RESUMO
The rate of cholesterol synthesis from [14C]acetate was low in circulating blood lymphocytes freshly isolated from 17 normal subjects and 4 subjects with homozygous FH. On the other hand, the rate of cholesterol synthesis was two to fourfold above normal in freshly isolated lymphocytes from two subjects with abetalipoproteinemia. When the lymphocytes from subjects with all three genotypes were incubated for 48-72 h in the absence of lipoproteins, the rate of cholesterol synthesis increased by 5-15-fold. The subsequent addition of plasma LDL, but not HDL, rapidly suppressed cholesterol synthesis in the lymphocytes from normal subjects. In contrast, lymphocytes from the FH homozygotes, which have been shown previously to be deficient in cell surface LDL receptors, were resistant to LDL-mediated suppression of cholesterol synthesis. In addition to its ability to suppress cholesterol synthesis after it had been elevated by incubation of the cells in the absence of lipoproteins, LDL was able to suppress the induction of the enhanced rate of sterol synthesis when added to normal lymphocytes immediately after their isolation from the bloodstream. In contrast to the former action of LDL, the latter action of LDL-i.e., the suppression of induction of sterol synthesis-also occurred to a limited extent in lymphocytes from FH homozygotes. However, the FH lymphocytes, but not the normal cells, could be made resistant to this action of LDL by inclusion in the incubation medium of lipoprotein-deficient serum (30 percent, vol/vol) plus HDL (1 mg protein/ml). Considered together with previous data demonstrating a deficiency of LDL receptors in freshly isolated lymphocytes from FH homozygotes, the current studies provide evidence in support of the hypothesis that the interaction of plasma LDL with its cell surface receptor serves to regulate cholesterol synthesis in human lymphocytes.
Assuntos
Abetalipoproteinemia/genética , Colesterol/biossíntese , Hipercolesterolemia/genética , Lipoproteínas LDL/fisiologia , Linfócitos/metabolismo , Abetalipoproteinemia/sangue , Acetatos/metabolismo , Adolescente , Adulto , Criança , Depressão Química , Retroalimentação , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Hipercolesterolemia/sangue , Lanosterol/biossíntese , Lipoproteínas HDL/fisiologia , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , Masculino , Ácidos Oleicos/metabolismo , Receptores de DrogaRESUMO
Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol.
Assuntos
LDL-Colesterol/metabolismo , Doenças de Niemann-Pick/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Cinética , Lipoproteínas LDL/fisiologia , Lisossomos/metabolismo , Ácido Mevalônico/farmacologia , Valores de Referência , Frações Subcelulares/metabolismo , TrítioRESUMO
The transfer of normal human fibroblasts from medium containing whole serum to medium devoid of lipoproteins produced a 90 percent decrease in the cellular content of cholesteryl esters and a 30 percent decrease in the free cholesterol content. When these lipoprotein-deprived cells were subsequently incubated with human low density lipoprotein (LDL), there was a 7-fold increase in the cellular content of esterified cholesterol and a 1.6-fold increase in the cellular content of free cholesterol. The concentration at which LDL produced its half-maximal effect in elevating cellular sterol content (30 mug/ml of LDL-cholesterol) was similar to the half-maximal concentration previously reported for high affinity binding of LDL to its cell surface receptor. High density lipoprotein (HDL) and whole serum from a patient with abetalipoproteinemia (neither of which contains a component that binds to the LDL receptor) did not produce a significant increase in the content of either cholesterol or cholesteryl esters in normal cells. Furthermore, in fibroblasts from patients with the homozygous form of familial hypercholesterolemia, which lack functional LDL receptors, LDL had no effect in raising the cellular content of either free or esterified cholesterol even when present in the medium at concentrations as high as 450 mug sterol/ml. It is concluded that LDL-receptor interactions constitute an important biochemical mechanism for the regulation of the cholesterol content of normal human fibroblasts. Moreover, when considered in light of current concepts of LDL metabolism in intact mammals, the present data suggest that a major function of plasma LDL may be to transport cholesterol from its site of synthesis in liver and intestine to its site of uptake in peripheral tissues.
