Microsomal Triacylglycerol Transfer Protein Prevents Presecretory Degradation of Apolipoprotein B‐100

Abstract

The role of microsomal triacylglycerol transfer protein (MTP) in the secretion of apolipoprotein B-1 00 (apoB-100) has been studied using an inhibitor of MTP: 4'-bromo-3'-methylmetaqualone. In vitro, this compound inhibits trioleoylglycerol transfer between lipid vesicles mediated by MTP with an IC,, of 0.9 pM whereas it does not inhibit the lipid transfer mediated by the cholesteryl ester transfer protein.In HepG2 cells, 4'-bromo-3'-methylmetaqualone inhibits the secretion of apoB-100 with an IC,, of 0.3 pM, without affecting the secretion of several other proteins like apoA-I or albumin. Moreover, there is no accumulation of apoB-100 in treated cells. Oleic acid, which increases apoB-100 secretion, only slightly modifies the IC,, of 4'-bromo-3'-methylmetaqualone (0.5 pM). The latter has no effect on the synthesis of major lipids within the cell, but decreases the secretion of triacylglycerol into apoB-100-containing lipoproteins. Pulsekhase experiments reveal that 4'-bromo-3'-methylmetaqualone acts on apoB-I00 production either at the co-translational or post-translational level. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal does not protect apoB-100 from the 4'-bromo-3'-methylmetaqualone effect but seems to be involved in a later step of apoB-100 intracellular degradation. By contrast, dithiothreitol can totally reverse the effect of the MTP inhibitor on apoB-100 production. The mechanism of MTP-mediated lipid assembly with apoB-100 is discussed.Keywords: lipoprotein assembly ; lipid transfer; microsomal triacylglycerol-transfer protein ; N-acetylleucinylleucinyl-2-aminohexanol; hepatocyte.Microsomal triacylglycerol transfer protein (MTP) mediates the transfer of triacylglycerol and cholesteryl ester between membranes 111. MTP displays a clear preference for transporting neutral lipids compared to polar lipids [2]. The transfer activity has only been reported to be present in liver and intestinal mucosa. MTP is a heterodimer containing two polypeptides of molecular masses 58 and 97 kDa. The 58-kDa subunit has been identified as a multifunctional protein, protein disulfide isomerase, whereas the 97-kDa subunit confers lipid transfer activity to the complex 131. In patients with abetalipoproteinemia, a rare human genetic disease characterized by a defect in very low-density lipoprotein (VLDL) and chylomicron production, mutations in the gene encoding the 97-kDa subunit prevent the expression of active MTP [4-71.Apolipoprotein B-100 (apoB-100), the structural protein of VLDL and low-density lipoprotein (LDL) cholesteryl esters are synthesized and assembled in the endoplasmic reticulum to form lipoprotein particles. The particles are subsequently transported to the Golgi apparatus and secreted. However, the sequence of events leading to the formation of mature lipoprotein particles in the lumen of the endoplasmic reticulum is still under investigation (for a review see 1191). Increasing evidence suggests that, at least in some cellular models, several steps of lipidation are required for l...

Co-translational Degradation of Apolipoprotein B100 by the Proteasome Is Prevented by Microsomal Triglyceride Transfer Protein

“…This compound inhibited secretion of apoB100 in a dose-dependent manner but had no effect on secretion of other proteins from HepG2 or primary human hepatocytes (41). As shown in Fig.…”

“…we demonstrated that 4Ј-bromo-3Ј-methylmetaqualone specifically inhibited MTP-mediated lipid transfer (41). This compound inhibited secretion of apoB100 in a dose-dependent manner but had no effect on secretion of other proteins from HepG2 or primary human hepatocytes (41).…”

“…Using HepG2 cells treated with a potent and specific MTP inhibitor 4Ј-bromo-3Ј-methylmetaqualone, we have observed a decrease in the secretion of apoB100 and neutral lipids associated with lipoproteins (41). The decreased secretion of apoB and lipids by the MTP inhibitor has been suggested to reflect mainly an early presecretory degradation of apoB100 (41). We hypothesize that the apparent presecretory degradation of apoB upon MTP inhibition could be attributable to alteration of apoB polypeptide elongation.…”

Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly

“…In Chinese hamster ovary cells, in addition to MTP, cholesterol 7 ␣ -hydroxylase has been shown to be necessary for apoB secretion (54). Third, the importance of the lipid transfer activity in the lipidation of apoB polypeptides was reinforced by using specific inhibitors (55)(56)(57)(58)(59)(60). MTP inhibitors that inhibited lipid transfer activity in vitro decreased apoB secretion in vivo.…”

Insights from human congenital disorders of intestinal lipid metabolism

“…Once inside the enterocyte, the lipolytic products are bound to cytoplasmic FABPs (I-and L-FABP) in order to migrate to the ER where they are reesterifi ed by various enzymes: monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) for the conversion of 2-MG to TG, ACAT-2 for the esterifi cation of(Ping Pong Bi Bi kinetics), MTP acts as a carrier of lipids from their site of synthesis to nascent lipoproteins within the ER ( 66 ). Additional evidence for the critical role of MTP in CM and VLDL production in the enterocyte and hepatocyte, respectively, has been provided with pharmacological approaches utilizing specifi c MTP inhibitors that simultaneously inhibit lipid transfer activity in situ while blocking Apo B secretion (67)(68)(69)(70). Treatment with MTP inhibitors results in a dose-dependent decrease in Apo B secretion, suggesting that MTP is rate-limiting for TG-rich lipoprotein secretion ( 67 ).…”