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Ta-Yuan Chang

Publications and source records attributed to Ta-Yuan Chang.

At least 19 recordsLinked to original sources
PubMed2006

Roles of endogenously synthesized sterols in the endocytic pathway.

The effect(s) of endogenously synthesized cholesterol (endo-CHOL) on the endosomal system in mammalian cells has not been examined. Here we treated Chinese hamster ovary cell lines with lovastatin (a hydroxymethylglutaryl-CoA reductase inhibitor) and mevalonate (a precursor for isoprenoids) to block endo-CHOL synthesis and then examined its effects on the fate of cholesterol liberated from low density lipoprotein (LDL-CHOL). The results showed that blocking endo-CHOL synthesis for 2 h or longer does not impair the hydrolysis of cholesteryl esters but partially impairs the transport of LDL-CHOL to the plasma membrane. Blocking endo-CHOL synthesis for 2 h or longer also alters the localization patterns of the late endosomes/lysosomes and retards their motility, as monitored by time-lapse microscopy. LDL-CHOL overcomes the effect of blocking endo-CHOL synthesis on endosomal localization patterns and on endosomal motility. Overexpressing Rab9, a key late endosomal small GTPase, relieves the endosomal cholesterol accumulation in Niemann-Pick type C1 cells but does not revert the reduced endosomal motility caused by blocking endo-CHOL synthesis. Our results suggested that endo-CHOL contributes to the cholesterol content of late endosomes and controls its motility, in a manner independent of NPC1. These results also supported the concept that endosomal motility plays an important role in controlling cholesterol trafficking activities.

Animals↗
PubMed2006

Human acyl-CoA:cholesterol acyltransferase 2 gene expression in intestinal Caco-2 cells and in hepatocellular carcinoma.

Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.

Adult↗
PubMed2006

Human acyl-CoA:cholesterol acyltransferase (ACAT) and its potential as a target for pharmaceutical intervention against atherosclerosis.

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty-acyl-coenzyme A. At the single-cell level, ACAT serves as a regulator of intracellular cholesterol homeostasis. In addition, ACAT supplies cholesteryl esters for lipoprotein assembly in the liver and small intestine. Under pathological conditions, the accumulation of cholesteryl esters produced by ACAT in macrophages contributes to foam cell formation, a hallmark of the early stage of atherosclerosis. Several reviews addressing various aspects of ACAT and ACAT inhibitors are available. This review briefly outlines the current knowledge on the biochemical properties of human ACATs, and then focuses on discussing the merit of ACAT as a drug target for pharmaceutical interventions against atherosclerosis.

Acyl Coenzyme A↗
PubMed2006

Cholesterol sensing, trafficking, and esterification.

Mammalian cells acquire cholesterol from low-density lipoprotein (LDL) and from endogenous biosynthesis. The roles of the Niemann-Pick type C1 protein in mediating the endosomal transport of LDL-derived cholesterol and endogenously synthesized cholesterol are discussed. Excess cellular cholesterol is converted to cholesteryl esters by the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 or is removed from a cell by cellular cholesterol efflux at the plasma membrane. A close relationship between the ACAT substrate pool and the cholesterol efflux pool is proposed. Sterol-sensing domains (SSDs) are present in several membrane proteins, including NPC1, HMG-CoA reductase, and the SREBP cleavage-activating protein. The functions of SSDs are described. ACAT1 is an endoplasmic reticulum cholesterol sensor and contains a signature motif characteristic of the membrane-bound acyltransferase family. The nonvesicular cholesterol translocation processes involve the START domain proteins and the oxysterol binding protein-related proteins (ORPs). The properties of these proteins are summarized.

Amino Acid Sequence↗
PubMed2005

Investigating the allosterism of acyl-CoA:cholesterol acyltransferase (ACAT) by using various sterols: in vitro and intact cell studies.