Assuntos
Colesterol/análise , Fibroblastos/análise , Lipoproteínas LDL/metabolismo , Receptores de Droga , Abetalipoproteinemia/sangue , Transporte Biológico , Criança , Colesterol/biossíntese , Colesterol/metabolismo , Ésteres , Feminino , Fibroblastos/efeitos dos fármacos , Homozigoto , Humanos , Hipercolesterolemia/genética , Recém-Nascido , Mucosa Intestinal/metabolismo , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/sangue , Fígado/metabolismo , Masculino , Ácidos Oleicos/farmacologia , Ligação ProteicaRESUMO
A recombinant plasmid containing a full-length cDNA for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase was introduced by calcium phosphate-mediated transfection into UT-2 cells, a mutant line of Chinese hamster ovary cells that lack 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus require low density lipoprotein-cholesterol and mevalonate for growth. We selected a line of permanently transfected cells, designated TR-36 cells, that expressed high levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thus grew in the absence of low density lipoprotein and mevalonate. Constitutive synthesis of reductase mRNA in TR-36 cells was driven by the simian virus 40 early promoter, and therefore the mRNA was not suppressed by sterols, such as 25-hydroxycholesterol or cholesterol derived from low density lipoprotein, which normally suppresses transcription of reductase mRNA when the reductase gene is driven by its own promoter. Although TR-36 cells continued to synthesize large amounts of reductase mRNA and protein in the presence of sterols, reductase activity declined by 50 to 60%. This decline was caused by a twofold increase in the rate of degradation of preformed enzyme molecules. The current data demonstrate that sterols accelerate the degradation of reductase protein independently of any inhibitory effect on the synthesis of the protein.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Lovastatina/análogos & derivados , Esteróis/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Colesterol/farmacologia , Cricetinae , Cricetulus , DNA/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Peso Molecular , Naftalenos/farmacologia , OvárioRESUMO
The mechanism of biosynthesis of 4-methyl-5-beta-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various 14C-labeled precursors was determined. We found that;e1Me-14C]methionine, [2-14C]methionine, [U-14C]alanine, and [2-14C]glycine were not incorporated whereas [2-14C]tyrosine was incorporated. Degradation of the 4-methyl-5-beta-hydroxyethyl thiazole obtained after [2-14C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-beta-hydroxyethyl thiazole biosynthesis.
Assuntos
Salmonella typhimurium/metabolismo , Tiamina/biossíntese , Tiazóis/metabolismo , Alanina/metabolismo , Glicina/metabolismo , Metionina/metabolismo , Tirosina/metabolismoRESUMO
Endoplasmic reticulum-like membranes (MAM) that are associated with mitochondria have been implicated as intermediates in the import of lipids, particularly phosphatidylserine, from the endoplasmic reticulum to mitochondria (Vance, J.E. (1990) J. Biol. Chem. 265, 7248-7256; Shiao, Y.-J. et al. (1995) J. Biol. Chem. 270, 11190-11198). We have now examined further the role of MAM in lipid metabolism using the mnd/mnd mouse, a model for the human degenerative disease neuronal ceroid lipofuscinosis. The biochemical phenotype of the mnd/mnd mutant mouse (in which lipids and proteins accumulate abnormally in storage bodies in cells of affected tissues) suggested that the mutation might lead to impaired mitochondrial import of lipids and proteins as a result of a defective linkage between MAM and mitochondria. We, therefore, investigated the status of MAM and phospholipid metabolism in mnd/mnd mice livers. Separation of MAM from livers of older, but not younger, mnd/mnd mice was aberrant. In addition, the amount of the MAM-specific protein, phosphatidylethanolamine N-methyltransferase-2 (PEMT2), was greatly reduced in homogenates and MAM from livers of mnd/mnd mice of all ages, although PEMT2 mRNA abundance was normal. Moreover, PEMT activity in MAM from mnd/mnd mice was 60% less than in control mice. Activities of two additional phospholipid biosynthetic enzymes-CTP:phosphocholine cytidylyltransferase and phosphatidylserine synthase-were also reduced by > 50% in mnd/mnd microsomes. Radiolabeling experiments in hepatocytes indicated that neither the mitochondrial import nor the subsequent metabolism of phosphatidylserine was grossly affected in mnd/mnd mice. However, 3 proteins (cytochrome b5, NADH:cytochrome b5 reductase and mitochondrial F1Fzero-ATP synthase c subunit) which are normally present in mitochondria were partially redistributed to microsomes in mnd/mnd mouse liver. These studies indicate that MAM are defective in the mnd/mnd mutant mouse in which the biochemical phenotype includes an abnormal accumulation of lipids and proteins in storage bodies.
