Christopher J. Fielding

A protein cofactor of lecithin:Cholesterol acyltransferase

Abstract The effect of proteins isolated from human high density lipoprotein on cholesterol esterification by purified lecithin:cholesterol acyltransferase in the presence of dispersed lipids was studied. One of the major proteins present gave a maximal rate of esterification similar to that found with the native lipoprotein. The other major lipoprotein protein was inactive as a cofactor but significantly reduced activity produced by the cofactor protein.

Cellular cholesterol efflux.

Efflux of free cholesterol (FC) continues even when cellular FC mass is unchanged. This reflects a recirculation of preformed FC between cells and extracellular fluids which has multiple functions in cell biology including receptor recycling and signaling as well as cellular FC homeostasis. Total FC efflux is heterogeneous. Simple diffusion to mature high density lipoprotein (HDL), mainly via albumin as intermediate, initiates FC net transport driven by plasma lecithin:cholesterol acyltransferase activity. A second major efflux component reflects protein-facilitated transport from cell surface domains (caveolae, rafts) driven by FC binding to lipid-poor, pre-beta-migrating HDL (pre-beta-HDL). Facilitated efflux from caveolae, unlike simple diffusion, is highly regulated. Neither ABC1 (the protein defective in Tangier disease) nor other ATP-dependent transporters now appear likely to contribute directly to FC efflux. Their role is limited to the initial formation of a particle precursor to circulating pre-beta-HDL, which recycles without further lipid input from ATP-dependent transporter proteins. Lipid-free apolipoprotein A-I, previously considered a surrogate for pre-beta-HDL, has a reactivity much lower than that of native lipoprotein FC acceptors.

Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virions

HIV buds from lipid rafts and requires cholesterol for its egress from and entry into cells. Viral accessory protein Nef plays a major role in this process. In this study, it not only increased the biosynthesis of lipid rafts and viral particles with newly synthesized cholesterol, but also enriched them. Furthermore, via the consensus cholesterol recognition motif at its C terminus, Nef bound cholesterol. When this sequence was mutated, Nef became unable to transport newly synthesized cholesterol into lipid rafts and viral particles. Interestingly, although its levels in lipid rafts were not affected, this mutant Nef protein was poorly incorporated into viral particles, and viral infectivity decreased dramatically. Thus, Nef also transports newly synthesized cholesterol to the site of viral budding. As such, it provides essential building blocks for the formation of viruses that replicate optimally in the host.

Cholesterol net transport, esterification, and transfer in human hyperlipidemic plasma.

Cholesterol esterification, cholesteryl ester transfer between lipoproteins, and cholesterol transport between lipoproteins and cultured cells have been measured in the plasma of 22 patients with primary hyperlipidemia and 10 normolipidemic subjects. In hyperbetalipoproteinemia, increase in plasma low density lipoprotein levels was associated with a reduction of cholesteryl ester transfer rates, and with a reversal of the normal direction of sterol transport between fibroblasts and their plasma culture medium. Instead of net transport from cells to medium there was a net uptake of sterol from plasma by the cells, despite a level of plasma lecithin/cholesterol acyltransferase activity that was within the normal range. In dysbetalipoproteinemia, esterification rates were increased above normal levels, but cholesteryl ester transfer was reduced and the direction of sterol transport between the cells and plasma medium was reversed, as in the hyperbetalipoproteinemic group. In hypertriglyceridemia, those subjects with cardiovascular disease showed a metabolic pattern similar to the hyperbetalipoproteinemic group. The subjects in this group without symptoms of cardiovascular disease showed a normal direction of sterol transport, normal or raised rates of cholesteryl ester transfer between lipoproteins, and an increased rate of sterol esterification in plasma that decreased towards normal levels as plasma triglyceride levels decreased. Despite their quite distinct metabolic patterns there was no consistent difference between the two hypertriglyceridemic groups in triglyceride or cholesterol levels, very low density lipoprotein composition, or electrophoretic or isoelectric focussing patterns. All hypertriglyceridemic subjects with documented cardiovascular disease showed reversed cell-plasma sterol transport and all subjects without such disease showed a normal direction of cell-plasma sterol transport. The results of this study indicate major and reproducible abnormalities in plasma cholesterol metabolism in several groups of subjects with genetically distinct hyperlipidemias, who are at risk for atherosclerotic vascular disease. The possible predictive value of sterol metabolic measurements in the analysis of cardiovascular disease is discussed.

Lecithin : Cholesterol acyltransferase : Effects of substrate composition upon enzyme activity

Abstract 1. 1. The action of purified lecithin : cholesterol acyltransferase against synthetic dispersions of lecithin and cholesterol in the presence of proteins isolated from high density lipoprotein was investigated. Maximal enzymatic activity was obtained at a lecithin/cholesterol molar ratio of 4, in the presence of a specific protein cofactor. 2. 2. The optimal substrate ratio was not changed by addition of a protein component of high density lipoproteins which inhibited enzyme activity. 3. 3. Cholesterol esters, at proportions similar to those present in high density lipoproteins, reduced enzyme activity to about 20% of that obtained in the presence of only cholesterol and lecithin as lipid components. This lower rate was similar to that found using the native lipoprotein. 4. 4. Triglyceride inhibited the enzyme under the same conditions, but to a lesser extent than did cholesterol ester. 5. 5. Lysolecithin reduced enzyme activity only in the absence of albumin.

