Evidence for coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase ad low density lipoprotein binding activity.

Chin, Jean; Chang, Ta−Yuan

doi:10.1016/s0021-9258(19)69163-5

Cited by 44 publications

(29 citation statements)

References 32 publications

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“…Both media were supplemented with antibiotics as previously described (4, 5) and 10% FCS (Sigma Chemical Co.). When delipidated FCS was used, it was prepared according to a published procedure (6) as modified by Chin and Chang (14). Human LDL (d = 1.019-1.063 g/ml) was prepared from human plasma by sequential flotation in the presence of protease inhibitors as previously described (5).…”

Section: Cell Culturementioning

confidence: 99%

Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

Cadigan

Chang

1989

The Journal of Cell Biology

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Abstract. We have previously reported the isolation of Chinese hamster ovary cell mutants deficient in acylcoenzyme A/cholesterol acyltransferase (ACAT) activity (Cadigan, K. M., J. G. Heider, and T. Y. Chang. 1988. J. Biol. Chem. 263:274-282). We now describe a procedure for isolating cells from these mutants that have regained the ability to synthesize cholesterol esters. The protocol uses the fluorescent stain Nile red, which is specific for neutral lipids such as cholesterol ester. After ACAT mutant populations were subjected to chemical mutagenesis or transfected with human fibroblast whole genomic DNA, two revertants and one primary transformant were isolated by virtue of their higher fluorescent intensities using flow cytofluorimetry. Both the revertants and transformant have regained large amounts of intracellular cholesterol ester and ACAT activity. However, heat inactivation experiments revealed that the enzyme activity of the transformant had heat stability properties identical to that of human fibroblasts, while the ACAT activities of the revertants were similar to that of other Chinese hamster ovary cell lines. These results suggest that the molecular lesion in the ACAT mutants resides in the structural gene for the enzyme, and the transformant has corrected this defect by acquiring and stably expressing a human gene encoding the ACAT polypeptide. Secondary transformants were isolated by transfection of ACAT mutant cells with primary transformant genomic DNA. Genomic Southern analysis of the secondary transformants using a probe specific for human DNA revealed several distinct restriction fragments common to all the transformants which most likely comprise part or all of the human ACAT gene. The cell lines described here should facilitate the cloning of the gene encoding the human ACAT enzyme.vL-coenzyme A/cholesterol acyltransferase (ACAT) ~ is an intracellular enzyme that uses cholesterol and fatty acyl-coenzyme A (CoA) to form cholesterol esters (10, 50). The enzyme is localized to the rough endoplasmic reticulum in rat liver (2, 24); is highly regulated in many cell types and tissues; and is believed to play an important role in cholesterol metabolism in various cells and tissues such as the small intestinal mucosa, hepatocytes, and the steroid hormone-producing tissues (10, 50).Although ACAT has been studied intensively, little is known about its molecular structure. In rat liver, the active site of the enzyme has been localized to the cytoplasmic surface of the microsomal vesicles using a combination of detergent and protease treatments (24, 34), but whether the enzyme spans the entire membrane could not be determined. Recent chemical modification studies have demonstrated that an essential histidyl and sulfhydryl residue(s) may reside at or near the active site of the enzyme. ACAT activities from different rabbit tissues have different sensitivities to the histidyl-modifying reagents, suggesting the existence of different ACAT subtypes (31, 32). ACAT activity has been solubilized and reconst...

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Section: Cell Culturementioning

confidence: 99%

Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

Cadigan

Chang

1989

The Journal of Cell Biology

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“…sists of sterol auxotrophs that cannot synthesize choles-Such drugs have been shown to reduce heart attacks terol or LDL receptors in response to sterol depletion and to prolong life in humans with atherosclerosis. (Chin and Chang, 1981;Evans and Metherall, 1993). The central molecules in the cholesterol feedback sys-These cells fail to release the NH2-terminal segment of tem are a pair of membrane-bound transcription factors SREBP-1 or -2 from membranes because they cannot designated sterol regulatory element binding proteins-1 cleave the SREBPs at site 2 (Sakai et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Sterol Resistance in CHO Cells Traced to Point Mutation in SREBP Cleavage–Activating Protein

