Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists.This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening. -Athar, H., J. Iqbal, X-C. Jiang, and M. M. Hussain. A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein.
Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.
To Recent studies showing that a defect in the MTP is the proximal cause of abetalipoproteinemia indicate that this protein is required for assembly and secretion of apoBcontaining lipoproteins (10, 11). Abetalipoproteinemic patients have only trace amounts of plasma apoB-containing lipoproteins, resulting in extremely low TG and cholesterol levels (12). The cause of this phenotype is a defect in the pathway responsible for assembly and secretion of apoBcontaining lipoproteins since the apoB gene (13, 14) and lipid synthesis (12) have been shown to be normal. Thus, it is clear that MTP is required for the efficient assembly and secretion of apoB-containing lipoprotein particles.Although studies of abetalipoproteinemic patients indicate that MTP is required for the production of plasma lipoproteins containing apoB, the role of MTP in this process remains unclear. Also, it is not known whether MTP is the only tissue-specific factor needed by hepatocytes and enterocytes to synthesize and secrete these particles. To address these issues, apoB-53 was expressed in either a nonlipoprotein-producing cell line (HeLa) or a derivative cell line stably expressing the large subunit ofMTP and MTP activity. These cells were evaluated for their ability to secrete apoBcontaining lipoproteins into the tissue culture medium. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 7628The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-lH-isoindol-1-one (BMS-200150). The similarity of the IC50 for inhibition of bovine MTPmediated TG transfer (0.6 ,iM) Genetic studies (1)(2)(3)(4) have demonstrated that an absence of microsomal triglyceride (TG) transfer protein (MTP) causes abetalipoproteinemia, a disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing plasma lipoproteins. These studies indicate that MTP is required for the production of the apoB containing lipoproteins, very low density lipoproteins and chylomicrons by the liver and intestine. The requirement for MTP for lipoprotein production is further supported by studies using cell lines that are not of hepatic or intestinal origin. When a truncated form of apoB representing 53% of the full-length apoB-100 is expressed in HeLa cells, virtually no apoB is secreted (5). However, when MTP is coexpressed with apoB, apoB is secreted as a lipoprotein particle. Similar findings have been observed in COS cells (6).MTP is found in the lumen of microsomes isolated from liver and intestine (7). The protein purified from bovine liver is a heterodimer consisting of the multifunctional enzyme, protein disulfide isomerase, and a unique, large 97-kDa subunit (8-10). In vitro, MTP A mixture of compound A (62.5 mmol) and Zn dust (438 mmol) in AcOH (250 ml) was heated at reflux for 18 h, cooled to room temperature, filtered through Celite (Aldrich), and concentrated. The residue was dissolved in CH2Cl2 (500 ml), washed with saturated NaHCO3 (2 x 100 ml) and brine (100 ml), dried over MgSO4, and concentrated. The crude product was recrystallized from i-PrOH to yield 2,3-dihydro-2-[1-
Highly active anti-retroviral therapies, which incorporate HIV protease inhibitors, resolve many AIDS-defining illnesses. However, patients receiving protease inhibitors develop a marked lipodystrophy and hyperlipidemia. Using cultured human and rat hepatoma cells and primary hepatocytes from transgenic mice, we demonstrate that protease inhibitor treatment inhibits proteasomal degradation of nascent apolipoprotein B, the principal protein component of triglyceride and cholesterol-rich plasma lipoproteins. Unexpectedly, protease inhibitors also inhibited the secretion of apolipoprotein B. This was associated with inhibition of cholesteryl-ester synthesis and microsomal triglyceride transfer-protein activity. However, in the presence of oleic acid, which stimulates neutral-lipid biosynthesis, protease-inhibitor treatment increased secretion of apolipoprotein B-lipoproteins above controls. These findings suggest a molecular basis for protease-inhibitor-associated hyperlipidemia, a serious adverse effect of an otherwise efficacious treatment for HIV infection.
