In HepG2 Cells, Translocation, Not Degradation, Determines the Fate of the de Novo Synthesized Apolipoprotein B

Bonnardel, Julie A.; Davis, Roger J.

doi:10.1074/jbc.270.48.28892

Cited by 74 publications

(51 citation statements)

References 39 publications

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“…OA does not affect the ability of ALLN to protect apoB in cells not treated with BFA; in fact, OA and ALLN have additive effects on apoB secretion (14). The results suggested, therefore, that (1) ALLN reduced apoB degradation in BFAtreated cells, possibly by inhibiting the initial degradation of partially translocated apoB (14,15) and (2) OA, by increasing apoB translocation across the ER membrane (see experiment below), exposed apoB to an ER luminal proteolytic process that was ALLN-resistant. The failure of OA to protect apoB in BFA-treated cells was not the result of failure to stimulate new triglyceride (11).…”

Section: Resultsmentioning

confidence: 95%

“…OA-induced translocation is probably the result of enhanced interaction between apoB and the microsomal triglyceride transfer protein (16). ALLN does not seem to facilitate the translocation of apoB across the ER membrane (14,15). Direct protection of apoB, either by inhibition of a neutral cystine protease or proteasome activity (17), is the likely mechanism underlying the effect of ALLN.…”

Section: Apobmentioning

confidence: 99%

“…A major portion of newly synthesized apoB undergoes rapid intracellular degradation in a pre-Golgi or ER compartment (10 -12) in HepG2 cells as well as in apoB cDNA-transfected Chinese hamster ovary cells (13). ApoB can be protected from early intracellular degradation by incubation of cells with OA (9) or with the aldehydic tripeptide ALLN (13)(14)(15). OA treatment seems to stimulate apoB secretion by providing triglyceride to newly synthesized apoB and facilitating apoB translocation across the ER membrane (14).…”

Section: Apobmentioning

confidence: 99%

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A Two-site Model for ApoB Degradation in HepG2 Cells

Sakata

Lele

et al. 1997

Journal of Biological Chemistry

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Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi compartment in HepG2 cells. A major site of this early degradation seems to be on the cytosolic side of the endoplasmic reticulum (ER) membrane and is sensitive to N-acetylleucinyl-leucinyl-norleucinal (ALLN), which can inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates translocation of nascent apoB across the ER membrane, also reduces early degradation. In the present studies, we have used brefeldin A (BFA), which inhibits vesicular transport from the ER to the Golgi, to demonstrate that apoB can also be degraded by an ER luminal proteolytic activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were cotreated with ALLN, which protects apoB but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was inhibited. Furthermore, addition of DTT resulted in the accumulation of a 70-kDa amino-terminal fragment of apoB. Both full-length and amino-terminal apoB were degraded if DTT was removed from the incubation media; both were secreted if only BFA was removed. Thus, even after apoB is translocated into the ER lumen (thereby avoiding the initial proteolytic pathway), it can potentially be degraded by a lumenal proteolytic process that is ALLN-resistant but DTT-sensitive. The present results, together with previous studies, suggest that at least two distinct steps may be involved in the posttranslational degradation of apoB: 1) the first occurs while apoB is partially translocated and is ALLNsensitive; and 2) the second occurs in the ER lumen and is DTT-sensitive. Finally, our results support the hypothesis that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a DTT-sensitive pathway. ApoB1 secretion from cultured liver cells is regulated mainly at the posttranslational level. Thus, apoB mRNA levels are relatively stable under many conditions, whereas secretion of apoB-containing lipoproteins is altered (1-5). The impact of this posttranslational regulation is demonstrated by the observations that only a small to moderate proportion of the newly synthesized apoB is eventually secreted from primary rat hepatocytes (6, 7), McArdle cells (8), and HepG2 cells (9). A major portion of newly synthesized apoB undergoes rapid intracellular degradation in a pre-Golgi or ER compartment (10 -12) in HepG2 cells as well as in apoB cDNA-transfected Chinese hamster ovary cells (13). ApoB can be protected from e...

