[Show abstract][Hide abstract] ABSTRACT: ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P(3) and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.
[Show abstract][Hide abstract] ABSTRACT: Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.
[Show abstract][Hide abstract] ABSTRACT: Caveolae (small plasma membrane invaginations) and their coat proteins, caveolins, have attracted the attention of researchers in diverse fields, including cell biology, cardiovascular and cancer research. The tight association between caveolin and cholesterol governs the biochemical behaviour of caveolae and is emerging as an important characteristic in a number of processes assigned to these multifunctional organelles. In this review, selected aspects of the caveolin-cholesterol association and its potential functional implications are discussed.
[Show abstract][Hide abstract] ABSTRACT: Previous work demonstrates that the biosynthetic precursor of cholesterol, desmosterol, is released from cells and that its efflux to high density lipoprotein or phosphatidylcholine vesicles is greater than that of newly synthesized cholesterol (Johnson, W. J., Fischer, R. T., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 25037-25046). Here we report that the release of individual precursor sterols varies with the efflux of newly synthesized zymosterol being greater than that of lathosterol and both exceeding that of newly synthesized cholesterol when using either methyl-beta-cyclodextrin or complete serum as acceptors. The transfer of newly synthesized lathosterol to methyl-beta-cyclodextrin was inhibited by actin polymerization but not by Golgi disassembly whereas that of newly synthesized cholesterol was inhibited by both conditions. Newly synthesized lathosterol associated with cellular detergent-resistant membranes more rapidly than newly synthesized cholesterol. Upon efflux to serum, newly synthesized cholesterol precursors associated with both high and low density lipoproteins. Stimulation of the formation of direct endoplasmic reticulum-plasma membrane contacts was accompanied by enhanced efflux of newly synthesized lathosterol but not of newly synthesized cholesterol to serum acceptors. The data indicate that the efflux of cholesterol precursors differs not only from that of cholesterol but also from each other, with the more polar zymosterol being more avidly effluxed. Moreover, the results suggest that the intracellular routing of cholesterol precursors differs from that of newly synthesized cholesterol and implicates a potential role for the actin cytoskeleton and endoplasmic reticulum-plasma membrane contacts in the efflux of lathosterol.
[Show abstract][Hide abstract] ABSTRACT: In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.
[Show abstract][Hide abstract] ABSTRACT: In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15 degrees C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.
Proceedings of the National Academy of Sciences 08/2000; 97(15):8375-80. DOI:10.1073/pnas.140218797 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have investigated whether pyrene-labelled cholesterol esters (PyrnCEs) (n indicates the number of aliphatic carbons in the pyrene-chain) can be used to observe the degradation of low-density lipoprotein (LDL)-derived cholesterol esters (CEs) in the lysosomes of living cells. To select the optimal substrates, hydrolysis of the PyrnCE species by lysosomal acid lipase (LAL) in detergent/phospholipid micelles was compared. The rate of hydrolysis varied markedly depending on the length of the pyrenyl chain. Pyr10CE was clearly the best substrate, while Pyr4CE was practically unhydrolysed. Pyr10CE and [3H]cholesteryl linoleate, the major CE species in LDL, were hydrolysed equally by LAL when incorporated together into reconstituted LDL (rLDL) particles, thus indicating that Pyr10CE is a reliable reporter of the lysosomal degradation of native CEs. When rLDL particles containing Pyr4CE or Pyr10CE were incubated with fibroblasts, the accumulation of bright intracellular vesicular fluorescence was observed with the former fluorescent derivative, but not with the latter. However, when the cells were treated with chloroquine, an inhibitor of lysosomal hydrolysis, or when cells with defective LAL were employed, Pyr10CE also accumulated in vesicular structures. HPLC analysis of cellular lipid extracts fully supported these imaging results. It is concluded that PyrnCEs can be used to observe degradation of CEs directly in living cells. This should be particularly useful when exploring the mechanisms responsible for the accumulation of lipoprotein-derived CEs in complex systems such as the arterial intima.
[Show abstract][Hide abstract] ABSTRACT: The effect of the physical state of low density lipoprotein (LDL) core and the selectivity of the degradation of LDL cholesterol esters (CEs) by the lysosomal acid lipase (LAL) in vitro were investigated. The physical state of LDL was modulated by varying temperature or the triglyceride content of the core. Normal LDL showed an abrupt increase of CE hydrolysis at 24 degrees C and another deviation occurred close to 36 degrees C. 1H-NMR measurements showed that these temperatures coincide with the onset and end temperatures of the LDL core lipid transition, respectively. Enrichment of LDL with triglycerides abolished the abrupt changes both in the CE hydrolysis and in the physical state of LDL lipids. These findings show that there is a correlation between the physical state of LDL lipids and the rate of LAL-mediated hydrolysis of the CEs in the particle. The relative rates of hydrolysis of different CE species were also compared. With native LDL, increasing the length of a saturated acyl chain from 14 to 20 carbons reduced the rate of degradation of CE modestly, while increasing acyl chain unsaturation increased the rate of degradation markedly. However, cholesterol oleate was hydrolyzed more slowly than cholesterol stearate. Essentially the same order of hydrolytic susceptibility was observed when the CE species were incorporated into triglyceride-enriched LDL, reconstituted high density lipoprotein particles or in detergent/phospholipid micelles. These results indicate that the selective hydrolysis of CE species in LDL is determined mainly by the ease with which the CE molecule can emerge from the surface layer reach the active site of LAL. Slower degradation of the more saturated CEs by LAL could lead, under certain conditions, to their accumulation in lysosomes and eventually, to cell death, lysis and deposition of crystalline, poorly mobilizable lipids to the arterial intima.
