Research Paper	Pdf file on Synergy Omments
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 YAO^1,2#, Can-Hua WANG^1,2#, 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 �antibio�tics 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 �se�quences 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 �indivi�dually 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 al�ternatively spliced ACAT2mRNA variants may be specific to some developmental stages, particular tissues or di�sease 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 Alzheimer�s 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 A�s 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