The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.     

Overexpression of the endoplasmic reticulum 60 protein ER-60 downregulates apoB100 secretion by inducing its intracellular degradation via a nonproteasomal pathway: evidence for an ER-60-mediated and pCMB-sensitive intracellular degradative pathway

Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway.     

Efficient glycosylation site utilization by intracellular apolipoprotein B. Implications for proteasomal degradation

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) may control the secretion of apoB-containing lipoproteins. In one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation behavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-terminal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide cleavage, all of the sites were glycosylated irrespective of cell type, labeling time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn residues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-linked glycosylation occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2. 5-fold increase in intracellular apoB but failed to cause accumulation of an unglycosylated form. These results indicate that 1) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2) despite its large size and affinity for lipid, delivery of misassembled apoB to the proteasome requires retrograde translocation from the ER lumen to cytosol.     

Ubiquitin-dependent and -independent proteasomal degradation of apoB associated with endoplasmic reticulum and Golgi apparatus,respectively, in HepG2 cells

Studies in hepatocyte cultures indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the proteasome pathway is a major mechanism for the degradation. In the present study, we have examined the detailed itinerary of apoB degradation through its secretory pathway in HepG2 cells. We found that ubiquitin-dependent proteasomal degradation of apoB largely occurred on the cytosolic surface of rough and smooth endoplasmic reticulum (ER) and that a small proportion of apoB was dislodged from the secretory organelles into the cytosolic compartment where it underwent ubiquitination for proteasomal degradation. The transmembrane conformation of apoB persisted as the protein was transported through the Golgi apparatus. We further demonstrated that proteasomal degradation of apoB was associated the Golgi apparatus but Golgi-associated apoB was not ubiquitinated, indicating an ubiquitin-independent proteasomal degradation of apoB is associated with this organelle. We conclude that apoB undergoes proteasomal degradation while going through different compartments of the secretory pathway; further, ER-associated proteasomal degradation of apoB in the ER is ubiquitin-dependent whereas that occurring in the Golgi is ubiquitin-independent.     

Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 micro M) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation. We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Amino acid sequences within the beta1 domain of human apolipoprotein B can mediate rapid intracellular degradation

Apolipoprotein B (apoB)-48 contains a region termed the beta1 domain that is predicted to be composed of extensive amphipathic beta-strands. Analysis of truncated apoB variants revealed that sequences between the carboxyl termini of apoB-37 and apoB-42 governed the secretion efficiency and intracellular stability of apoB. Although apoB-37, apoB-34, and apoB-29 were stable and secreted efficiently, apoB-42 and apoB-100 were secreted poorly and were degraded by an acetyl-leucyl-leucyl-norleucinal (ALLN)-sensitive pathway. Amino acid sequence analysis suggested that a segment between the carboxyl termini of apoB-38 and apoB-42 was 63% homologous to fatty acid binding proteins (FABPs), which contain orthogonal beta-sheets. To test the hypothesis that sequences from the beta1 domain are involved in apoB degradation, fusion proteins were created that contained apoB-29 linked to fragments derived from the beta1 domain of apoB or to liver FABP. Fusion proteins containing the beta1 domain segments apoB-34-42 or apoB-37-42 were degraded rapidly, whereas other fusion proteins were stable and secreted efficiently. Degradation was ALLN-sensitive, and the apoB-34-42 segment increased the association of the apoB protein with the cytosolic surface of the microsomal membrane. Our data suggest that the presence of specific sequences in the beta1 domain of human apoB increases degradation by promoting the cytosolic exposure of the protein, although not all regions of the beta1 domain are functionally equivalent.     