[Show abstract][Hide abstract] ABSTRACT: Low density lipoprotein (LDL) particles can undergo fusion in the arterial intima, where they are bound to proteoglycans. Here we studied the effect of human arterial proteoglycans on proteolytic fusion of LDL in vitro. For this purpose, an assay was devised based on fluorescence resonance energy transfer that allowed continuous monitoring of fusion of proteoglycan-bound LDL particles. We found that addition of human arterial proteoglycans markedly increased the rate of proteolytic fusion of LDL. The glycosaminoglycans isolated from the proteoglycans also increased the rate of fusion, demonstrating that this effect was produced by the negatively charged sulfated polysaccharides in the proteoglycans. Furthermore, heparin, chondroitin 6-sulfate, and dextran sulfate, three commercially available sulfated polysaccharides, also increased the rate of LDL fusion, with heparin and chondroitin 6-sulfate being as effective as and dextran sulfate more effective than human proteoglycans. The ability of the sulfated polysaccharides to increase the rate of proteolytic fusion of LDL depended critically on their ability to form insoluble complexes with LDL, which, in turn, resulted in an increased rate of LDL proteolysis and, in consequence, in an increased rate of LDL fusion. The results reveal a novel mechanism regulating LDL fusion and point to the potentially important role of arterial proteoglycans in the generation of LDL-derived lipid droplets in the arterial intima during atherogenesis.
[Show abstract][Hide abstract] ABSTRACT: The hydrolysis of pyrenylacyl phosphatidylcholines (PyrnPCs)(n indicates the number of aliphatic carbons in the pyrene-chain) by crude lysosomal phospholipases in vitro was investigated. PyrnPCs consist of several sets in which the length of the pyrene-labelled or the unlabelled acyl chain, linked to the sn-1 or sn-2 position, was systematically varied. Lysophosphatidylcholine and fatty acid were the only fluorescent breakdown products detected, thus indicating that PyrnPCs were degraded by A-type phospholipases and lysophospholipases. Of these, mainly A1-type phospholipases appear to be involved, as determined from the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and position of the pyrene-labelled and unlabelled chains it is suggested that (1) the lysosomal A-type phospholipases acting on PyrnPCs recognize the carboxy-terminal part of the lipid acyl chains and (2) the relevant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are peroxidized and polymerized, may not be good substrates for the lysosomal phospholipases mentioned. The impaired hydrolysis of the most hydrophobic PyrnPCs indicates that lysosomal phospholipases may not be able to penetrate significantly into the substrate interphase, but upward movement of the lipid may be required for efficient hydrolysis. Finally, the rate of hydrolysis of many pyrenyl derivatives was found to be comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivatives can be used as faithful reporters of lysosomal degradation of natural lipids in vivo and in vitro.
[Show abstract][Hide abstract] ABSTRACT: 1. Phospholipid transfer protein (PLTP) mediates conversion of high-density lipoprotein (HDL3) to large particles, with concomitant release of apolipoprotein A-I (apoA-I). To study the mechanisms involved in this conversion, reconstituted HDL (rHDL) particles containing either fluorescent pyrenylacyl cholesterol ester (PyrCE) in their core (PyrCE-rHDL) or pyrenylacyl phosphatidylcholine (PysPC) in their surface lipid layer (PyrPC-rHDL) were prepared. Upon incubation with PLTP they behaved as native HDL3, in that their size increased considerably. 2. When PyrPC-rHDL was incubated with HDL3 in the presence of PLTP, a rapid decline of the pyrene excimer/monomer fluorescence ratio (E/M) occurred, demonstrating that PLTP induced mixing of the surface lipids of PyrPC-rHDL and HDL3. As this mixing was almost complete before any significant increase in HDL particle size was observed, it represents PLTP-mediated phospholipid transfer or exchange that is not directly coupled to the formation of large HDL particles. 3. When core-labelled PyrCE-rHDL was incubated in the presence of PLTP, a much slower, time-dependent decrease of E/M was observed, demonstrating that PLTP also promotes mixing of the core lipids. The rate and extent of mixing of core lipids correlated with the amount of PLTP added and with the increase in particle size. The enlarged particles formed could be visualized as discrete, non-aggregated particles by electron microscopy. Concomitantly with the appearance of enlarged particles, lipid-poor apoA-I molecules were released. These data, together with the fact that PLTP has been shown not to mediate transfer of cholesterol esters, strongly suggest that particle fusion rather than (net) lipid transfer or particle aggregation is responsible for the enlargement of HDL particles observed upon incubation with PLTP.4.ApoA-I rHDL, but not apoA-II rHDL, were converted into large particles, suggesting that the presence of apoA-I is required for PLTP-mediated HDL fusion. A model for PLTP-mediated enlargement of HDL particles is presented.