Assuntos
Membranas Intracelulares/enzimologia , Mitocôndrias Hepáticas/enzimologia , Lipofuscinoses Ceroides Neuronais/enzimologia , Lipofuscinoses Ceroides Neuronais/patologia , Transferases de Grupos Nitrogenados , Fosfolipídeos/biossíntese , Animais , Fracionamento Celular , Diacilglicerol Colinofosfotransferase/metabolismo , Modelos Animais de Doenças , Etanolaminofosfotransferase/metabolismo , Humanos , Membranas Intracelulares/ultraestrutura , Lipídeos/biossíntese , Fígado/enzimologia , Metiltransferases/metabolismo , Camundongos , Camundongos Mutantes , Mitocôndrias Hepáticas/ultraestrutura , Fosfatidiletanolamina N-Metiltransferase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/enzimologia , Transferases/metabolismoRESUMO
Functioning bovine adrenocortical cells in monolayer culture were shown to obtain cholesterol for steroid synthesis from plasma low density lipoprotein (LDL). When grown in medium devoid of lipoproteins, the cells developed a minimal enhancement in steroid secretion in response to ACTH or cholera toxin. However, when LDL was available, steroid secretion was stimulated 4- to 9-fold. To determine the mechanism for this effect, we used LDL in which the protein component was labeled with 125I and the cholesteryl ester component was labeled with [3H]cholesteryl linoleate. These studies demonstrated that the cells derived cholesterol from LDL by binding the lipoprotein at a high affinity receptor site, internalizing it, and hydrolyzing its cholesteryl esters within lysosomes. The resultant free cholesterol was used for steroid synthesis and also acted to suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis within the cell. LDL receptor activity was enhanced several-fold by treatment of the cells with ACTH or cholera toxin. High density lipoprotein, which did not bind to the LDL receptor, was not degraded with high affinity by the cells and did not support steroid synthesis. The current data suggest that the bovine adrenal cortex can obtain cholesterol for steroid hormone secretion from circulating LDL by means of a high affinity LDL receptor pathway. In a subsequent paper in this series, a similar high affinity LDL-binding site is demonstrated in membranes prepared from fresh bovine adrenocortical tissue.
Assuntos
Córtex Suprarrenal/metabolismo , Colesterol/metabolismo , Lipoproteínas LDL , Lipoproteínas LDL/metabolismo , Receptores de Droga/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Animais , Transporte Biológico , Bovinos , Células Cultivadas , Toxina da Cólera/farmacologia , Ésteres do Colesterol/metabolismo , Humanos , Cinética , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/sangue , EsteroidesRESUMO
The neuronal ceroid lipofuscinoses (NCLs) comprise a set of at least 6 distinct human and an unknown number of animal diseases characterized by storage of proteolipids in lysosomes of many cell types. By unknown mechanisms, this accumulation leads to or is associated with severe neuronal and retinal degeneration. The genes for 3 human NCLs, infantile, late infantile, and juvenile, have been cloned. The first murine form of NCL, the motor neuron degeneration (mnd) mouse, has been described and mapped to proximal Chromosome 8. Here we describe a second genetic variant of NCL in the mouse, neuronal ceroid lipofuscinosis, nclf. These mice exhibited a phenotype that was almost exactly the same as that observed in mnd/mnd mice. Homozygous nclf mice developed progressive retinal atrophy early in life and become paralyzed at around 9 months of age. They accumulated luxol fast blue staining material in cytoplasm of neurons and many other cell types. Ultrastructurally, affected lysosomes had a "finger print pattern" with membranous material arranged in "pentalaminar" patterns. Affected mice developed severe cerebral gliosis in late stages of their disease. They also had severe Wallerian degeneration of long tracts in spinal cord and brain stem, lesions that accounted for the distinctive upper motor neuron signs displayed by both nclf/nclf and mnd/mnd mice. By crossing nclf/nclf mice with CAST/Ei mice, linkage analysis of nclf with respect to SSLP markers was performed, showing that nclf is located on Chromosome 9 between D9Mit164 and D9Mit165, in a region that is homologous with human Ch 15q21, where the gene for one variant of late infantile NCL, CLN6, recently has been mapped. The genes for two proteolipids known to be stored in lysosomes of animals and people with NCL were also mapped in this study and found not to map to the mnd or nclf loci nor to any mouse locus homologous to any known human NCL disease locus.