ACAT1 (acyl-CoA:cholesterol acyltransferase 1) is thought to have two distinct sterol-binding sites: a substrate-binding site and an allosteric-activator site. In the present work, we investigated the structural features of various sterols as substrates and/or activators in vitro. The results show that without cholesterol, the plant sterol sitosterol is a poor substrate for ACAT. In the presence of cholesterol, ACAT1-mediated esterification of sitosterol is highly activated while ACAT2-mediated esterification of sitosterol is only moderately activated. For ACAT1, we show that the stereochemistry of the 3-hydroxy group at steroid ring A is a critical structural feature for a sterol to serve as a substrate, but less critical for activation. Additionally, enantiomeric cholesterol, which has the same biophysical properties as cholesterol in membranes, fails to activate ACAT1. Thus ACAT1 activation by cholesterol is the result of stereo-specific interactions between cholesterol and ACAT1, and is not related to the biophysical properties of phospholipid membranes. To demonstrate the relevance of the ACAT1 allosteric model in intact cells, we showed that sitosterol esterification in human macrophages is activated upon cholesterol loading. We further show that the activation is not due to an increase in ACAT1 protein content, but is partly due to an increase in the cholesterol content in the endoplasmic reticulum where ACAT1 resides. Together, our results support the existence of a distinct sterol-activator site in addition to the sterol-substrate site of ACAT1 and demonstrate the applicability of the ACAT1 allosteric model in intact cells.

Allosteric Regulation↗
PubMed2005

Workers' exposures and potential health risks to air toxics in a petrochemical complex assessed by improved methodology.

OBJECTIVE: This study was designed to comprehensively evaluate workers' potential health risks of exposure to 39 air toxics in the Ta-sher Petrochemical Complex. METHODS: Open-Path Fourier Transform Infrared Spectroscopy (OP-FTIR) was used to measure concentrations of air toxics. We used the measured worksite concentrations between 1997 and 1999 at 11 companies in the petrochemical complex, employing 3,100 on-site workers. The 39 measured air toxics included 10 chemicals with acute reference exposure levels (RELa), 19 chemicals with chronic reference exposure levels (RELc), and 3 chemicals classified as Class 1 or 2A human carcinogens by the International Agency for Research on Cancer (IARC). We then used RELa to calculate the hazard index of acute health effects (HI ( A )) for workers in individual plants. We also calculated the hazard index of chronic health effects (HIc) and cancer risks for all workers in the entire petrochemical complex. RESULTS: Workers in five companies had HI ( A ) greater than 1 because of toluene, benzene, methyl ethyl ketone, chloroform and isopropanol exposures. Workers in this petrochemical complex had HIc greater than 1 because of acrylonitrile, 1,3-butadiene, hydrogen cyanide, and n,n-dimethylformamide exposures. Risk of hematopoietic system cancer because of benzene and ethylene oxide exposure, and respiratory system cancer because of 1,3-butadiene exposure was estimated to be 3.1-6.1 x 10(-4) and 5.2-7.1 x 10(-4), respectively. CONCLUSIONS: Our findings indicated that workers in the petrochemical complex might have excess cancer and noncancer risks due to acute or chronic exposures to air toxics from multiple emission sources.

Air Pollutants↗
PubMed2005

The active site His-460 of human acyl-coenzyme A:cholesterol acyltransferase 1 resides in a hitherto undisclosed transmembrane domain.

Human acyl-coenzyme A:cholesterol acyltransferase 1 (hACAT1) esterifies cholesterol at the endoplasmic reticulum (ER). We had previously reported that hACAT1 contains seven transmembrane domains (TMD) (Lin, S., Cheng, D., Liu, M. S., Chen, J., and Chang, T. Y. (1999) J. Biol. Chem. 274, 23276-23285) and nine cysteines. The Cys near the N-terminal is located at the cytoplasm; the two cysteines near the C-terminal form a disulfide bond and are located in the ER lumen. The other six free cysteines are located in buried region(s) of the enzyme (Guo, Z.-Y., Chang, C. C. Y., Lu, X., Chen, J., Li, B.-L., and Chang, T.-Y. (2005) Biochemistry 44, 6537-6548). In the current study, we show that the conserved His-460 is a key active site residue for hACAT1. We next performed Cys-scanning mutagenesis within the region of amino acids 354-493, expressed these mutants in Chinese hamster ovary cells lacking ACAT1, and prepared microsomes from transfected cells. The microsomes are either left intact or permeabilized with detergent. The accessibility of the engineered cysteines of microsomal hACAT1 to various maleimide derivatives, including mPEG(5000)-maleimide (large, hydrophilic, and membrane-impermeant), N-ethylmaleimide, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (small, hydrophilic, and ER membrane-permeant), and N-phenylmaleimide (small, hydrophobic, and ER membrane-permeant), were monitored by Western blot analysis. The results led us to construct a revised, nine-TMD model, with the active site His-460 located within a hitherto undisclosed transmembrane domain, between Arg-443 and Tyr-462.