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Section: Resultsmentioning

confidence: 95%

Section: Apobmentioning

confidence: 99%

Section: Apobmentioning

confidence: 99%

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A Two-site Model for ApoB Degradation in HepG2 Cells

Sakata

Lele

et al. 1997

Journal of Biological Chemistry

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“…[17][18][19][20]22,57 Evidence that the actual process of apoB translocation across the ER may be a crucial regulatory point in the production of lipoproteins has recently been suggested by Rusiñol and Vance. 22 They have shown that the supplementation of primary rat hepatocytes with phosphatidyl-monomethylethanolamine decreased the secretion of apoB.…”

Section: Discussionmentioning

confidence: 99%

Effects of Atorvastatin on the Intracellular Stability and Secretion of Apolipoprotein B in HepG2 Cells

Mohammadi

Macri

Newton

et al. 1998

ATVB

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Abstract-We investigated the effects of atorvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the biogenesis of apolipoprotein B (apoB) in intact and permeabilized HepG2 cells. Intact cells were pretreated either with single or multiple doses of atorvastatin (0.1 to 20 mol/L) for periods of 6 to 20 hours and pulsed with [ 35 S]methionine. In some cases the cells were permeabilized with digitonin. Experiments were performed to investigate the effects of atorvastatin on (1) the rates of lipid synthesis and secretion, (2) the synthesis and accumulation of apoB, (3) the intracellular stability of apoB, (4) the amount of apoB-containing lipoprotein particles assembled in HepG2 microsomes, and (5) 35 S]methionine by the cells nor did it influence the synthesis of apoB or a control protein, albumin. However, atorvastatin reduced the secretion of apoB into the culture medium, apparently by enhancing the degradation of apoB in the cell under basal and induced conditions with oleate. The stability of apoB associated with the lipoprotein particles was also significantly lowered by atorvastatin. The stimulated degradation of apoB in atorvastatin-treated cells was sensitive to MG132, a proteasome inhibitor. The net effect of atorvastatin was a reduction in the number of apoB-containing lipoprotein particles of different sizes isolated from microsomes and a reduction in apoB secretion into the culture medium. The data suggest that atorvastatin may impair the translocation of apoB into the lumen of the endoplasmic reticulum, thus increasing the amount of apoB degraded intracellularly. It is hypothesized that atorvastatin alters these parameters primarily as a result of inhibiting cholesterol synthesis and limiting the availability of cholesterol and/or cholesterol ester for the normal assembly of apoB-containing lipoprotein particles. (Arterioscler Thromb Vasc Biol. 1998;18:783-793.)

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“…Trypsin Digestion-Trypsin digestion of microsomes was done according to Bonnardel and Davis (16). Microsomes were suspended in 0.25 M sucrose and 10 mM HEPES, pH 7.4.…”

Section: Reagents-trypsinmentioning

confidence: 99%

Proteasome-mediated Degradation of Apolipoprotein B Targets Both Nascent Peptides Cotranslationally Before Translocation and Full-length Apolipoprotein B After Translocation into the Endoplasmic Reticulum

Liao

Yeung

Chan

1998

Journal of Biological Chemistry

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A major portion of newly synthesized apolipoprotein B (apoB) is degraded intracellularly. This degradation has been demonstrated to be mediated largely by the ubiquitin-proteasome pathway. We examined whether nascent apoB polypeptides or full-length apoB is selectively retrotranslocated from the endoplasmic reticulum into the cytosol for degradation. Herein, we found that full-length apoB as well as partial-length apoB peptides are ubiquitinated in HepG2 cells, and ubiquitination is an exclusively cytosolic process. Calnexin, which binds specifically to glycoproteins, has been postulated to promote apoB folding and complete translocation; we found that ubiquitinated apoB is bound to calnexin, suggesting that ubiquitinated apoB is glycosylated. In addition to calnexin binding, we have other pieces of evidence that the full-length intracellular ubiquitinated apoB is glycosylated, because (i) it binds to concanavalin A, and (ii) glycan can be demonstrated in the fulllength ubiquitinated apoB by a chemical detection method involving oxidation of adjacent hydroxyl groups in the glycan moiety. Because glycosylation occurs inside the endoplasmic reticulum, the full-length glycosylated apoB must have been retrotranslocated into the cytosol for ubiquitination and proteasome-mediated degradation. Next we synchronized translation in HepG2 cells by puromycin treatment. A pulse-chase experiment using [35 S]methionine labeling of intracellular apoB in these synchronized cells demonstrated that nascent partial-length apoB peptides are also ubiquitinated cotranslationally. We conclude that the ubiquitin proteasome-mediated degradation of apoB targets both nascent peptides cotranslationally before translocation as well as full-length apoB after its translocation into the endoplasmic reticulum.

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In HepG2 Cells, Translocation, Not Degradation, Determines the Fate of the de Novo Synthesized Apolipoprotein B

Cited by 74 publications

References 39 publications

A Two-site Model for ApoB Degradation in HepG2 Cells

A Two-site Model for ApoB Degradation in HepG2 Cells

Effects of Atorvastatin on the Intracellular Stability and Secretion of Apolipoprotein B in HepG2 Cells

Proteasome-mediated Degradation of Apolipoprotein B Targets Both Nascent Peptides Cotranslationally Before Translocation and Full-length Apolipoprotein B After Translocation into the Endoplasmic Reticulum

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