Assuntos
Mapeamento Cromossômico , Lipofuscinoses Ceroides Neuronais/genética , Animais , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Lipofuscinoses Ceroides Neuronais/patologia , Proteolipídeos/genéticaRESUMO
One characteristic of type C Niemann-Pick (NPC) disease is the substantial intracellular accumulation of unesterified cholesterol. The increased cholesterol content in NPC fibroblasts which are grown in the presence of low density lipoproteins (LDL) has been postulated to be due to a deficiency in cellular cholesterol esterification. We have examined several aspects of LDL metabolism in NPC fibroblasts. We observe that LDL binding, internalization, and lysosomal hydrolysis of LDL cholesteryl esters are normal in NPC cells. As reported by Pentchev et al. (Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251), we find that LDL does not stimulate cholesterol esterification. However, we also show that LDL does not down-regulate cholesterol synthesis or LDL receptor activity as normal. In NPC cells, these processes are regulated normally by nonlipoprotein effectors, such as 25-hydroxycholesterol or mevalonate. Since NPC cells are not defective in lysosomal hydrolysis of LDL-derived cholesteryl esters, they must exhibit a different defect than Wolman's or cholesteryl ester storage diseases. We conclude that NPC cells are defective specifically in LDL-mediated regulation of cellular cholesterol metabolism. We suggest that the intracellular processing of LDL-derived cholesterol may be defective in NPC fibroblasts.
Assuntos
Colesterol/biossíntese , Lipoproteínas LDL/metabolismo , Doenças de Niemann-Pick/metabolismo , Ésteres do Colesterol/metabolismo , Fibroblastos/metabolismo , Humanos , Hidroxicolesteróis/metabolismo , Fatores de TempoRESUMO
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.
Assuntos
Androstenos/farmacologia , Anticolesterolemiantes/farmacologia , LDL-Colesterol/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , LDL-Colesterol/antagonistas & inibidores , Cricetinae , Cricetulus , Depressão Química , Hidrólise , Hidroxicolesteróis/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes , Receptores de LDL/metabolismoRESUMO
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.
Assuntos
Compartimento Celular/fisiologia , Colesterol/metabolismo , Animais , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Humanos , Fígado/metabolismoRESUMO
We have prepared antibodies that recognize isopentenyladenosine (i6A), a modified nucleoside derived from mevalonic acid (MVA). In immunoblot assays, affinity-purified anti-i6 A antibodies specifically bound to a 26-kDa protein (i6A26) in Chinese hamster ovary cells. Anti-i6A recognition of i6A26 was blocked with i6A but not adenosine or isopentenol. Employing immunoblot analysis we have quantitated the level of i6A26 in cells expressing various rates of DNA synthesis. The cellular content of i6A26 was reduced 4-fold in quiescent cells cultured in the absence of serum. When serum-deprived cells were stimulated to enter the cell cycle, the amount of i6A26 increased in the cells during the G1 phase. However, when synchronized cells were stimulated with serum-containing medium in the presence of mevinolin (an inhibitor of cellular MVA synthesis), we observed impaired G1 expression of i6A26 and delayed onset of S phase DNA synthesis. Mevinolin addition to asynchronously growing cells resulted in low rates of cellular DNA synthesis and suppressed levels of i6A26 which were reversed by coincubation with MVA. The ability of MVA to restore DNA synthesis and the cellular content of i6A26 in mevinolin-treated cells showed similar MVA concentration and time dependences. Regenerating liver tissue also exhibited elevated levels of i6A26. Thus, the expression of i6A26 correlates with cellular proliferation and growth. We speculate that i6A26 contains isopentenyladenine moieties and mediates isoprenoid regulation of DNA synthesis. Isopentenyladenylated proteins may also function in cytokinin regulation of proliferation and differentiation in plants.
