A Two-site Model for ApoB Degradation in HepG2 Cells

Wu, Xujun; Sakata, Nobuhiro; Lele, Karen M.; Zhou, Mingyue; Jiang, Hongshi; Ginsberg, Henry N.

doi:10.1074/jbc.272.17.11575

Cited by 89 publications

(77 citation statements)

References 31 publications

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“…There is conflicting evidence in the literature on the effects of increased hepatic cholesteryl ester concentration on the secretion of VLDL. Wu et al [20] showed that a variety of manipulations which altered cholesteryl ester concentration in HepG2 cells had no effect on VLDL secretion, while Fungwe et al [13,21] have described a stimulation of VLDL formation in the livers of rats and hamsters fed cholesterol-enriched diets. Recent data from human subjects also emphasise the importance of available cholesterol in regulating VLDL secretion [22].…”

Section: Resultsmentioning

confidence: 99%

Hepatic microsomal triglyceride transfer protein messenger RNA concentrations are increased by dietary cholesterol in hamsters

et al. 1996

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Section: Resultsmentioning

confidence: 99%

Hepatic microsomal triglyceride transfer protein messenger RNA concentrations are increased by dietary cholesterol in hamsters

et al. 1996

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“…The intracellular mechanisms responsible for degradation of nascent apoB chains have been the subject of intense investigation in the past few years (5, 6, 9 -30, 35). Many recent reports have shown evidence for the involvement of the proteasome in the co-translational degradation of apoB in HepG2 cells (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). This evidence includes the sensitivity of apoB degradation to various proteasome inhibitors (12, 13, 15, 18 -22), the detection of ubiquitinated apoB (12, 18 -20, 22), and more recently the association of ubiquitinated apoB with the Sec61 complex of the translocon (18,19).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, we have reported that the apoB associated with luminal lipoproteins is also degraded post-translationally by an ALLN-sensitive degradation mechanism and could be rescued from degradation by cytosolic factors and metabolic energy, perhaps by inducing transport of apoB out of the degradation compartment (30). Furthermore, Wu et al (14) have reported a two-site model for the degradation of apoB in HepG2 cells, suggesting that after the initial rapid degradation process, apoB that is fully translocated into the ER lumen can still undergo degradation via a second proteolytic system that is ALLN-resistant but sensitive to DTT.…”

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“…Inefficient translocation has been suggested to lead to the formation of a pool of membrane-associated apoB that becomes ubiquitinated (12-20, 22) and ultimately destined for intracellular degradation (6, 8 -11). It is evident that co-translational degradation of membrane-associated apoB is mediated by the cytosolic proteasome based on its sensitivity to proteasome inhibitors such as ALLN, lactacystin, and MG132 (9,[12][13][14][15][16][17][18][19][20][21][22].…”

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Studies on Degradative Mechanisms Mediating Post-translational Fragmentation of Apolipoprotein B and the Generation of the 70-kDa Fragment

Cavallo¹,

Rudy²,

Mohammadi³

et al. 1999

Journal of Biological Chemistry

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It has been well established that the biogenesis of apoB is mediated co-translationally by the cytosolic proteasome. Here, however, we investigated the role of both the cytosolic proteasome as well as non-proteasome-mediated degradation systems in the post-translational degradation of apoB. In pulse-chase labeling experiments, co-translational (0-h chase) apoB degradation in both intact and permeabilized cells was sensitive to proteasome inhibitors. Interestingly, turnover of apoB in intact cells over a 2-h chase was partially inhibitable by lactacystin, thus suggesting a role for the cytosolic proteasome in the post-translational degradation of apoB. In permeabilized cells, however, there was no posttranslational protection of apoB by lactacystin. Further investigations of proteasomal activity in HepG2 cells revealed that, following permeabilization, there was a dramatic loss of the 20 S proteasomal subunits, and consequently the cells exhibited no detectable lactacystininhibitable activity. Thus, apoB fragmentation and the generation of the 70-kDa apoB degradation fragment, characteristic of permeabilized cells, continued to occur in these cells despite the absence of functional cytosolic proteasome. Similar results were observed when we used a derivative of lactacystin, clastolactacystin ␤-lactone, which represents the active species of the inhibitor. Interestingly, however, the abundance of the 70-kDa fragment could be modulated by the microsomal triglyceride transfer protein inhibitor, BMS-197636, as well as by pretreatment of the permeabilized cells with dithiothreitol. These data thus suggest that although the cytosolic proteasome appears to be involved in the posttranslational turnover of apoB in intact cells, the specific post-translational fragmentation of apoB generating the 70-kDa fragment observed in permeabilized cells occurs independent of the cytosolic proteasome.Hepatic apolipoprotein B100 (apoB) 1 secretion appears to be regulated post-transcriptionally (1-4). More specifically, efficient translocation of newly synthesized apoB molecules across the membrane of the endoplasmic reticulum (ER) is believed to be an important event that contributes to the formation of secretion competent apoB-containing lipoproteins (5-8). Inefficient translocation has been suggested to lead to the formation of a pool of membrane-associated apoB that becomes ubiquitinated (12-20, 22) and ultimately destined for intracellular degradation (6, 8 -11). It is evident that co-translational degradation of membrane-associated apoB is mediated by the cytosolic proteasome based on its sensitivity to proteasome inhibitors such as ALLN, lactacystin, and MG132 (9,[12][13][14][15][16][17][18][19][20][21][22].The involvement of the proteasome in degradation of apoB raises a number of intriguing questions. Clearly, the proteasome is involved in co-translational degradation of membraneassociated apoB, which is expected to have cytosolic exposure. However, recent evidence has suggested that the proteasome may also be involved in the ...