Influence of peroxisome proliferator-activated receptor alpha agonists on the intracellular turnover and secretion of apolipoprotein (Apo) B-100 and ApoB-48

The peroxisome proliferator-activated receptor (PPAR) alpha agonist WY 14,643 increased the secretion of apolipoprotein (apo) B-100, but not that of apoB-48, and decreased triglyceride biosynthesis and secretion from primary rat hepatocytes. These effects resulted in decreased secretion of apoB-100-very low density lipoprotein (VLDL) and an increased secretion of apoB-100 on low density lipoproteins/intermediate density lipoproteins. ApoB-48-VLDL was also replaced by more dense particles. The proteasomal inhibitor lactacystin did not influence the recovery of apoB-100 or apoB-48 in primary rat hepatocytes, indicating that co-translational (proteasomal) degradation is of less importance in these cells. Treatment with WY 14,643 made the recovery of apoB-100 sensitive to lactacystin, most likely reflecting the decreased biosynthesis of triglycerides. The PPAR alpha agonist induced a significant increase in the accumulation of pulse-labeled apoB-100 even after a short pulse (2-5 min). There was also an increase in apoB-100 nascent polypeptides, indicating that the co-translational degradation of apoB-100 was inhibited. However, a minor influence on an early posttranslation degradation cannot be excluded. This decreased co-translational degradation of apoB-100 explained the increased secretion of the protein. The levels of apoB-48 remained unchanged during these pulse-chase experiments, and albumin production was not affected, indicating a specific effect of PPAR alpha agonists on the co-translational degradation of apoB-100. These findings explain the difference in the rate of secretion of the two apoB proteins seen after PPAR alpha activation. PPAR alpha agonists increased the expression and biosynthesis of liver fatty acid-binding protein (LFABP). Increased expression of LFABP by transfection of McA-RH7777 cells increased the secretion of apoB-100, decreased triglyceride biosynthesis and secretion, and increased PPAR alpha mRNA levels. These findings suggest that PPAR alpha and LFABP could interact to amplify the effect of endogenous PPAR alpha agonists on the assembly of VLDL.     

Adenovirus-mediated overexpression of microsomal triglyceride transfer protein (MTP): mechanistic studies on the role of MTP in apolipoprotein B-100 biogenesis.

The intracellular concentration of the microsomal triglyceride transfer protein large subunit (lMTP), the abetalipoproteinemia gene product, is tightly controlled. To date, attempts at overexpressinglMTP in vivo or in vitro have been unsuccessful. We successfully overexpressed lMTP in HepG2 cells using an adenoviral vector containing an lMTP cDNA, AdMTP. AdMTP-transduced HepG2 cells overexpressed MTP activity. They secreted increased amounts of apoB-100 lipoproteins with LDL and HDL density into the medium. lMTP overexpression alone minimally changed the density profile of apoB-containing lipoproteins, but addition of oleic acid shifted the profile toward lower densities. Oleic acid had a greater stimulatory effect on apoB-100 secretion in control HepG2 cells than in AdMTP-transduced cells, because (i) adenoviral transduction per se suppressed protein synthesis, affecting apoB-100 and albumin equally, and (ii) adenoviral transduction partially attenuated the increase in triglyceride synthesis in response to oleic acid supplementation. AdMTP treatment greatly diminished the intracellular degradation of apoB-100, but in comparison with recombinant virus containing luciferase cDNA (AdLuc), it caused no change in its biosynthetic rate. It greatly reduced, but did not eliminate, its proteasomal degradation. Our study constitutes the initial demonstration that adenovirus-mediated transfer of lMTP markedly stimulates MTP expression which in turn stimulates apoB-100 production. The mechanism involves a downregulation of ubiquitin-proteasome-mediated degradation without any change in synthetic rate.     

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.     

