringe fitted with an 18-G needle to
homogeneity. Protein concentrations of the cell lysates were determined by a
modified Lowry method [28]. The protein samples were then subjected to 12%
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After
gel separation, the proteins were transferred to a nitrocellulose membrane. The
transferred membrane was treated successively at room temperature with the
following solutions: 5% milk in TBST (50 mM Tris-HCl, pH 7.6, 0.15 M NaCl, and
0.05% Tween-20) for 2 h, the affinity-purified rabbit anti-ACAT2 IgGs or anti-b-actin
antibodies (control) for 3 h, and HRP-conjugated goat anti-rabbit antibodies
for an additional 1 h. After incubation, the membrane was washed extensively
with TBST, then with TBS (50 mM Tris-HCl, pH 7.6, and 0.15 M NaCl). The
immunoreactive bands were visualized using ECL detection reagent and developed
with Hyperfilm-ECL (Amersham Pharmacia Biotech).
In vitro phosphorylation assay
The in vitro phosphorylation assay was performed as described
previously with slight modifications [29]. Briefly, AC29 cells were cultured in
a 60 mm dish, washed twice with PBS in an ice-bath, then incubated with 350 ml of a lysis buffer
(10 mM Tris-HCl, pH 7.4, 1.0% Triton X-100, 0.5% NP-40, 150 mM NaCl, 1.0 mM
EDTA, and 0.2 mM PMSF) at 4 ?C for 30 min. The cell lysates were harvested,
sheared with a syringe fitted with a 26-G needle to homogeneity, and
centrifuged at 16,000 g at 4 ?C for 30 min. The supernatant obtained was
mixed with an equal volume of dilution buffer (10 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.2 mM PMSF, 20 mM MgCl2 and 1 mM DTT) as a casein kinase II (CK2)
mixture. The fusion proteins GST-ACAT2aN and GST-ACAT2bN were expressed in Escherichia
coli harboring the GST (227 amino acids) fusion expression plasmids and
purified with the GSH-Sepharose 4B column (Amersham Pharmacia Biotech) as
described previously [30]. The phosphorylation reactions were performed by
adding the purified fusion protein (2 mg GST-ACAT2aN or 6 mg GST-ACAT2aN), [g–32P]-ATP
(50 mCi) and cold ATP (final concentration 25 mM) to 200 ml of CK2
mixture, and incubating at 37 ?C for 15 min. The 20 ml of GSH-Sepharose 4B beads
were added to the phosphorylated reaction mixture and incubated at 4 ?C for 1
h. The precipitate was harvested by centrifugation and washed four times using
PBS. The pellets were run on SDS-PAGE, and then the gel was
subsequently stained with Coomassie brilliant blue and autoradiographed with the
PhosphorImager scanning system (Molecular Dynamics, Sunnyvale, USA).
ACAT activity assay
The ACAT activity assay using the in vitro reconstitution
method was performed as described previously [25].
Results
Multiple human ACAT2 mRNAs
detected by RT-PCR
We have reported the organization of human ACAT2 genomic DNA and
cloned its promoter [16]. It was also noticed that the different-sized fragments
of human ACAT2 cDNA were amplified using total RNA from intestinal Caco-2
cells by RT-PCR (data not shown), suggesting that there might be more than one
kind of human ACAT2 mRNA produced in the tissue cells. To address this
possibility, RT-PCR using various primers [Table 1 and Fig. 1(A)]
was performed with the total RNAs prepared from human cell lines and tissues.
As shown in Fig. 1(B), using a forward/reverse primer set C31/C21, two
cDNA fragments, one with the predicted 894 bp and another smaller (arrowed),
were respectively amplified from the RT products of HepG2, Caco-2, fetal lung,
fetal liver and cancer liver; only one bigger band (894 bp) with very low
amount was observed from that of THP-1; and there are no detectable from those
of HEK293, HeLa, adult liver and adjacent benign liver matched with the cancer
liver. Significantly, comparable amounts of the two kinds of ACAT2 mRNA were
detected in the fetal tissues and cancer liver. Further experiments using the
other forward/reverse primer set C41/C14 or C41/C21 for amplification
consistently revealed the fragments with the predicted size (489-bp or
1372-bp) and smaller size (arrowed) [Fig. 1(C), lanes 2, 4 or 7, 8],
confirming that multiple ACAT2 mRNAs were produced in human cells.