Amino Acid Sequence↗
PubMed2005

The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A:cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiol-specific modifications by mPEG(5000)-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACAT1s. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACAT1 with a hemagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACAT1 present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme.

Alanine↗
PubMed2005

Two human ACAT2 mRNA variants produced by alternative splicing and coding for novel isoenzymes.

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 intestinecells. 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.

Alternative Splicing↗
PubMed2004

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.

A rare form of human ACAT1 mRNA, containing the optional long 5'-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7. To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5'-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1(ORF). The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

Amino Acid Sequence↗
PubMed2004

Binding between the Niemann-Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain.

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [(3)H]7,7-azocholestanol ([(3)H]AC). After immunoprecipitation, (3)H-labeled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [(3)H]AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [(3)H]AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-alpha-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1.

Animals↗
PubMed2004

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts.

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.

ATP Binding Cassette Transporter 1↗
PubMed2004

A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain.

Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by progressive accumulation of cholesterol, gangliosides, and other lipids in the central nervous system and visceral organs. In the NPC1 mouse model, neurodegeneration and neuronal cell loss occur before postnatal day 21. Whether neuronal cholesterol accumulation occurs in vivo before the first signs of neuronal cell loss has not been demonstrated. In this report, we used the NPC1 mouse model and employed a novel cholesterol binding reagent, BC theta, that enabled us to visualize cellular cholesterol accumulation at a level previously unattainable. The results demonstrate the superiority of BC theta staining over conventional filipin staining in confocal microscopy and highlight several new findings. We show that at postnatal day 9, although only mild signs of neurodegeneration are detectable, significant neuronal cholesterol accumulation has already occurred throughout the NPC1 brain. In addition, although NPC1 Purkinje neurons exhibit a normal morphology at day 9, significant cholesterol accumulation within their extensive dendritic trees has occurred. We also show that in the thalamus and cortex of NPC1 mice, activated glial cells first appear at postnatal day 9 and heavily populate by day 22, suggesting that in NPC1 mice, neuronal cholesterol accumulation precedes neuronal injury and neuronal cell loss.

Animals↗
PubMed2004

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.

Human ACAT 1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG(1397-1399) and AUG(1415-1417), at 5'-terminus of the open reading frame (ORF, nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5'-terminus of the predicted ORF (5'-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5'-ORF-AUGs can be selected as start codons but the first 5'-ORF-AUG(1397-1399) is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5'-ORF-AUG(1397 -1399) as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5'-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5'-ORF-AUGs.

5' Untranslated Regions↗
PubMed2003

Distinct endosomal compartments in early trafficking of low density lipoprotein-derived cholesterol.

We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. Biol. Chem. 275, 4013-4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5-10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum.

Animals↗
PubMed2003

Human acyl-coenzyme A:cholesterol acyltransferase expressed in chinese hamster ovary cells: membrane topology and active site location.

Acyl-CoA:cholesterol acyltransferase (ACAT) is a membrane-bound enzyme that produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been identified. The expression of ACAT1 is ubiquitous, whereas that of ACAT2 is tissue restricted. Previous research indicates that ACAT1 may contain seven transmembrane domains (TMDs). To study ACAT2 topology, we inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophilic regions flanking each of its predicted TMDs, and expressed the recombinant proteins in mutant Chinese hamster ovary cells lacking endogenous ACAT. Each tagged ACAT2 was expressed in the endoplasmic reticulum as a single undegraded protein band and was at least partially active enzymatically. We then used cytoimmunofluorescence and protease protection assays to monitor the sidedness of the hemagglutinin and Mab1 tags along the ER membranes. The results indicated that ACAT2 contains only two detectable TMDs, located near the N terminal region. We also show that a conserved serine (S245), a candidate active site residue, is not essential for ACAT catalysis. Instead, a conserved histidine (H434) present within a hydrophobic peptide segment, may be essential for ACAT catalysis. H434 may be located at the cytoplasmic side of the membrane.

Animals↗