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Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers. [ABSTRACT FROM AUTHOR]

Lipid homeostasis in animal cells is maintained by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose proteolytic activation requires the cholesterolsensing membrane protein Scap. In endoplasmic reticulum (ER) membranes, the carboxyl-terminal domain (CTD) of SREBPs binds to the CTD of Scap. When cholesterol levels are low, Scap escorts SREBPs from the ER to the Golgi, where the actions of two proteases release the amino-terminal domains of SREBPs that travel to the nucleus to up-regulate expression of lipogenic genes. The CTD of SREBP remains bound to Scap but must be eliminated so that Scap can be recycled to bind and transport additional SREBPs. Here, we provide insights into how this occurs by performing a detailed molecular dissection of the CTD of SREBP2, one of three SREBP isoforms expressed in mammals. We identify a degradation signal comprised of seven noncontiguous amino acids encoded in exon 19 that mediates SREBP2's proteasomal degradation in the absence of Scap. When bound to the CTD of Scap, this signal is masked and SREBP2 is stabilized. Binding to Scap requires an arginine residue in exon 18 of SREBP2. After SREBP2 is cleaved in Golgi, its CTD remains bound to Scap and returns to the ER with Scap where it is eliminated by proteasomal degradation. The Scap-binding motif, but not the degradation signal, is conserved in SREBP1. SREBP1's stability is determined by a degradation signal in a different region of its CTD. These findings highlight a previously unknown role for the CTD of SREBPs in regulating SREBP activity. [ABSTRACT FROM AUTHOR]

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an 125I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to 125I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The 125I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane. [ABSTRACT FROM AUTHOR]

A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-β-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2. [ABSTRACT FROM AUTHOR]

Cholesterol synthesis in animals is controlled by the regulated transport of sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum to the Golgi, where the transcription factors are processed proteolytically to release active fragments. Transport is inhibited by either cholesterol or oxysterols, blocking cholesterol synthesis. Cholesterol acts by binding to the SREBP-escort protein Scap, thereby causing Scap to bind to anchor proteins called Insigs. Here, we show that oxysterols act by binding to Insigs, causing Insigs to bind to Scap. Mutational analysis of the six transmembrane helices of Insigs reveals that the third and fourth are important for Insig's binding to oxysterols and to Scap. These studies define Insigs as oxysterol-binding proteins, explaining the long-known ability of oxysterols to inhibit cholesterol synthesis in animal cells. [ABSTRACT FROM AUTHOR]

A general theoretical model is described for the NMR spectra of mixtures of sterols and deuterium-labeled phospholipids. In the case of homogeneous membranes, the average quadrupole splittings are determined by equilibria between lipids in cholesterol-phospholipid complexes and lipids not in complexes. Chemical exchange of lipids between those in the free state and those in the complex state affects the deuterium resonance line shapes. The lifetime of a phospholipid molecule in an ergosterol-dipalmitoylphosphatidylcholine complex is estimated to be of the order of 10-5 s on the basis of the observed line broadenings. In the vicinity of a critical point of a cholesterol-phospholipid mixture, fluctuations in the concentration of complexes also can contribute to the deuterium nuclear resonance line broadening. At the critical point, the temperature derivative of the concentration of complexes is discontinuous. There is a corresponding jump in the calculated heat capacity as well as in the temperature derivative of the deuterium NMR first moment. [ABSTRACT FROM AUTHOR]

Animal cell membranes pose conceptual problems related to the physical chemistry of liquids. An avenue to the solution of some of these problems has been opened by the discovery of liquid-liquid immiscibility in synthetic membranes composed of cholesterol and phospholipids. This discovery has led to the development of a thermodynamic model involving condensed complexes. In this model, the phospholipids with longer fatty-acid chains react reversibly with cholesterol to form complexes. The complexes themselves can have a repulsive interaction with other phospholipids, leading to immiscibility. A striking example of this effect is revealed in the phase diagrams of ternary mixtures of cholesterol, a saturated phosphatidylcholine (or sphingomyelin), and an unsaturated phosphatidylcholine. As found by a number of investigators, all binary pairs are miscible in bilayers, whereas the ternary mixture can form two liquid phases. The model of condensed complexes accounts for this effect. Condensed complexes also have a major effect on the chemical activity of cholesterol and on the ordering of phospholipid acyl chains both in the presence and absence of phase separations. Model calculations of phospholipid order parameters account for several features of the deuterium NMR spectra of labeled phospholipid molecules in bilayer mixtures with cholesterol. [ABSTRACT FROM AUTHOR]

The polytopic membrane protein SCAP transports sterol regulatory element-binding proteins (SREBP5) from the endoplasmic reticulum (ER) to the Golgi, thereby activating cholesterol synthesis. Choles- terol accumulation in the ER membranes changes SCAP to an alternate conformation in which it binds ER retention proteins called Insigs, thereby terminating cholesterol synthesis. Here, we show that the conserved Asp-428 in the sixth transmembrane helix of SCAP is essential for SCAP's dissociation from Insigs. In transfected hamster cells, mutant SCAP in which Asp-428 is replaced by alanine (D428A) remained in an Insig-binding conformation when cells were depleted of sterols. As a result mutant SCAP failed to dissociate from Insigs, and it failed to carry SREBPs to the Golgi. These data identify an important functional residue in SCAP, and they provide genetic evidence that the conformation of SCAP dictates the rate of cholesterol synthesis in animal cells. [ABSTRACT FROM AUTHOR]

Presents critical points in charged membranes containing cholesterol. Technique used to determine phase diagrams for lipid monolayers containing binary mixtures of cholesterol; Association of critical points with the formation of condensed complexes between lipids and cholesterol.

Describes a liquid-state model for theoretical approximation of nonideal thermodynamic properties of coexisting liquid phases of cholesterol and phospholipids at the air-water interface of cell membranes. Theoretical pressure-composition phase diagrams; Role of cholesterol and phospholipids on cellular functions.

Presents a study which examined electric field effect on dihydrocholesterol and phospholipids. Challenges in understanding the chemical activity of cholesterol in cell membranes; Materials and methods used in the study; Results.

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers., Competing Interests: Competing interests statement:The authors declare no competing interest.

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann-Pick C1 (NPC1) and soluble Niemann-Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25-264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [(3)H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.