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T Y Chang

Publications and source records attributed to T Y Chang.

At least 37 records · Page 2Linked to original sources
PubMed2001

Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis.

Two ACAT sharing protein sequence homology near their C termini have been identified. Both proteins may span the endoplasmic reticulum (ER) membrane several times. There is good evidence implicating the role of ACAT1 in macrophage foam cell formation, and ACAT2 in intestinal cholesterol absorption. On the other hand, the functional roles of ACAT1 and ACAT2 in the VLDL or chylomicron assembly process are less clear. It is possible that both enzymes are able to form lipid droplets (which are present in the cytoplasm), and participate in lipoprotein assembly (which occurs in the ER lumen). To link the site of ACAT catalysis with its function, we propose that part of the ACAT catalytic site may reside within the lipid bilayer, allowing catalysis to be completed within the plane of the membrane. Cholesteryl esters (CE) produced in situ may burst into cytoplasmic lipid droplets, carrying phospholipid monolayers as their outer coats. In cells engaged in lipoprotein assembly and secretion, CE in the bilayer may be recognized by the specific protein microsomal triacylglycerol transfer protein (MTP), reaching out from the lumenal side of the membrane. MTP then lipidates the growing apolipoprotein B (apoB) chain with CE and TG during the early stages of apoB lipoprotein assembly.

Acetyl Coenzyme A↗
PubMed2000

Fate of endogenously synthesized cholesterol in Niemann-Pick type C1 cells.

Mammalian cells obtain cholesterol via two pathways: endogenous synthesis in the endoplasmic reticulum and exogenous sources mainly through the low density lipoprotein (LDL) receptor pathway. We performed pulse-chase experiments to monitor the fate of endogenously synthesized cholesterol and showed that, after reaching the plasma membrane from the endoplasmic reticulum, the newly synthesized cholesterol eventually accumulates in an internal compartment in Niemann-Pick type C1 (NPC1) cells. Thus, the ultimate fate of endogenously synthesized cholesterol in NPC1 cells is the same as LDL-derived cholesterol. However, the time required for endogenous cholesterol to accumulate internally is much slower than LDL-derived cholesterol. Different pathways thus govern the post-plasma membrane trafficking of endogenous cholesterol and LDL-derived cholesterol to the internal compartment. Results using the inhibitor N-butyldeoxynojirimycin, which depletes cellular complex glycosphingolipids, demonstrates that the cholesterol trafficking defect in NPC1 cells is not caused by ganglioside accumulation. The ultimate cause of death in NPC disease is progressive neurological deterioration in the central nervous system, where the major source of cholesterol is derived from endogenous synthesis. Our current study provides a plausible link between defects in intracellular trafficking of endogenous cholesterol and the etiology of Niemann-Pick type C disease.

Animals↗
PubMed2000

Accumulation and aggregation of amyloid beta-protein in late endosomes of Niemann-pick type C cells.

There is growing evidence suggesting that cholesterol metabolism is linked to susceptibility to Alzheimer's disease by influencing amyloid beta-protein (Abeta) metabolism. However, the precise cellular linkage sites between cholesterol and Abeta have not yet been clarified. To address this issue, we investigated Niemann-Pick type C (NPC) model cells and NPC mutant cells, which showed aberrant cholesterol trafficking. We observed a remarkable Abeta accumulation in late endosomes of both NPC model cells and mutant cells where cholesterol accumulates and a significant accumulation in the NPC mouse brain. This Abeta accumulation was independent of its constitutive secretion and production through an endocytic pathway. In addition, it is characterized by a marked predominance of Abeta42 and insolubility in SDS, suggesting the presence of aggregated Abeta in late endosomes. Most importantly, Abeta accumulation is coupled with the cholesterol levels in late endosomes. Thus, late endosomes of NPC cells are a novel pool of aggregated Abeta42 as well as cholesterol, suggesting a direct interaction between aggregated Abeta and cholesterol.

Amyloid beta-Peptides↗
PubMed2000

Towards a quantitative model of immunogenicity: counting pathways in sequence space.

One of the fundamental aims of structural biology is the identification of high-affinity ligands for arbitrary receptors. The maturation of the antibody repertoire elegantly and robustly solves this problem through an evolutionary mechanism comprising repeated cycles of mutation and preferential replication. To understand better the limitations and biases of this process, we developed an interpretation of antibody maturation within the framework of sequence space and fitness landscapes. Several well-described phenomena can be directly derived from this framework, and new predictions can be made. Ultimately, this reconceptualization of the clonal selection process suggests a quantitative, testable model of immunogenicity.

Antibodies↗
PubMed2000

Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine.

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

Adolescent↗
PubMed2000

Embryonic striatal neurons from niemann-pick type C mice exhibit defects in cholesterol metabolism and neurotrophin responsiveness.