Relationship between cholesterol trafficking and signaling in rafts and caveolae

Caveolae and lipid rafts are two distinct populations of free cholesterol, sphingolipid (FC/SPH)-rich cell surface microdomains. They differ in stability, shape, and the presence or absence of caveolin (present in caveolae) or GPI-anchored proteins (enriched in lipid rafts). In primary cells, caveolae and rafts support the assembly of different signaling complexes, though signal transduction from both is strongly dependent on the presence of FC. It was initially thought that FC promoted the formation of inactive reservoirs of signaling proteins. Recent data supports the concept of a more dynamic role for FC in caveolae and probably, also lipid rafts. It is more likely that the FC content of these domains is actively modulated as protein complexes are formed and, following signal transduction, disassembled. In transformed cell lines with few caveolae, little caveolin and a preponderance of rafts, complexes normally assembled on caveolae may function in rafts, albeit with altered kinetics. However, caveolae and lipid rafts appear not to be interconvertible. The presence of non-caveolar pools of caveolin in recycling endosomes (RE), the trans-Golgi network (TGN) and in mobile chaperone complexes is now recognized. A role in the uptake of microorganisms by cells ascribed to caveolae now seems more likely to be mediated by cell surface rafts.

Caveolae and intracellular trafficking of cholesterol.

Caveolae, free cholesterol (FC)-rich microdomains of the plasma membrane, are both a terminus for the intracellular transit of newly synthesized and recycling cellular FC, and a site for FC efflux to the extracellular medium. The same domains play key roles as locations for the assembly of signaling complexes and for the endocytosis of selected ligands. Caveolin, the major structural protein of caveolae, plays a regulatory role in growth, the cell cycle, and cell adhesion. Each of these functions is FC-dependent. Caveolae appear to act as both sensors and regulators of cellular FC content, and in this way mediate an array of membrane-dependent cell functions.

Cholesterol Transport Between Cells and Body Fluids: Role of Plasma Lipoproteins and the Plasma Cholesterol Esterification System

The manner in which cells retain their sterol content is reviewed. Although most of what is known at the molecular level has been defived from studies in continuous cell culture, the findings appear to be broadly applicable to conditions in vivo. The main impetus to this research has come from the potential direct relevance of cell sterol balance to lipid disorders.

Further characterisation of lipoprotein lipase and hepatic post-heparin lipase from rat plasma

Abstract 1. 1. The pre-heparin plasma of the rat, and the perfusate obtained from isolated rat liver following perfusion with heparin, showed lipolytic activities principally against monoglyceride and tributyrin substrates, and minor triglyceride and diglyceride hydrolase activities. Rates of activity with 1- and 2-monoglycerides were similar. 2. 2. Each activity in these preparations was characterised by resistance to inhibition by NaCl and protamine, and by sensitivity to inhibition by diethyl p-nitrophenyl phosphate. The pH optimum was 7.25–7.4 for the different lipid substrates. 3. 3. Purified lipoprotein lipase, the post-heparin perfusate of extrahepatic tissues and whole post-heparin plasma were characterised by major lipolytic activities against di- and triglycerides, and by monoglyceride hydrolase activity directed mainly against the I-isomer. Each activity was strongly inhibited by NaCI and by protamine, and was comparatively resistant to inhibition by diethyl p-nitrophenyl phosphate. pH optima were 8.0–8.3 for the different lipid substrates, in the presence of whole serum as activator. 4. 4. A principal function of the monoglyceride hydrolase activity of pre-heparin plasma, and released from the liver by heparin, may be to hydrolyse the 2-monoglyceride residue, which is the major product of lipoprotein lipase activity against triglyceride substrates.

Endotoxin and Cytokines Decrease Serum Levels and Extra Hepatic Protein and mRNA Levels of Cholesteryl Ester Transfer Protein in Syrian Hamsters

Endotoxin alters the metabolism of lipoproteins, including that of high density lipoprotein (HDL). Cholesteryl ester transfer protein (CETP) facilitates exchange of HDL cholesterol for very low density lipoprotein (VLDL) triglyceride, leading to catabolism of HDL. We investigated the effects of endotoxin and cytokines on CETP in Syrian hamsters. Endotoxin induced a rapid and progressive decrease in serum CETP levels, by 48 h CETP had decreased to < 20% of control levels. Endotoxin also decreased CETP mRNA and protein levels in adipose tissue, heart, and muscle, the tissues with highest levels of CETP mRNA, providing a plausible mechanism for the endotoxin-induced decrease in circulating CETP. Dexamethasone did not mimic the effects of endotoxin on CETP, but the combination of tumor necrosis factor and interleukin-1 did, indicating that these cytokines may in part mediate the effects of endotoxin on CETP. The endotoxin-induced decrease in CETP may help maintain HDL cholesterol levels during infection and inflammation when increased triglyceride levels could drive the exchange of HDL cholesteryl ester for VLDL triglyceride. Maintaining circulating HDL may be important because HDL protects against the toxic effects of endotoxin and provides cholesterol for peripheral cells involved in the immune response and tissue repair.