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L Liscum

Publications and source records attributed to L Liscum.

At least 19 recordsLinked to original sources
PubMed2000

Niemann-Pick type C mutations cause lipid traffic jam.

The Niemann-Pick C protein (NPC1) is required for cholesterol transport from late endosomes and lysosomes to other cellular membranes. Mutations in NPC1 cause lysosomal lipid storage and progressive neurological degeneration. Cloning of the NPC1 gene has given us tools with which to investigate the function of this putative cholesterol transporter. Here, we discuss recent studies indicating that NPC1 is not a cholesterol-specific transport molecule. Instead, NPC1 appears to be required for the vesicular shuttling of both lipids and fluid-phase constituents from multivesicular late endosomes to destinations such as the trans-Golgi network.

Animals↗
PubMed1998

Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.

We have studied the movement of low density lipoprotein (LDL)-derived cholesterol in cultured Chinese hamster ovary cells. Our hypothesis is that when LDL cholesterol is effluxed from lysosomes, the bulk of LDL cholesterol is mobilized to the plasma membrane, while another pathway delivers LDL cholesterol from lysosomes to acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum. Three lines of evidence support this model. First, LDL cholesterol transport to ACAT can be blocked without inhibiting the movement of cholesterol from lysosomes to plasma membrane or from plasma membrane to endoplasmic reticulum. Second, LDL cholesterol transport to ACAT is normal in a Chinese hamster ovary mutant with defective plasma membrane-to-ACAT movement. Third, LDL cholesterol is not diluted by the plasma membrane cholesterol pool before reaching ACAT. Our evidence supports a vesicular model of cholesterol transport from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane.

Amphotericin B↗
PubMed1998

Niemann-Pick disease type C.

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disease. Fibroblasts from individuals with Niemann-Pick type C exhibit defective intracellular cholesterol transport. Linkage analysis has led to the recent cloning of the NPC1 gene on human chromosome 18, which is the major disease locus. Analysis of NPC1 reveals homologies with key regulators of cholesterol homeostasis and a Drosophila morphogen receptor.

Carrier Proteins↗
PubMed1997

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Amino Acid Sequence↗
PubMed1997

Analysis of a Chinese hamster ovary cell mutant with defective mobilization of cholesterol from the plasma membrane to the endoplasmic reticulum.

The factors involved in shuttling cholesterol among cellular membranes have not been defined. Using amphotericin B selection, we previously isolated Chinese hamster ovary cell mutants expressing defects in intracellular cholesterol transport. Complementation analysis among seven mutants identified one cell line, mutant 3-6, with a unique defect. The present analysis revealed three key features of mutant 3-6. First, the movement of cholesterol both from the endoplasmic reticulum and through lysosomes to the plasma membrane was normal. However, when intact 3-6 cells were treated with sphingomyelinase, movement of plasma membrane cholesterol to acyl CoA:cholesterol acyltransferase in the endoplasmic reticulum was defective. Cellular cholesterol was mobilized to this enzyme upon activation by 25-hydroxycholesterol. Second, mutant 3-6 did not utilize endogenously synthesized sterol or low density lipoprotein-derived cholesterol for growth as effectively as parental Chinese hamster ovary cells. Finally, despite normal movement of cholesterol to the plasma membrane, mutant 3-6 was amphotericin B resistant. The plasma membrane cholesterol content was normal as assessed by cholesterol oxidase treatment and Semliki Forest virus fusion, which suggests that the 3-6 mutation alters the organization of cholesterol in the plasma membrane. Our characterization of this mutant cell line should facilitate the identification of gene(s) required for this transport pathway.

Amphotericin B↗
PubMed1996

Quantitative analysis of hydrophobic amine inhibition of intracellular cholesterol transport.

U18666A and imipramine are hydrophobic amines that inhibit intracellular cholesterol transport pathways. In this study, we conducted dose-response curves for each of the cholesterol transport pathways. Our analyses indicate that hydrophobic amine inhibition of LDL-stimulated cholesterol esterification is much more sensitive to inhibition than either the combined bulk movement of cholesterol from lysosomes to the plasma membrane and from the plasma membrane to the endoplasmic reticulum. Hydrophobic amines must inhibit a previously uncharacterized pathway from lysosomes to the endoplasmic reticulum or a signaling event that activates acyl CoA:cholesterol acyltransferase. Possible mechanisms for U18666A action were evaluated. The function of p-glycoprotein, which has been implicated in cholesterol transport, was unaffected by U18666A. We have evidence for a specific membrane U18666A binding site, which we hypothesize is involved in the plasma membrane to endoplasmic reticulum cholesterol transport pathway. Identification of the binding site and mechanism of hydrophobic amine action may provide information essential for understanding intracellular cholesterol transport.

