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

176

We previously showed that ⍀-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that ⍀-3-induced degradation preferentially reduces the secretion of large, assembled apoBlipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but ⍀-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that ⍀-3 fatty acids induced a striking loss of apoB 100 from the Golgi, while sparing apoB 100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the ⍀-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.Apolipoprotein B (apoB), 1 the major protein of atherogenic lipoproteins, is synthesized primarily by hepatic and intestinal cells. Most studies have focused on apoB metabolism in the liver, given the greater contribution to the plasma apoB pool made by that organ and the availability of relatively convenient primary and transformed hepatic cell models. The apoB message level and translational rate in hepatic cells are largely constitutive, and so secretory control is achieved primarily through co-and post-translational degradation of the protein (e.g. see Refs. 2-4 for recent reviews). Two specific mechanisms for the destruction of newly synthesized apoB in hepatic cells have been characterized. The first is endoplasmic reticulum-associated degradation (ERAD). Newly synthesized apoB in the endoplasmic reticulum (ER) is initially complexed with small amounts of lipid that are thought to be shuttled by the microsomal triglyceride transfer protein (MTP) (5). During severe lipid deprivation (6, 7) or MTP deficiency (8, 9), this initial lipidation fails, and the apoB becomes ubiquitinylated, which targets it for degradation by proteasomes (10 -14).The second mechanism for degradation of newly synthesized apoB is the re-uptake pathway. Re-uptake can occur after fully assembled apoB-containing particles have been exported across the plasma membrane but before they have diffused away from the vicinity of the cell by traversing the unstirred water layer that is adjacent...

Section: Nature Of the Target For ⍀-3mentioning

confidence: 99%

The Triple Threat to Nascent Apolipoprotein B

Fisher¹,

Pan²,

Chen³

et al. 2001

176

“…The expression of apoB alone in cells without MTP resulted in the intracellular degradation of nascent apoB and no secretion; co-expression of apoB with MTP resulted in the secretion of apoB on lipoprotein particles (4 -6). Furthermore, the inhibition of MTP activity in cultured liver cells blocked the assembly and secretion of apoB-containing lipoproteins (7)(8)(9)(10)(11). We demonstrated that inhibition of MTP activity was associated with increased ubiquitination of apoB, particularly when proteasomal degradation was inhibited (12).…”

Section: Microsomal Triglyceride Transfer Protein (Mtp)mentioning

confidence: 99%

Microsomal Triglyceride Transfer Protein Binding and Lipid Transfer Activities Are Independent of Each Other, but Both Are Required for Secretion of Apolipoprotein B Lipoproteins from Liver Cells

Liang

Ginsberg

2001

Recent studies indicate that microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) interact physically via two specific binding sites located within the amino-terminal globular region of apoB100. The first site is thought to be within the first 5.8% of the amino-terminal sequence, and the second site is between 9 and 16% of the amino-terminal sequence. It is not clear from prior studies whether these sites have unique or overlapping functions. Furthermore, there are no data differentiating between lipid transfer and potential chaperone functions of MTP. In the present study we have attempted to further characterize the physiologic interaction between apoB and MTP and to determine the relationship between the binding and lipid transfer aspects of the interaction. HepG2 cells were transiently transfected with apoB cDNAs, and MTP binding to apoB polypeptides was determined by two-step immunoprecipitation. MTP bound equally well to apoB polypeptides with (apoB13, 16,␤, apoB34, and apoB42) or without (apoB16, apoB13, and 16 or apoB13, 13, and 16) ␤ sheet domains. When proteasomal degradation of newly synthesized apoB polypeptides was blocked, MTP binding to all of the apoB polypeptides was only modestly affected by lipid availability and was independent of MTP-associated lipid transfer. Furthermore, MTP did not bind directly to a portion of the first ␤ sheet domain. We created two apoB constructs (apoB16 del and apoB34 del ) by deleting the first 210 amino acids of apoB16 and apoB34. These apoB polypeptides, therefore, lacked the putative first MTP binding site. MTP binding to apoB16 del and apoB34 del was decreased significantly. However, the secretion of apoB16 del was not different from apoB16, whereas the secretion of apoB34 del was impaired significantly. Our results indicate that the interaction between MTP and apoB involves independent binding and lipid transfer activities but that both activities are required for the secretion of apolipoprotein B from liver cells. Microsomal triglyceride transfer protein (MTP)1 is a heterodimer consisting of protein disulfide isomerase and the 97-kDa-large subunit and is found mainly in the endoplasmic reticulum (ER) lumen of liver and intestinal cells (1-3). The essential role of MTP in the translocation of apolipoprotein B (apoB) across the ER membrane and the assembly of apoB with lipids has been demonstrated clearly by co-expressing apoB and MTP in cells that do not normally express either protein and by the characterization of mice lacking MTP. The expression of apoB alone in cells without MTP resulted in the intracellular degradation of nascent apoB and no secretion; co-expression of apoB with MTP resulted in the secretion of apoB on lipoprotein particles (4 -6). Furthermore, the inhibition of MTP activity in cultured liver cells blocked the assembly and secretion of apoB-containing lipoproteins (7-11). We demonstrated that inhibition of MTP activity was associated with increased ubiquitination of apoB, particularly when proteasomal degradation was ...