Assuntos
Adenina/análogos & derivados , Ciclo Celular , Adenina/química , Adenina/imunologia , Animais , Anticorpos/imunologia , Ligação Competitiva , Western Blotting , Cricetinae , Cricetulus , DNA/antagonistas & inibidores , DNA/biossíntese , Isopenteniladenosina , Fígado/efeitos dos fármacos , Lovastatina/farmacologia , Ácido Mevalônico/químicaRESUMO
Squalene synthetase (farnesyltransferase; farnesyl diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), the enzyme in the cholesterol biosynthetic pathway that converts farnesyl pyrophosphate into squalene, is subject to regulation in cultured human fibroblasts. When cholesterol-carrying low density lipoprotein (LDL) was removed from the serum of the culture medium, squalene synthetase activity increased 8-fold over 24 hr. When LDL was added back to the medium, squalene synthetase was slowly suppressed, 50% and 90% reduction occurring in 15 and 48 hr, respectively. Suppression of squalene synthetase required uptake of LDL via the LDL receptor; hence, it did not occur in mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack receptors. The addition of a mixture of 25-hydroxycholesterol and cholesterol suppressed squalene synthetase equally well in normal and mutant fibroblasts. Coupled with previous data, the current findings indicate that cholesterol derived from LDL regulates at least two enzymes in the cholesterol synthetic pathway in fibroblasts: (i) its primary action is to rapidly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP(+), oxidoreductase (CoA-acylating), EC 1.1.1.34], which reduces mevalonate production by 95% within 8 hr, and (ii) its secondary action is to slowly suppress squalene synthetase. The LDL-mediated suppression of squalene synthetase does not regulate de novo cholesterol synthesis; it occurs after 3-hydroxy-3-methylglutaryl coenzyme A reductase is already suppressed. Rather, we hypothesize that it may function to allow the pool size of farnesyl pyrophosphate to be maintained in the presence of LDL so that low levels of mevalonate can be shunted preferentially into nonsterol products, such as ubiquinone-10 and dolichol. This mechanism may explain the earlier observation that the synthesis of ubiquinone-10 in fibroblasts proceeds at a normal rate in the presence of LDL despite a 95% decrease in mevalonate production.
Assuntos
Farnesil-Difosfato Farnesiltransferase/metabolismo , Lipoproteínas LDL/metabolismo , Oxirredutases/metabolismo , Receptores de Droga/fisiologia , Pele/enzimologia , Células Cultivadas , Colesterol/farmacologia , Fibroblastos/enzimologia , Humanos , Hidroxicolesteróis/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Cinética , Lipoproteínas HDL/farmacologia , Lipoproteínas LDL/farmacologiaRESUMO
Human fibroblasts are shown to incorporate [3H]-mevalonolactone into 3H-labeled delta 2-isopentenyl tRNA. This incorporation was observed in cells that were incubated with compactin (ML-236B), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme. A reductase that blocks mevalonate production by cells. When incubated with low concentrations of [3H]mevalonolactone in the presence of compactin and in the absence of exogenous cholesterol, the cells incorporated small amounts of [3H]mevalonolactone into delta 2-isopentenyl tRNA and large amounts into cholesterol. In the presence of low density lipoprotein, which serves as a source of cholesterol, the incorporation of [3H]mevalonolactone into cholesterol was reduced by 90% and the incorporation into delta 2-isopentenyl tRNA was stimulated by 10-fold. Thus, cultured mammalian cells are now known to use mevalonate for synthesis of three nonsterol products, ubiquinone, dolichol, and delta 2-isopentenyl tRNA, as well as for synthesis of cholesterol.