Androstenes↗
PubMed1995

Marked heterogeneity in Niemann-Pick disease, type C. Clinical and ultrastructural findings.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lysosomal lipid storage disorder of unknown etiology. Diagnosis of NP-C is based on characteristic clinical findings and reduced fibroblast esterification of LDL-derived cholesterol. We describe three patients who demonstrate the NP-C spectrum of clinical heterogeneity in age of onset, presenting signs, pattern of organ system involvement, and natural history. In addition, electron microscopic analysis of skin biopsy specimens from these patients revealed marked variability in the extent and cellular distribution of intralysosomal storage and was suggestive of the correct diagnosis in only one case. These cases demonstrate both the limitations of electron microscopy for diagnosis of NP-C and the marked clinical variability in patients with this disorder. Practical clinical guidelines for appropriate suspicion of NP-C are presented.

Adolescent↗
PubMed1994

Compartmentation of cholesterol within the cell.

Mammalian cells tightly regulate their cholesterol content and the intracellular disposition of cholesterol. Most cellular free cholesterol resides in the plasma membrane, where it exists in lateral domains. Mechanisms governing cellular cholesterol levels and compartmentation are still largely unknown. In this review, we highlight recent studies documenting the compartmentation of cholesterol, especially those that have relevance to the regulation of cholesterol content.

Animals↗
PubMed1994

A second complementation class of cholesterol transport mutants with a variant Niemann-Pick type C phenotype.

We previously isolated Chinese Hamster ovary cell mutants that were defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol (Dahl, N.K., K.L. Reed, M.A. Daunais, J.R. Faust, and L. Liscum. 1992 J. Biol. Chem. 267: 4889-4896). Several of the mutants exhibited the same biochemical phenotype as classical Niemann-Pick type C (NPC) fibroblasts. Complementation analysis between these mutants and other cholesterol transport mutants with a variant biochemical phenotype has defined two complementation classes. One class is characterized by expression of the classical NPC phenotype and may represent a true cholesterol transport mutant, while the second is characterized by expression of a variant NPC phenotype and may represent a signaling defect in LDL-sensitive homeostatic responses.

Animals↗
PubMed1993

Abnormal regulation of low density lipoprotein-sensitive events in a cholesterol transport mutant.

We have isolated and characterized Chinese hamster ovary cell mutants defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol (Dahl, N. K., Reed, K. L., Daunais, M. A., Faust, J. R., and Liscum, L. (1992) J. Biol. Chem. 267, 4889-4896). Mutant 2-2, which exhibits a cholesterol transport defect indistinguishable from the Niemann-Pick C phenotype, shows impaired but not absent LDL-mediated suppression of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. In parental cells, LDL suppression of HMG-CoA reductase is modulated by two mechanisms, decreased gene transcription and accelerated protein turnover. Using the chimeric protein HMGal as a reporter protein for LDL-mediated turnover and Northern blot analysis to monitor HMG-CoA reductase mRNA levels, we have dissected the contributions of these two regulatory responses to LDL-mediated suppression of HMG-CoA reductase activity. Kinetic modeling using the kinlsq program showed the following. Mutant 2-2 exhibits normal LDL-mediated acceleration of HMGal degradation, coupled with relatively abnormal regulation of mRNA. This suggests that the LDL-cholesterol signaling pathway to the nucleus is defective relative to the signal that results in HMG-CoA reductase turnover. In addition, LDL-mediated acceleration of HMGal turnover occurs well before LDL stimulation of cholesterol esterification in mutant 2-2, whereas these events occur synchronously in the parental cell line. This suggests that more than one pathway or mechanism exists for LDL-cholesterol signaling to the endoplasmic reticulum.

Animals↗
PubMed1992

Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.

We have isolated clones of an established cell line which express defects in intracellular cholesterol metabolism. Chinese hamster ovary cells were mutagenized, and clones unable to mobilize low density lipoprotein (LDL)-derived cholesterol to the plasma membrane were selected. Biochemical analysis of two mutant clones revealed a phenotype characteristic of the lysosomal storage disease, Niemann-Pick type C. The mutant cell lines were found to be defective in the regulatory responses elicited by LDL-derived cholesterol. LDL-mediated stimulation of cholesterol esterification was grossly defective, and LDL suppression of 3-hydroxy-3-methylglutaryl-CoA reductase was impaired. However, the mutants modulated these activities normally in response to 25-hydroxycholesterol or mevalonate. The LDL-specific defects were predicated by the inability of these mutants to mobilize LDL-derived cholesterol from lysosomes. Cell fractionation studies showed that LDL-derived, unesterified cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, commensurate with defective movement of cholesterol to other cellular membranes. Characterization of cell lines defective in intracellular cholesterol transport will facilitate identification of the gene(s) required for intracellular cholesterol movement and regulation.