Relation of the size and intracellular sorting of apoB to the formation of VLDL 1 and VLDL 2

In this study, we tested the hypothesis that two separate pathways, the two-step process and an apolipoprotein B (apoB) size-dependent lipidation process, give rise to different lipoproteins. Expression of apoB-100 and C-terminally truncated forms of apoB-100 in McA-RH7777 cells demonstrated that VLDL particles can be assembled by apoB size-dependent linear lipidation, resulting in particles whose density is inversely related to the size of apoB. This lipidation results in a LDL-VLDL 2 particle containing apoB-100. VLDL 1 is assembled by the two-step process by apoB-48 and larger forms of apoB but not to any significant amount by apoB-41. The major amount of intracellular apoB-80 and apoB-100 banded with a mean density of 1.10 g/ml. Its formation was dependent on the sequence between apoB-72 and apoB-90. This dense particle, which is retained in the cell, possibly by chaperones or association with the microsomal membrane, is a precursor of secreted VLDL 1. The intracellular LDL-VLDL 2 particles formed during size-dependent lipidation appear to be the precursors of intracellular VLDL 1. We propose that the dense apoB-100 intracellular particle is converted to LDL-VLDL 2 by size-dependent lipidation. LDL-VLDL 2 is secreted or converted to VLDL 1 by the uptake of the major amount of triglycerides.     

The production of 85 kDa N-terminal fragment of apolipoprotein B in mutant HepG2 cells generated by targeted modification of apob gene occurs by ALLN-inhibitable protease cleavage during translocation

To study the mechanism of low levels of full length and truncated apoB in individuals heterozygous for apoB truncation, a non-sense mutation was introduced in one of the three alleles of apob gene of HepG2 cells by homologous recombination. Despite very low levels of apoB-82 (1-2%) in the media, a prominent N-terminal apoB protein of 85 kDa (apoB-15) was secreted that fractionated at d > 1.065 in density gradient ultracentrifugation. The mechanism of production of this short protein was studied by ³⁵S-methionine pulse-chase experiment. Oleate prevented presecretory degradation of apoB-100 in the cell and resulted in increased secretion of newly synthesized apoB-100 with decreases in the apoB-15, suggesting that rescue of pre-secretary intracellular degradation of apoB restricted the production and secretion of apoB-15. Further investigation on the degradation of transmembrane forms of apoB, in the presence and absence of a cysteine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), showed appearance of detectable levels of newly synthesized apoB-82 in the cell and the media together with increased apoB-100 secretion, and reduction in the secretion of apoB-15. Compared to ER membrane, the levels of apoB were higher in the luminal content, and presence of both oleate and ALLN had additive effect on apoB secretion. These results suggest that the presence of improper folding of apoB during translocation led to the cleavage of both apoB-100 and apoB-82 by ALLN-sensitive protease and generation of 85 kDa N-terminal fragment of apoB.     

Characterization of a mutant form of human apolipoprotein B (Thr26_Tyr27del) associated with familial hypobetalipoproteinemia

We have previously identified a deletion mutant of human apoB [apoB (Thr26_Tyr27del)] in a subject with primary hypobetalipoproteinemia. The present study determined the effect of Thr26_Tyr27del mutation on apoB secretion using transfected McA-RH7777 cells. Transient or stable transfection of apoB-48 containing the Thr26_Tyr27del mutation showed drastically reduced secretion of the mutant as compared to wild-type apoB-48. No lipoproteins containing the mutant apoB-48 were secreted into the medium. Incubation of transfected cells in a lipid-rich medium in the presence of cycloheximide showed rapid turnover of cell-associated mutant apoB-48 as compared to that of wild-type apoB-48. Immunofluorescence experiments showed that the mutant apoB-48 was mostly localized in the endoplasmic reticulum. Treatment with the proteasomal inhibitor MG132 markedly attenuated the turnover of cell-associated mutant apoB-48, whereas treatment with inhibitors of autophagosomal/lysosomal function (e.g. 3-MA or ammonium chloride) had no effect. Taken together, these results indicated that the defective secretion of the Thr26_Tyr27del mutant was associated with increased intracellular degradation of apoB through the proteasome-dependent pathway.     

