Haris Jamil

Microsomal triglyceride transfer protein

Nucleic acid sequences, particularly DNA sequences, coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, expression vectors containing the DNA sequences, host cells containing the expression vectors, and methods utilizing these materials. The invention also concerns polypeptide molecules comprising all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, and methods for producing these polypeptide molecules. The invention additionally concerns novel methods for preventing, stabilizing or causing regression of atherosclerosis and therapeutic agents having such activity. The invention concerns further novel methods for lowering serum liquid levels and therapeutic agents having such activity.Nucleic acid sequences, particularly DNA sequences, coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, expression vectors containing the DNA sequences, host cells containing the expression vectors, and methods utilizing these materials. The invention also concerns polypeptide molecules comprising all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, and methods for producing these polypeptide molecules. The invention additionally concerns novel methods for preventing, stabilizing or causing regression of atherosclerosis and therapeutic agents having such activity. The invention concerns further novel methods for lowering serum liquid levels and therapeutic agents having such activity.

HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia.

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.

Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly

The microsomal triglyceride transfer protein (MTP) is necessary for the proper assembly of the apolipoprotein B containing lipoproteins, very low density lipoprotein and chylomicrons. Recent research has significantly advanced our understanding of the role of MTP in these pathways at the molecular and cellular level. Biochemical studies suggest that initiation of lipidation of the nascent apolipoprotein B polypeptide may occur through a direct association with MTP. This early lipidation may be required to allow the nascent polypeptide to fold properly and therefore avoid ubiquitination and degradation. Concerning the addition of core neutral lipids in the later stages of lipoprotein assembly, cell culture studies show that MTP lipid transfer activity is not required for this to occur for apolipoprotein B-100 containing lipoproteins. Likewise, MTP does not appear to directly mediate addition of core neutral lipid to nascent apoB-48 particles. However, new data indicate that MTP is required to produce triglyceride rich droplets in the smooth endoplasmic reticulum which may supply the core lipids for conversion of nascent, dense apoB-48 particles to mature VLDL. In addition, assembly of dense apolipoprotein B-48 containing lipoproteins has been observed in mouse liver in the absence of MTP. As a result of these new data, an updated model for the role of MTP in lipoprotein assembly is proposed.

Inhibition of the Microsomal Triglyceride Transfer Protein Blocks the First Step of Apolipoprotein B Lipoprotein Assembly but Not the Addition of Bulk Core Lipids in the Second Step

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 ~30%, 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 [35S]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 [35S]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.

A Novel Abetalipoproteinemia Genotype IDENTIFICATION OF A MISSENSE MUTATION IN THE 97-kDa SUBUNIT OF THE MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN THAT PREVENTS COMPLEX FORMATION WITH PROTEIN DISULFIDE ISOMERASE

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 → Gln, Asp384 → Ala, and Arg540 → His. Transient expression of each mutant showed that only the Arg540 → His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 → His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.

In Vitro Reconstitution of Assembly of Apolipoprotein B48-containing Lipoproteins

Human apolipoprotein B48 (apoB48) and apoB15 (the NH2-terminal 48 and 15% of apoB100, respectively) were translated in vitro from their respective mRNAs using a rabbit reticulocyte lysate and microsomes derived from rat liver or dog pancreas. Synthesis of phosphatidylcholine and triacylglycerols was reconstituted in freshly isolated microsomes by the addition of precursors of these glycerolipids (acylcoenzyme A, glycerol 3-phosphate, and CDP-choline) before, during, or after translation. Assembly of apoB15 and apoB48 with newly synthesized phospholipids and triacylglycerols was favored by active, co-translational lipid synthesis. Moreover, translocation of apoB48 but not B15 into the microsomal lumen was increased in the presence of co-translational lipid synthesis. When apoB48 was translated in vitro, approximately 50% of apoB48 was buoyant at a density of <1.10 g/ml in the lumen of liver microsomes only when lipid synthesis was reconstituted during translation. Microsomal triacylglycerol transfer protein has been proposed to be essential for lipidation and/or translocation of apoB48. However, apoB48 was translocated into the lumen of dog pancreas microsomes in which the activity of the microsomal triacylglycerol transfer protein was not detectable. These data indicate that (i) apoB15 and apoB48 bind newly synthesized phosphatidylcholine during translocation; (ii) apoB48 but not apoB15 associates co-translationally with triacylglycerols; (iii) translocation of apoB48 but not apoB15 is stimulated by lipid synthesis; (iv) assembly of buoyant apoB48-containing lipoproteins can be reconstituted in vitro in the presence of active lipid synthesis; and (v) even in microsomes lacking microsomal triacylglycerol transfer protein activity, apoB48 is translocated into the lumen.

Crystallization of microsomal triglyceride transfer protein from bovine liver

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein required for the assembly of plasma very low density lipoproteins in the liver and chylomicrons in the intestine. Bovine MTP was purified by a modification of a previously published procedure and crystals of MTP were grown reproducibly with polyethylene glycol as a precipitant at pH 7.0. MTP crystals, which diffract to Bragg spacings of better than 3.2 A, have the symmetry of space group P2(1)2(1)2(1) with refined lattice constants of a = 88.7, b = 100.9 and c = 201.1 A, with one heterodimer per asymmetric unit.