Amphotericin B↗
PubMed1992

Intracellular cholesterol transport.

The intracellular movement of cholesterol in mammalian cells may involve complex pathways by which the sterol moves to various cellular sites and mediates transcriptional regulation, enzyme activation, and protein degradation. Current evidence indicates that there are three distinct pathways modulating intracellular cholesterol trafficking. The movement of endogenously synthesized cholesterol from the endoplasmic reticulum appears to be distinct from movement of exogenous, low density lipoprotein (LDL)-derived cholesterol to the plasma membrane. In addition, steroidogenic cells possess a third mechanism by which cholesterol is transported to the mitochondria to initiate steroid hormone synthesis. In this review, we have outlined the current knowledge of cholesterol transport mechanisms and pathways and have described approaches that may help define cholesterol trafficking mechanisms in molecular detail. The use of genetic and molecular biologic techniques can potentially reveal gene products that are involved in intracellular cholesterol transport and regulation as well as those that may secondarily affect this process.

Animals↗
PubMed1991

Characterization of Chinese hamster ovary cells that are resistant to 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibition of low density lipoprotein-derived cholesterol metabolism.

The pharmacological agent U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one inhibits the intracellular transport of low density lipoprotein (LDL)-derived cholesterol in Chinese hamster ovary (CHO) cells. LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated cells causing delayed LDL-mediated regulation of cellular cholesterol metabolism and impaired movement of LDL-derived cholesterol to other cell membranes. As a result of impaired LDL-derived cholesterol transport, LDL-dependent growth of CHO cells is also inhibited by U18666A. By selecting for cell growth in the presence of U18666A, we have identified a CHO cell line, designated U18R, that is resistant to U18666A-inhibition of LDL-derived cholesterol trafficking. When compared to parental CHO cells, U18R cells are relatively resistant to U18666A inhibition of LDL-derived cholesterol transport as well as LDL-mediated regulation of cellular cholesterol metabolism. In cell fusion experiments, the U18666A resistance observed in U18R cells displays a dominant phenotype. Identification of the U18666A-resistant factor may provide important insights toward the understanding of intracellular LDL-derived cholesterol regulation and trafficking.

Androstenes↗
PubMed1990

Pharmacological inhibition of the intracellular transport of low-density lipoprotein-derived cholesterol in Chinese hamster ovary cells.

Mammalian cells, cultured in the presence of serum lipoproteins, acquire cholesterol necessary for growth from the uptake and lysosomal hydrolysis of low-density lipoproteins (LDL). The mechanism(s) of intracellular transport of LDL-derived cholesterol from lysosomes to other cellular sites is unknown. In this study, various pharmacological agents were assessed for their ability to inhibit the movement of LDL-cholesterol from lysosomes to the plasma membrane. The only pharmacological agent tested in these experiments that specifically inhibited LDL-cholesterol movement was U18666A. Ketoconazole impaired the intracellular transport of LDL-cholesterol; however, ketoconazole also had a general effect on cholesterol movement, since it impeded the desorption of endogenously synthesized cholesterol into the medium. Other drugs that affected cholesterol movement appeared to be nonspecific. Cholesterol transport from lysosomes to plasma membranes was not significantly altered by agents that affect lysosomal function or cytoskeletal organization, as well as energy poisons and cycloheximide.

Androstenes↗
PubMed1989

The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one.

In mammalian cells, low density lipoprotein (LDL) is bound, internalized, and delivered to lysosomes where LDL-cholesteryl esters are hydrolyzed to unesterified cholesterol. The mechanisms of intracellular transport of LDL-cholesterol from lysosomes to other cellular sites and LDL-mediated regulation of cellular cholesterol metabolism are unknown. We have identified a pharmacological agent, U18666A (3-beta-[2-diethyl-amino)ethoxy]androst-5-en-17-one), which impairs the intracellular transport of LDL-derived cholesterol in cultured Chinese hamster ovary (CHO) cells. U18666A blocks the ability of LDL-derived cholesterol to stimulate cholesterol esterification, and to suppress 3-hydroxy-3-methylglutaryl-coenzyme A reductase and LDL receptor activities. However, U18666A does not impair 25-hydroxycholesterol-mediated regulation of these processes. In addition, U18666A impedes the ability of LDL-derived cholesterol to support the growth of CHO cells. However, U18666A has only moderate effects on growth supported by non-lipoprotein cholesterol. LDL binding, internalization, and lysosomal hydrolysis of LDL-cholesteryl esters are not affected by the presence of U18666A. Analysis of intracellular cholesterol transport reveals that LDL-derived cholesterol accumulates in the lysosomes of U18666A-treated CHO cells which results in impaired movement of LDL-derived cholesterol to other cell membranes.

Androstenes↗