Human ACAT2 mRNA variants
matured by alternative splicing
Next, the forward (C41) and reverse (C42) primers, which are matched
with the sequences in the 1st and 15th exons [Fig. 1(A)], were used to
amplify the whole coding sequence of human ACAT2 cDNA. The PCR products were
cloned into pGEM-T easy vector. The inserted plasmids were identified by
digestion with the restriction enzymes and DNA sequencing. Three
different-sized fragments (arrowed) were cloned, as shown in Fig. 2(A).
DNA sequencing of these three cloned fragments demonstrated that three
distinct human mRNA species containing different exons [Fig. 2(B),
right] were matured from the same ACAT2 pre-mRNA [Fig. 2(B), left] by comparing
with the published sequences of human ACAT2 genomic and complementary DNAs. The
bigger one (designated ACAT2a mRNA) is the same as the ACAT2 cDNA reported
previously with all 15 exons [16,17]; the other smaller two (named ACAT2b and
ACAT2c mRNAs) lack the exon 4 (60 nt) and the exons 4-5 plus 8-9-10 (429 nt), respectively.
ACAT2a and ACAT2b mRNAs are very similar in size, so they are sometimes
observed as one band in agarose gel electrophoresis. These data clearly show
that alternative splicing produces human ACAT2 mRNA variants.
Two novel isoforms encoded by
human ACAT2 mRNA variants
The deduced amino acid sequences of human ACAT2a, ACAT2b and ACAT2c
from the alternatively spliced mRNA variants are shown in Fig. 3. The
full-length human ACAT2a is a 522 amino acid protein, as described [14,16,17],
and the two novel isoforms ACAT2b and ACAT2c have 502 and 379 amino acids in
which the absences of 20 and 143 amino acids are led by the in-frame
deficiencies of the exon 4 (60-nt) of ACAT2b mRNA and the exons 4-5 plus 8-9-10
(429-nt) of ACAT2c mRNA, respectively. The expression plasmids for human ACAT2
isoforms were then constructed by inserting the cloned human ACAT2 cDNAs into
the pcDNA3 vector [Fig. 4(A)] and transiently transfected into the
mutant Chinese hamster ovary cell line AC29 without endogenous ACAT. Western
blot analysis with b-actin as the control showed that the transfected AC29 cells highly
expressed human ACAT2a, ACAT2b and ACAT2c with different molecular weights [Fig.
4(B)], indicating that the alternatively spliced human ACAT2 mRNA variants
could encode two novel isoforms ACAT2b and ACAT2c in the cells besides the
reported full-length human ACAT2a. The endogenous expression of human ACAT2
isoforms in HepG2 and Caco-2 cells was also observed by Western blot [Fig.
4(C)]. But ACAT2a and ACAT2b were so similar in size that they might form
one band in SDS-PAGE. Also, the human ACAT2 isoforms were not detected in
HEK293, HeLa and THP-1 cells, similar to the results of RT-PCR [Fig. 1(B)].
Two novel isoenzymes without
phosphorylatable site SLLD and with lower enzymatic activity
A comparison between the N-terminal amino acid sequences of human
ACAT2 isoforms showed that the two novel identified isoforms ACAT2b and ACAT2c
lacked a potential CK2 phosphorylation site SLLD located at the 20 amino acid
region (Fig. 3, underlined) encoded by exon 4 which was in-frame
deficiency in both ACAT2b and ACAT2c mRNAs. This phosphorylation site SLLD for
the CK2 has been found at N-terminal regions of all the reported ACAT2 and
ACAT1 species [Fig. 5(A)], implying that it might be phosphorylatable
and have more important roles in the cells. For the phosphorylation assay, the
fusion proteins GST-ACAT2aN and GST-ACAT2bN were expressed by fusion of GST
with the N-terminal region of ACAT2a (121 amino acids) and ACAT2b (101 amino
acids), respectively, as shown in Fig. 5(B). The results demonstrated
that GST-ACAT2aN could be highly phosphorylatable, but GST-ACAT2bN had no
labeling signal even when a large amount of protein was used [Fig. 5(C)].
Furthermore, the enzymatic activities of human ACAT2 isoforms were analyzed by
the transient transfection of human ACAT2 expression plasmids into AC29 cells.
ACAT activity analysis using the methods of the reconstitution in vitro (Fig.
6) and [3H]oleate pulse in vivo (data not shown) indicated that the enzymatic
activities of the two novel ACAT2 isoenzymes were lower than that of the
full-length ACAT2a, approximately 25%–35% of ACAT2a activity.