Adipocyte lipid-binding protein (A-LBP) and muscle fatty acid-binding protein (M-FABP) are members of a family of small ( approximately 15 kDa) cytosolic proteins that are involved in the metabolism of fatty acids and other lipid-soluble molecules. Although highly homologous (65%) and structurally very similar, A-LBP and M-FABP display distinct ligand binding characteristics. Since ligand binding may be influenced by intrinsic protein dynamical properties, we have characterized the backbone and side chain dynamics of uncomplexed (apo) human A-LBP and M-FABP. Backbone dynamics were characterized by measurements of 15N T1 and T2 values and ¿1H¿-15N NOEs. These data were analyzed using model-free spectral density functions and reduced spectral density mapping. The dynamics of methyl-containing side chains were charaterized by measurements of 2H T1 and T1rho relaxation times of 13C1H22H groups. The 2H relaxation data were analyzed using the model-free approach. For A-LBP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 42 methyl groups. For M-FABP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 53 methyl groups. The intrinsic flexibilities of these two proteins are compared, with particular emphasis placed on binding pocket residues. There are a number of distinct dynamical differences among corresponding residues between the two proteins. In particular, many residues display greater backbone picosecond to nanosecond and/or microsecond to millisecond time scale mobility in A-LBP relative to M-FABP, including F57, K58, and most residues in alpha-helix 2 (residues 28-35). Variations in the dynamics of this region may play a role in ligand selectivity. The side chains lining the fatty acid binding pocket display a wide range of motional restriction in both proteins. Side chains showing distinct dynamical differences between the two proteins include those of residues 20, 29, and 51. This information provides a necessary benchmark for determining dynamical changes induced by ligand binding and may ultimately lead to an enhanced understanding of ligand affinity and selectivity among fatty acid-binding proteins.
The microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of the lipoproteins containing apolipoprotein B (apoB): very low density lipoproteins and chylomicrons. Evidence indicates that the subclasses of these lipoproteins that contain apoB-48 are assembled in a distinct two-step process; first a relatively lipid-poor primordial lipoprotein precursor is produced, and then bulk neutral lipids are added to form the core of these spherical particles. To determine if either step is mediated by MTP, a series of clonal cell lines stably expressing apoB-53 and MTP was established in non-lipoprotein-producing HeLa cells. MTP activity in these cells was ϳ30%, and apoB secretion was 7-33% of that in HepG2 cells on a molar basis. Despite having robust levels of triglyceride and phospholipid synthesis, these cell lines, as exemplified by HLMB53-59, secreted >90% of the apoB-53 on relatively lipid-poor particles in the density range of 1.063-1.21 g/ml. These results suggested that coexpression of MTP and apoB only reconstituted the first but not the second step in lipoprotein assembly. To extend this observation, additional studies were carried out in McArdle RH-7777 rat hepatoma cells, in which the second step of apoB-48 lipoprotein assembly is well defined. Treatment of these cells with the MTP photoaffinity inhibitor BMS-192951 before pulse labeling with [ 35 S]methionine/cysteine led to an 85% block of both apoB-48 and apoB-100 but not apoAI secretion, demonstrating inhibition of the first step of lipoprotein assembly. After a 30-min [ 35 S]methioneine/cysteine pulse labeling and 120 min of chase, all of the nascent apoB-48 was observed to have a density of high density lipoproteins (1.063-1.21 g/ml), indicating that only the first step of lipoprotein assembly had occurred. The addition of oleic acid to the cell culture media activated the second step as evidenced by the conversion of the apoB-48 high density lipoproteins to very low density lipoproteins (d < 1.006 g/ml) during an extended chase period. Inactivation of MTP after completion of the first step, but before stimulation of the second step by the addition of oleic acid, did not block this conversion. Thus, inhibition of MTP did not hinder the addition of bulk core lipid to the primordial lipoprotein precursor particles, indicating that MTP is not required for the second step of apoB-48 lipoprotein assembly. Lipoproteins containing apolipoprotein B (apoB)1 are the main carriers of triglyceride and cholesteryl esters in the circulatory system (1). Very low density lipoprotein (VLDL) is a triglyceride-rich particle produced by the liver. In humans, VLDL contains one copy of apoB-100 per particle. In other species, such as rats, mice, rabbits, and dogs, the liver also produces VLDL containing one copy of a smaller form of apoB generated by post-transcriptional deamination of cytidine residue 6666 of the mRNA, converting it to a uridine, changing codon 2153 from a glutamine to a translation stop signal (2). The resulting translation p...
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