Hua

Nohturfft

Goldstein

et al. 1996

Cell

453

467

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Through expression cloning we have isolated a cDNA-encoding SREBP cleavage-activating protein (SCAP), which regulates cholesterol metabolism by stimulating cleavage of transcription factors SREBP-1 and -2, thereby releasing them from membranes. The cDNA was isolated from Chinese hamster ovary cells with a dominant mutation that renders them resistant to sterol-mediated suppression of cholesterol synthesis and uptake. Sterol resistance was traced to a G-->A transition at codon 443 of SCAP, changing aspartic acid to asparagine. The D443N mutation enhances the cleavage-stimulating ability of SCAP and renders it resistant to inhibition by sterols. SCAP has multiple membrane-spanning regions, five of which resemble the sterol-sensing domain of HMG CoA reductase, an endoplasmic reticulum enzyme whose degradation is accelerated by sterols. SCAP appears to be a central regulator of cholesterol metabolism in animal cells.

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“…Besides the degradation of the LDL protein moiety, the hydrolysis of LDL-cholesterol ester also occurs in the lysosomes, via a specific acid lipase (26,28). Of the total cholesterol in LDL, 75-80% is estedfied (53), and the hydrolysis of this sterol ester is necessary for the regulatory effects of LDL to be observed in human fibtoblast (26,28) and CHO cells (22). By labeling the LDL with [3H]cholesteryl linoleate, it is possible to measure the hydrolysis of [3H]cholesteryl linoleate to free [3H]cholesterol.…”

Section: Ldl Receptor Pathway Analysismentioning

confidence: 99%

“…During the mutant selections all cells were maintained in F-12 medium minus linoleic acid supplemented with antibiotics as previously de-scribed (11) plus 10% FCS (Sigma Chemical Co.). When delipidated FCS was used, it was prepared according to a published procedure (14), as modified by Chin and Chang (22). After the mutant selections, all of the CHO cell lines used in this report were maintained on a 1:1 mixture of the above mentioned F-12 medium and MEM (Gibco Laboratories, Grand Island, NY) supplemented with 2 mM glutamine and antibiotics, plus 10% bovine calf serum (Hyclone Laboratories, Sterile Systems, Inc., Logan UT).…”

Section: Cell Culturementioning

confidence: 99%

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

Cadigan

Spillane

Chang

1990

The Journal of Cell Biology

105

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This paper reports the isolation and characterization of Chinese hamster ovary cell mutants defective in low density lipoprotein (LDL)- cholesterol trafficking. The parental cell line was 25-RA, which possesses LDL receptors and various cholesterogenic enzyme activities that are partially resistant to down regulation by exogenous sterols (Chang, T. Y., and J. S. Limanek. 1980. J. Biol. Chem. 255:7787-7795). Because these cells accumulate a large amount of intracellular cholesteryl ester when grown in medium containing 10% fetal calf serum, mutagenized populations of 25-RA cells were grown in the presence of a specific inhibitor of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which depleted their cholesteryl ester stores. Without this cholesterol ester storage, 99% of 25-RA cells die after 5-d growth in cholesterol starvation medium, while the mutant cells, which accumulate free cholesterol intracellularly, survived. In two mutant clones chosen for characterization, activation of cholesteryl ester synthesis by LDL was markedly reduced in the mutant cells compared with 25-RA cells. This lack of activation of cholesterol ester synthesis in the mutant cells could not be explained by defective uptake and/or processing of LDL or by a decreased amount of ACAT, as determined by in vitro enzyme activity. Mutant cells grown in the presence of LDL contain numerous cytosolic particles that stain intensely with the fluorescent compound acridine orange, suggesting that they are acidic. The particles are also stained with filipin, a cholesterol-specific fluorescent dye. Indirect immunofluorescence with a monoclonal antibody specific for a lysosomal/endosomal fraction revealed a staining pattern that colocalized with the filipin signal. The mutant phenotype was recessive. The available evidence indicates that the mutant cells can take up and process LDL normally, but the hydrolyzed cholesterol accumulates in an acidic compartment, probably the lysosomes, where it can not be transported to its normal intracellular destinations.

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Evidence for coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase ad low density lipoprotein binding activity.

Cited by 44 publications

References 32 publications

Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

Isolation of Chinese hamster ovary cell lines expressing human acyl-coenzyme A/cholesterol acyltransferase activity.

Sterol Resistance in CHO Cells Traced to Point Mutation in SREBP Cleavage–Activating Protein

Isolation and characterization of Chinese hamster ovary cell mutants defective in intracellular low density lipoprotein-cholesterol trafficking.

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