Discussion
In this study, the multiple human ACAT2 mRNAs in human liver and intestine
cells were analyzed by RT-PCR, cloning and DNA sequencing. Two mRNA variants
were found to be matured by alternative splicing, and to code for the novel
isoenzymes ACAT2b and ACAT2c without phosphorylation and with lower enzymatic
activity. These findings might help to understand the regulation and roles of
the ACAT2 gene expression under certain physiological and pathological
conditions.With respect to cell type- or tissue-specific expression of human
ACAT2 mRNAs, some human cell lines, such as HepG2, HEK293, HeLa, THP-1 and
Caco-2 were used in this study. The intestine cell line Caco-2 and liver cell
line HepG2 expressed a high level of human ACAT2 mRNAs, but no or very
low-level expression was observed in HEK293, HeLa and THP-1 cells. Comparable
amounts of different kinds of ACAT2 mRNAs were significantly detected in the
fetal tissues and the cancer liver [Fig. 1(B)]. The identification of
multiple human ACAT2 mRNAs, which are at high levels in the fetal tissues (liver
and lung) but very low levels in adult liver and the adjacent benign liver
matched with the cancer liver [Fig. 1(B)], supports the premise that the
ACAT2 gene is highly expressed in human fetal liver but greatly diminished in
the human adult liver [20]. However, whether the mechanistic regulation of the
multiple human ACAT2 mRNA expression takes place in a cell type- or
tissue-specific manner remains unknown.By comparing the sequences of the cloned human ACAT2 cDNA fragments with
ACAT2 genomic DNA [16,17], it was found that alternative splicing matured
ACAT2b and ACAT2c mRNA variants. The alternatively spliced mRNA variants differ
from the ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and the exons 4-5 plus
8-9-10 (ACAT2c mRNA). Consistent results have been obtained with several sets
of primers [Fig. 1(B,C)], therefore it is believed that the ACAT2 mRNA
variants are authentic in human liver and intestine cells. The nucleotide
sequences of the alternatively spliced mRNA variants lacking some coding
regions does not cause the shifting of the open reading frame, implying that
certain functional regulation might have existed during the ACAT2 mRNA
maturation. It will be of interest to study whether the alternatively spliced
ACAT2mRNA variants may be specific to some developmental stages, particular
tissues or disease states.Due to the in-frame deficiencies of exon 4 (60 nt) in ACAT2b mRNA
and exons 4-5 plus 8-9-10 (429 nt) in ACAT2c mRNA [Fig. 2(B)], the two
novel isoforms ACAT2b, with 502 amino acids, and ACAT2c, with 379 amino acids,
are smaller than the full-length ACAT2a, with 522 amino acids (Fig. 3).
Western blot analysis showed that the novel ACAT2 isoforms present in human
liver and intestine cells [Fig. 4(C)], suggesting that they may fulfill
common essential roles under certain physiological or pathological conditions.
So far, the subunit structure and functional domains of the ACAT2 holoenzyme
are not known. In this study, phosphorylation and enzymatic activity analyses
demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the
phosphorylatable site SLLD (Fig. 3, underlined) and their enzymatic
activities reduced to 25%–35% of ACAT2a activity (Fig. 6). The phosphorylatable site
SLLD has been found at N-terminal regions of all the reported ACAT2 and ACAT1
species [Fig. 5(A), underlined] and can be recognized by CK2, a
ubiquitous protein kinase responsible for the phosphorylation of Ser and Thr
residues at its recognition motif S/TXXD/E [31]. Thus, it is postulated
that the phosphorylation and dephosphorylation of ACATs might be important for
the regulation activity of these aligomeric and allosteric enzymes, and that
the lower enzymatic activity of the novel ACAT2 isoenzymes lacking the
phosphorylatable site SLLD could not be regulated as protection against
certain stress conditions. In addition, human ACAT2c, with abundant amino acid
deficiency, may cause a great change in the membrane, according to the published
membrane topology of ACAT1 and ACAT2 [32–35]. Further investigation
on regulation of ACAT activity for common essential roles will be conducted in
the future.
Acknowledgements
We gratefully acknowledge our colleagues Han-Hui MA, Li YANG, Lei
LEI, Jia CHEN and Yi WANG for their helpful discussion and technical
assistance during the course of this study.
References
1 Chang TY, Chang CCY, Cheng D. Acyl-coenzyme
A:cholesterol acyltransferase. Annu Rev Biochem 1997, 66: 613–638
2 Suckling KE, Stange EF. Role of
acyl-CoA:cholesterol acyltransferase in cellular cholesterol metabolism.