Niemann-Pick type C (NP-C) disease is a progressive and fatal neuropathological disorder previously characterized by abnormal cholesterol metabolism in peripheral tissues. Although a defective gene has been identified in both humans and the npc(nih) mouse model of NP-C disease, how this leads to abnormal neuronal function is unclear. Here we show that whereas embryonic striatal neurons from npc(nih) mice can take up low density lipoprotein-derived cholesterol, its subsequent hydrolysis and esterification are significantly reduced. Given the importance of cholesterol to a variety of signal transduction mechanisms, we assessed the effect of this abnormality on the ability of these neurons to respond to brain-derived neurotrophic factor (BDNF). In contrast to its effects on wild type neurons, BDNF failed to induce autophosphorylation of the TrkB receptor and to increase neurite outgrowth in npc(nih) neurons, despite expression of TrkB on the cell surface. The results suggest that abnormal cholesterol metabolism occurs in neurons in the brain during NP-C disease, even at embryonic stages of development prior to the onset of phenotypic symptoms. Moreover, this defect is associated with a lack of TrkB function and BDNF responsiveness, which may contribute to the loss of neuronal function observed in NP-C disease.

Animals↗
PubMed2000

Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol.

Niemann-Pick type C (NPC) is a disease that affects intracellular cholesterol-trafficking pathways. By cloning the hamster ortholog of NPC1, we identified the molecular lesions in two independently isolated Chinese hamster ovary cell mutants, CT60 and CT43. Both mutants lead to premature translational terminations of the NPC1 protein. Transfecting hamster NPC1 cDNA complemented the defects of the mutants. Investigation of the CT mutants, their parental cells, and an NPC1-stable transfectant allow us to present evidence that NPC1 is involved in a post-plasma membrane cholesterol-trafficking pathway. We found that the initial movement of low density lipoprotein (LDL)-derived cholesterol to the plasma membrane (PM) did not require NPC1. After reaching the PM and subsequent internalization, however, cholesterol trafficking back to the PM did involve NPC1. Both LDL-derived cholesterol and cholesterol originating from the PM accumulated in a dense, intracellular compartment in the CT mutants. Cholesterol movement from this compartment to the PM or endoplasmic reticulum was defective in the CT mutants. Our results functionally distinguish the dense, intracellular compartment from the early endocytic hydrolytic organelle and imply that NPC1 is involved in sorting cholesterol from the intracellular compartment back to the PM or to the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed2000

Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli.

It is difficult to express recombinant Candida rugosa lipases (CRLs) in heterologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG for leucine. In this study, recombinant LIP4 in which all 19 CUG codons had been converted to a universal serine codon was overexpressed in Escherichia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion bodies and showed a low catalytic activity. To increase the amount of soluble form and activity of recombinant LIP4, the DNA was fused to the gene for thioredoxin (TrxFus-LIP4) and then expressed in E. coli strain AD494(DE3). This strategy promotes the formation of disulfide bonds in the cytosol and yields enzymatically active forms of LIP4. The purified recombinant TrxFus-LIP4 and LIP4 expressed in AD494(DE3) had the same catalytic profiles. In addition, recombinant LIP4 had higher esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein, and olive oil. This system for the expression of fungal lipase in E. coli strain AD494(DE3) is reliable and may produce enzymatically active forms of recombinant lipase without an in vitro refolding procedure.

Candida↗
PubMed2000

Localization of human acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues.

To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues, we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages, antigen-presenting cells, steroid hormone-producing cells, neurons, cardiomyocytes, smooth muscle cells, mesothelial cells, epithelial cells of the urinary tracts, thyroid follicles, renal tubules, pituitary, prostatic, and bronchial glands, alveolar and intestinal epithelial cells, pancreatic acinar cells, and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages, steroid hormone-producing cells, followed by hepatocytes, and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however, on cholesterol loading, about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demonstrated extensive colocalization of ACAT-1 and GRP 78 signals in both the tubular and vesicular endoplasmic reticulum before and after cholesterol loading. These results raise the possibility that foam cell formation may activate an endoplasmic reticulum vesiculation process, producing vesicles enriched in the ACAT-1 protein.

Adult↗
PubMed1999

Emission characteristics of VOCs from athletic tracks.