The inhibition of microsomal triglyceride transfer protein activity in rat hepatoma cells promotes proteasomal and nonproteasomal degradation of apoprotein b100

In the human hepatic cell line, HepG2, apolipoprotein B100 (apoB100) degradation is increased by inhibiting lipid transfer mediated by the microsomal triglyceride transfer protein (MTP) and is predominantly accomplished by the ubiquitin-proteasome pathway. In the current study, we determined whether this degradative pathway was restricted to HepG2 cells or was of more general importance in hepatic apoB100 metabolism. Rat hepatoma McArdle RH7777 cells (McA), compared to HepG2 cells, secrete a large fraction of apoB100 associated with VLDL particles, as does the normal mammalian liver. In McA cells studied under basal conditions, the proteasome inhibitor lactacystin (LAC) increased apoB100 recovery, indicating that the role of the proteasome in apoB100 metabolism is not restricted to HepG2 cells. When apoB100 lipidation was blocked by an inhibitor of MTP (MTPI), recovery of cellular apoB100 was markedly reduced, but LAC was only partially ( approximately 50%) effective in reversing the induced degradation. This partial effectiveness of LAC may have represented either (1) incomplete inhibition by LAC of its preferred target, the chymotrypsin-like activity of the proteasome, (2) the presence of an apoB100 proteolytic activity of the proteasome resistant to LAC, or (3) a nonproteasomal proteolytic pathway of apoB100 degradation. By studying immunoisolated proteasomes and McA cells treated with LAC and/or MTPI and a variety of protease inhibitors, we determined that the proteasomal component of apoB100 degradation was entirely attributable to the chymotrypsin-like catalytic activity, but only accounted for part of apoB100 degradation induced by MTPI. The nonproteasomal apoB100 degradative pathway was nonlysosomal and resistant to E64d, DTT, and peptide aldehydes such as MG132 or ALLN but was partially sensitive to the serine protease inhibitor APMSF. Furthermore, when the protein trafficking inhibitor, brefeldin A, was used to block endoplasmic reticulum (ER) to Golgi transport in MTPI-treated McA cells, degradative activity resistant to LAC was increased, suggesting that the nonproteasomal pathway is associated with the ER.     

Cytosolic components are required for proteasomal degradation of newly synthesized apolipoprotein B in permeabilized HepG2 cells.

Recent studies have proposed that post-translational degradation of apolipoprotein B100 (apoB) involves the cytosolic ubiquitin-proteasome pathway. In this study, immunocytochemistry indicated that endoplasmic reticulum (ER)-associated proteasome molecules were concentrated in perinuclear regions of digitonin-permeabilized HepG2 cells. Signals produced by antibodies that recognize both alpha- and beta-subunits of the proteasome co-localized in the ER with specific domains of apoB. The mechanism of apoB degradation in the ER by the ubiquitin-proteasome pathway was studied using pulse-chase labeling and digitonin-permeabilized cells. ApoB in permeabilized cells incubated at 37 degrees C in buffer alone was relatively stable. When permeabilized cells were incubated with both exogenous ATP and rabbit reticulocyte lysate (RRL) as a source of ubiquitin-proteasome factors, >50% of [3H]apoB was degraded in 30 min. The degradation of apoB in the intact ER of permeabilized cells was much more rapid than that of extracted [3H]apoB incubated with RRL and ATP in vitro. The degradation of apoB was reduced by clasto-lactacystin beta-lactone, a potent proteasome inhibitor, and by ubiquitin K48R mutant protein, an inhibitor of polyubiquitination. ApoB in HepG2 cells was ubiquitinated, and polyubiquitination of apoB was stimulated by incubation of permeabilized cells with RRL. These results suggest that newly synthesized apoB in the ER is accessible to the cytoplasmic ubiquitin-proteasome pathway and that factors in RRL stimulate polyubiquitination of apoB, leading to rapid degradation of apoB in permeabilized cells.     

Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [³H]apoB, isolated from [³H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37°C in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin β-lactone (10 μM) or MG-132 (50 μM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.     

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures.

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.     

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.