J Lipid Res 1985, 26: 647–671
3 Dietschy JM, Turley SD, Spady DK. Role of
liver in the maintenance of cholesterol and low density lipoprotein homeostasis
in different animal species, including humans. J Lipid Res 1993, 34: 1637–1659
4 Wilson MD, Rudel LL. Review of cholesterol
absorption with emphasis on dietary and biliary cholesterol. J Lipid Res 1994,
35: 943–955
5 Bocan TM, Mueller SB, Uhlendorf PD, Newton
RS, Krause BR. Comparison of CI-976, an ACAT inhibitor, and selected
lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and
thoracic aortic lesions. A biochemical, morphological, and morphometric
evaluation. Arterioscler Thromb 1991, 11: 1830–1843
6 Miyazaki A, Sakashita N, Lee O, Takahashi K,
Horiuchi S, Hakamata H, Morganelli PM et al. Expression of ACAT-1
protein in human atherosclerotic lesions and cultured human
monocytes-macrophages. Arterioscler Thromb Vasc Biol 1998, 18: 1568–1574
7 Kusunoki J, Hansoty DK, Aragane K, Fallon JT,
Badimon JJ, Fisher EA. Acyl-CoA: cholesterol acyltransferase inhibition reduces
atherosclerosis in apolipoprotein E-deficient mice. Circulation 2001, 103: 2604–2609
8 Puglielli L, Konopka G, Pack-Chung E, Ingano
LA, Berezovska O, Hyman BT, Chang TY et al. Acyl-coenzyme A:cholesterol
acyltransferase modulates the generation of the amyloid b-peptide. Nat Cell
Biol 2001, 3: 905–912
9 Hutter-Paier B, Huttunen HJ, Puglielli L,
Eckman CB, Kim DY, Hofmeister A, Moir RD et al. The ACAT inhibitor
CP-113, 818 markedly reduces amyloid pathology in a mouse model of Alzheimers
disease. Neuron 2004, 44: 227–238
10 Yang L, Yang JB, Chen J, Yu GY, Zhou P, Lei L,
Wang ZZ et al. Enhancement of human ACAT1 gene expression to promote the
macrophage-derived foam cell formation by dexamethasone. Cell Res 2004, 14: 315–323
11 Chang CCY, Huh HY, Cadigan KM, Chang TY.
Molecular cloning and functional expression of human acyl-coenzyme
A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J Biol
Chem 1993, 268: 20747–20755
12 Anderson RA, Joyce C, Davis M, Reagan JW,
Clark M, Shelness GS, Rudel LL. Identification of a form of
acyl-CoA:cholesterol acyltransferase specific to liver and intestine in
nonhuman primates. J Biol Chem 1998, 273: 26747–26754
13 Cases S, Novak S, Zheng YW, Myers HM, Lear SR,
Sande E, Welch CB et al. ACAT-2, a second mammalian acyl CoA:cholesterol
acyltransferase. Its cloning, expression, and characterization. J Biol Chem
1998, 273: 26755–26764
14 Oelkers P, Behari A, Cromley D, Billheimer JT,
Sturley SL. Characterization of two human genes encoding acyl coenzyme
A:cholesterol acyltransferase related enzymes. J Biol Chem 1998, 273: 26765–26771
15 Li BL, Li XL, Duan ZJ, Lee O, Lin S, Ma ZM,
Chang CC et al. Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1)
gene organization and evidence that the 4.3-kilobase ACAT-1 mRNA is produced
from two different chromosomes. J Biol Chem 1999, 274: 11060–11071
16 Song BL, Qi W, Yang XY, Chang CCY, Zhu JQ,
Chang TY, Li BL. Organization of human ACAT-2 gene and its cell-type-specific
promoter activity. Biochem Biophys Res Commun 2001, 282: 580–588
17 Katsuren K, Tamura T, Arashiro R, Takata K,
Matsuura T, Niikawa N, Ohta T. Structure of the human acyl-CoA:cholesterol
acyltransferase-2 (ACAT-2) gene and its relation to dyslipidemia. Biochim
Biophys Acta 2001, 1531: 230–240
18 Lee O, Chang CCY, Lee W, Chang TY.
Immunodepletion experiments suggest that acyl-coenzyme A:cholesterol
acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human
liver, adrenal gland, macrophages, and kidney, but not in intestines. J Lipid
Res 1998, 39: 1722–1727
19 Sakashita N, Miyazaki A, Takeya M, Horiuchi S,
Chang CCY, Chang TY, Takahashi K. Localization of human acyl-coenzyme
A:cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues.