Dynamic and flow-through flux chambers are convenient tools for field measurements of gas or VOC emission flux from solid surfaces in the field. This study was undertaken to collect on site and quantify the emissions of volatile organic compounds (VOCs) released from athletic running tracks. Three typical types of tracks, one synthetic rubber and two tracks (types I and II) consisting mainly of polyurethane, were studied. They were all installed with adhesives and backings, both of which contributed significant amount of VOCs. VOCs released from the track surface were collected with a flux chamber and subsequently analyzed by a gas chromatograph/mass spectrometer (GC/MS). Also, for each track and at each selected time the emission flux and mass emission were measured on site under outdoor conditions over a period of 40 min. GC/MS analyses show that the VOCs emitted include 2-methyl furan, butanal, methyl ethyl ketone, benzene, heptane, methyl isobutyl ketone, toluene+octane, hexanal, nonane+ethylbenzene, xylenes+styrene, propyl benzene, decane, 1,3,5-trimethyl benzene, 1,2,4-trimethyl benzene, 1,2, 3-trimethyl benzene and undecane. Of these, hexanal was the common and principal compound for all three types of tracks. 2-Methyl furan and methyl isobutyl ketone were the characteristic compounds for the synthetic rubber and the type II of polyurethane tracks, respectively. In the field studies, no unique compounds were found in the type I of polyurethane tracks. For each of these three types of tracks the total-VOCs emission flux was correlated to the track age and track surface temperature. The results of multiple regression analysis showed good correlation. The type II polyurethane track had the highest decay rate, while the synthetic rubber track had the lowest decay rate. Two years after the track installation, the VOC concentrations measured at 1.5 m above the track, the breathing height of school children, were not significantly higher than the background levels.

Air Pollutants↗
PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro.

Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. ACAT-1 may function as an allosteric enzyme. We took a multifaceted approach to investigate the subunit composition of ACAT-1. When ACAT-1 with two different tags were co-expressed in the same Chinese hamster ovary cells, antibody specific to one tag caused co-immunoprecipitation of both types of ACAT-1 proteins. Radioimmunoprecipitations of cells expressing the untagged ACAT-1 or the 6-histidine-tagged ACAT-1 yielded a single radiolabeled band of predicted size on SDS-polyacrylamide gel electrophoresis. These results show that ACAT-1 exists as homo-oligomers in intact Chinese hamster ovary cells. We solubilized HisACAT-1 with the detergent deoxycholate or CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid), performed gel filtration chromatography and sucrose density gradient centrifugations in H(2)O and D(2)O, and determined the Stokes radii and sedimentation coefficients of the HisACAT1-detergent complexes. The estimated molecular mass of HisACAT-1 is 263 kDa, which is 4 times that of the HisACAT-1 monomer (69 kDa). Finally, cross-linking experiments in intact cells and in vitro show that the increase in cross-linker concentrations causes an increase in size of the HisACAT-1-positive signals, forming material(s) 4 times the size of the monomer, supporting the conclusion that ACAT-1 is a homotetrameric enzyme.

Animals↗
PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and is involved in atherosclerosis. ACAT-1 protein is located mainly in the ER. The hydropathy plot suggests that ACAT-1 protein contains multiple transmembrane segments. We inserted either the hemagglutinin tag or the HisT7 tag at various hydrophilic regions within the human ACAT-1 protein and used immunofluorescence microscopy to determine the topography of the tagged proteins expressed in mutant Chinese hamster ovary cells lacking endogenous ACAT. All of the tagged proteins are located mainly in the ER and retain full or partial enzyme activities. None of the tagged proteins produces detectable intracellular degradation intermediates. Treating cells with digitonin at 5 micrograms/ml permeabilizes the plasma membranes while leaving the ER membranes sealed; in contrast, treating cells with 0.25% Triton X-100 or with cold methanol permeabilizes both the plasma membranes and the ER membranes. After appropriate permeabilization, double immunostaining using antibodies against the N-terminal region and against the inserted tag were used to visualize various regions of the tagged protein. The results show that human ACAT-1 in the ER contains seven transmembrane domains.

Algorithms↗
PubMed1999

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.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Four human ACAT-1 mRNAs (7.0, 4.3, 3.6, and 2.8 kilobases (kb)) share the same short 5'-untranslated region (exon 1) and coding sequence (exons 2-15). The 4.3-kb mRNA contains an additional 5'-untranslated region (1289 nucleotides in length; exons Xa and Xb) immediately upstream from the exon 1 sequence. One ACAT-1 genomic DNA insert covers exons 1-16 and a promoter (the P1 promoter). A separate insert covers exon Xa (1277 base pairs) and a different promoter (the P7 promoter). Gene mapping shows that exons 1-16 and the P1 promoter sequences are located in chromosome 1, while exon Xa and the P7 promoter sequence are located in chromosome 7. RNase protection assays demonstrate three different protected fragments, corresponding to the 4.3-kb mRNA and the two other mRNAs transcribed from the two promoters. These results are consistent with the interpretation that the 4.3-kb mRNA is produced from two different chromosomes, by a novel RNA recombination mechanism involving trans-splicing of two discontinuous precursor RNAs.

5' Untranslated Regions↗
PubMed1999

Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses.

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell-independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.

Amino Acid Sequence↗
PubMed1998

Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol.

Acyl Coenzyme A↗
PubMed1998

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

Apoptosis↗