“…ApoB is marked with ubiquitin for degradation even before it is finished being translated (7,9,10) while the protein is still in the translocation channel (8,11). Furthermore, factors that slow the translocation of apoB increase the number of ubiquitin-conjugated apoB molecules marked for degradation by proteasomes (8) suggesting that translocation is a point of regulation of secretion.…”

mentioning

confidence: 99%

“…In HepG2 cells, unassembled apoB can be targeted for endoplasmic reticulum-associated degradation (ERAD) via the ubiquitin-proteasome pathway (5)(6)(7)(8). ApoB is marked with ubiquitin for degradation even before it is finished being translated (7,9,10) while the protein is still in the translocation channel (8,11).…”

mentioning

confidence: 99%

Ferritins Can Regulate the Secretion of Apolipoprotein B

Hevi

Chuck

2003

Apolipoprotein B is secreted with atherogenic lipids as lipoprotein particles from hepatocytes. Regulation of the secretion of apolipoprotein B is largely post-translational and reflects the balance between processes that leads to particle assembly or to intracellular degradation. Previously, we conducted a proteomic screen to find proteins that bind apolipoprotein B in rat liver microsomes. We identified ferritin heavy and light chains in this screen among other proteins and showed that the two ferritins bind apolipoprotein B directly in vitro. In hepatocytes and other cells, ferritin heavy and light chains form cytosolic cages that store iron. We now show that ferritin heavy or light chains post-translationally inhibit the secretion of apolipoprotein B without altering the export of other hepatic proteins including albumin, factor XIII, and apolipoprotein A-I. This inhibition of apolipoprotein B secretion is not due to diminished lipid synthesis and can be partially overcome by stimulating triglyceride synthesis. The block in apolipoprotein B secretion by ferritins leads to an increase in endoplasmic reticulum-associated degradation of the apolipoprotein. Thus, despite being cytosolic proteins without known chaperone activity, ferritins can specifically regulate the secretion of apolipoprotein B post-translationally. The metabolic pathways for iron storage and intercellular cholesterol and triglyceride transport could intersect. Apolipoprotein B (apoB)1 is the large (molecular mass greater than 500 kDa), hydrophobic protein that is secreted with cholesterol esters and triglycerides in very low density lipoprotein particles (1). Regulation of the secretion of apoB from hepatocytes occurs largely post-translationally (2). Newly synthesized apoB can be assembled with lipids into lipoprotein particles or degraded intracellularly (3, 4), and it is the balance assembly and degradation that determines secretion. Since cholesterol esters and triglycerides can only be secreted into the bloodstream with apoB, factors that regulate the secretion of this protein likewise impact plasma levels of these atherogenic lipids.In HepG2 cells, unassembled apoB can be targeted for endoplasmic reticulum-associated degradation (ERAD) via the ubiquitin-proteasome pathway (5-8). ApoB is marked with ubiquitin for degradation even before it is finished being translated (7, 9, 10) while the protein is still in the translocation channel (8, 11). Furthermore, factors that slow the translocation of apoB increase the number of ubiquitin-conjugated apoB molecules marked for degradation by proteasomes (8) suggesting that translocation is a point of regulation of secretion. Other factors also regulate secretion since some ubiquitin-conjugated apoB can be deubiquitinylated and secreted (11). In HepG2 cells, other non-ERAD routes for disposal exist but are not sensitive to agents that inhibit proteasomes (12-15).The intertwined processes of lipidation, folding, translocation, intracellular degradation, quality control, and regulation of the secret...