Am J Pathol 2000, 156: 227–236
20 Chang CCY, Sakashita N, Ornvold K, Lee O,
Chang ET, Dong R, Lin S et at. Immunological quantitation and localization
of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem 2000, 275:
28083–28092
21 Sakashita N, Miyazaki A, Chang CCY, Chang TY,
Kiyota E, Satoh M, Komohara Y et al. Acyl-coenzyme A: cholesterol
acyltransferase 2 (ACAT2) is induced in monocyte-derived macrophages: In
vivo and in vitro studies. Lab Invest 2003, 83: 1569–1581
22 Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang
XY, Sun X et al. Synergistic transcriptional activation of human
acyl-coenzyme A:cholesterol acyltransterase-1 gene by interferon-gamma and all-trans-retinoic
acid in THP-1 cells. J Biol Chem 2001, 276: 20989–20998
23 Yang L, Lee O, Chen J, Chen J, Chang CCY, Zhou
P, Wang ZZ et al. Human acyl-coenzyme A:cholesterol acyltransferase 1
(acat1) sequences located in two different chromosomes (7 and 1) are
required to produce a novel ACAT1 isoenzyme with additional sequence at the N
terminus. J Biol Chem 2004, 279: 46253–46262
24 Yang L, Chen J, Chang CCY, Yang XY, Wang ZZ,
Chang TY, Li BL. A stable upstream stem-loop structure enhances selection of
the first 5‘-ORF-AUG as a main start codon for translation initiation of
human ACAT1 mRNA. Acta Biochim Biophys Sin 2004, 36: 259–268
25 Chang CCY, Chen J, Thomas MA, Cheng D, del
Priore VA, Newton RS, Pape ME et al. Regulation and immunolocalization
of acyl-coenzyme A:cholesterol acyltransferase in mammalian cells as studied
with specific antibodies. J Biol Chem 1995, 270: 29532–29540
26 Bishop JE, Hajra AK. A method for the chemical
synthesis of 14C-labeled fatty acyl coenzyme As of high specific activity.
Anal Biochem 1980, 106: 344–350
27 Liu J, Streiff R, Zhang YL, Vestal RE, Spence
MJ, Briggs MR. Novel mechanism of transcriptional activation of hepatic LDL
receptor by oncostatin M. J Lipid Res 1997, 38: 2035–2048
28 Oosta GM, Mathewson NS, Catravas GN.
Optimization of Folin-Ciocalteu Reagent concentration in an automated Lowry
protein assay. Anal Biochem 1978, 89: 31–34
29 Torres J, Pulido R. The tumor suppressor PTEN
is phosphorylated by the protein kinase CK2 at its C terminus. Implications for
PTEN stability to proteasome-mediated degradation. J Biol Chem 2001, 276: 993–998
30 Kim ST, Lim DS, Canman CH, Kastan MB.
Substrate specificities and identification of putative substrates of ATM kinase
family members. J Biol Chem 1999, 274: 37538–37543
31 Ahmed K, Davis AT, Wang H, Faust RA, Yu S,
Tawfic S. Significance of protein kinase CK2 nuclear signaling in neoplasia. J
Cell Biochem 2000, 35 (Suppl): 130–135
32 Guo ZY, Lin S, Heinen JA, Chang CC, Chang TY.
The active site His460 of human acyl-coenzyme A: cholesterol acyltransferase 1
(ACAT1) resides in a hitherto undisclosed transmembrane domain. J Biol Chem
2005, 280: 37814–37826
33 Lin S, Lu X, Chang CCY, Chang TY. Human
acyl-coenzyme A:cholesterol acyltransferase expressed in chinese hamster ovary
cells: membrane topology and active site location. Mol Biol Cell 2003, 14: 2447–2460
34 Lin S, Cheng D, Liu MS, Chen J, Chang TY.
Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum
contains seven transmembrane domains. J Biol Chem, 1999, 274: 23276–23285
35 Joyce CW, Shelness GS, Davis MA, Lee RG,
Skinner K, Anderson RA, Rudel LL. ACAT1 and ACAT2 membrane topology segregates
a serine residue essential for activity to opposite sides of the endoplasmic
reticulum membrane. Mol Biol Cell 2000, 11: 3675–3687