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“…Several groups have shown that apoB can be marked for degradation by the ubiquitin-proteasome pathway even during its translocation into the ER (18 -20). ApoB can also enter other routes of intracellular degradation in hepatic cells (21,22). Remarkably, some molecules of ubiquitin-conjugated apoB can be rescued from degradation and secreted when hepatocytes are treated with lipids (23).…”

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A Proteomic Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B

Rashid¹,

Hevi

Yin³

et al. 2002

Journal of Biological Chemistry

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The biogenesis of apolipoprotein B is quite complex in view of its huge size, hydrophobicity, obligate association with lipids such as cholesterol and triglycerides prior to secretion, and intracellular degradation of a substantial proportion of newly synthesized molecules. Multiple proteins likely serve roles as molecular chaperones to assist in folding, assembly with lipids, and regulation of the secretion of apolipoprotein B. In these studies, we developed a strategy to isolate proteins associated with apolipoprotein B in rat livers. The purification consisted of two stages: first, microsomes were prepared from rat liver and treated with chemical crosslinkers, and second, the solubilized proteins were coimmunoprecipitated with antibody against apolipoprotein B. We found that several proteins were cross-linked to apolipoprotein B. The proteins were digested with trypsin, and the released peptides were sequenced by tandem mass spectrometry. The sequences precisely matched 377 peptides in 99 unique proteins. We show that at least two of the identified proteins, ferritin heavy and light chains, can directly bind apolipoprotein B. These and possibly other proteins identified by this proteomic approach are novel candidates for proteins that affect apolipoprotein B during its biogenesis. Apolipoprotein B (apoB)1 is secreted with lipids including cholesterol esters, phospholipids, cholesterol, and triglycerides, as very low density lipoproteins (1-5). The secretion of cholesterol from the liver in humans is tied to the export of apoB into plasma. Hepatic regulation of apoB secretion is chiefly posttranslational (6); secretion reflects the balance between assembly of this protein with lipids into a lipoprotein particle and intracellular degradation. Both of these competing processes appear to involve several other proteins.The biogenesis of this large (molecular mass greater than 500 kDa) (7, 8), hydrophobic protein requires the participation of several known chaperone proteins including some that are particular to the specialized physiologic role of apoB. Evidence suggests that calnexin, calreticulin, BiP, Erp72, GRP94, and protein disulfide isomerase (PDI) all interact with apoB during its translocation and further biogenesis once it has entered the endoplasmic reticulum (ER) (9 -11). These proteins also function in the proper folding and quality control of other secretory proteins. However, the assembly of apoB with lipids into a lipoprotein particle necessitates additional proteins that may be particular to apoB. The lack of solubility of apoB in an aqueous environment, such as the lumen of the ER, necessitates its co-translational association with lipids (12). This process is facilitated by microsomal triglyceride transport protein (MTP), which plays a crucial role in the initial assembly and regulation of secretion of apoB (13-15). In a second step that is sensitive to inhibition by brefeldin A, apoB is assembled with a full complement of lipids into a mature lipoprotein particle (16,17). The protein that mediat...

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A Two-site Model for ApoB Degradation in HepG2 Cells

Cited by 89 publications

References 31 publications

Hepatic microsomal triglyceride transfer protein messenger RNA concentrations are increased by dietary cholesterol in hamsters

Hepatic microsomal triglyceride transfer protein messenger RNA concentrations are increased by dietary cholesterol in hamsters

Studies on Degradative Mechanisms Mediating Post-translational Fragmentation of Apolipoprotein B and the Generation of the 70-kDa Fragment

A Proteomic Approach Identifies Proteins in Hepatocytes That Bind Nascent Apolipoprotein B

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