Assuntos
Alcenos/biossíntese , Ácido Mevalônico/metabolismo , RNA de Transferência/biossíntese , Células Cultivadas , Colesterol/biossíntese , Fibroblastos/metabolismo , Humanos , Cinética , Lipoproteínas LDL/farmacologia , Trítio , Ubiquinona/biossínteseRESUMO
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) is a single polypeptide chain with two contiguous domains: a soluble domain (548 amino acids) that catalyzes the rate-controlling step in cholesterol synthesis and a membrane-bound domain (339 amino acids) that anchors the protein to the endoplasmic reticulum (ER). HMG CoA reductase is degraded at least 10-fold more rapidly than other ER proteins; degradation is accelerated in the presence of cholesterol. To understand this controlled degradation, we transfected reductase-deficient Chinese hamster ovary (CHO) cells with a plasmid expression vector containing a reductase cDNA that lacks the segment encoding the membrane domain. The plasmid produced a truncated reductase (37 kd smaller than normal) that was enzymatically active with normal kinetics; most of the truncated enzyme was found in the cytosol. The truncated enzyme was degraded one-fifth as fast as the holoenzyme; degradation was no longer accelerated by sterols. We conclude that the membrane-bound domain of reductase plays a crucial role in the rapid and regulated degradation of this ER protein.
Assuntos
Colesterol/farmacologia , Retículo Endoplasmático/enzimologia , Hidroxicolesteróis/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Membranas Intracelulares/enzimologia , Lovastatina/análogos & derivados , Animais , Linhagem Celular , Cricetinae , Citosol/enzimologia , DNA Recombinante , Feminino , Hidroximetilglutaril-CoA Redutases/genética , Cinética , Naftalenos/farmacologia , Ovário , Plasmídeos , TransfecçãoRESUMO
We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER.
Assuntos
Colesterol/metabolismo , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Animais , Fracionamento Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Colesterol/biossíntese , Colesterol 7-alfa-Hidroxilase/metabolismo , Retículo Endoplasmático/enzimologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Lanosterol/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Esterol O-Aciltransferase/metabolismo , Frações Subcelulares/enzimologiaRESUMO
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.
Assuntos
LDL-Colesterol/metabolismo , Anfotericina B/farmacologia , Animais , Transporte Biológico , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus/genética , Hidroxicolesteróis/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases , Lisossomos/metabolismo , Ácido Mevalônico/farmacologiaRESUMO
To determine the presence and identity of isopentenyladenosine-containing transfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs. This method was based on 3' polyadenylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning and DNA sequencing. Using this unique procedure, we report the cloning and sequencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and other small RNAs where the primary sequence is unknown.
Assuntos
Células CHO/química , Isopenteniladenosina/metabolismo , RNA de Transferência/isolamento & purificação , RNA de Transferência/metabolismo , Animais , Cricetinae , Dados de Sequência Molecular , Análise de Sequência de RNA/métodosRESUMO
The adenine analogue 4-aminopyrazolopyrimidine has been reported previously to reduce the hepatic secretion of plasma lipoproteins in rats, thereby lowering the plasma cholesterol level. In the current studies, reduction of the plasma cholesterol level by 90% in rats through the administration of aminopyrazolopyrimidine was found to be associated with a 5- to 30-fold increase in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC1.1.1.34] in kidney and lung. In both tissues, the enhanced activity of this microsomal enzyme was associated with a 3-fold elevation in the rate of cholesterol synthesis from either [14C]acetate or [14C]octanoate. Comparable increases were not observed in the activities of several other microsomal enzymes or in the rates of [14C]acetate incorporation into saponifiable lipids or CO2. When administration of 4-aminopyrazolopyrimidine was terminated, plasma cholesterol levels rose and 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity declined in the kidney in a reciprocal manner. These data are consistent with the hypothesis that the low levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and cholesterol synthesis that are normally observed in certain nonhepatic tissues of the rat are due to an active form of feedback regulation mediated by cholesterol carried in plasma lipoproteins.