The Triple Threat to Nascent Apolipoprotein B

Abstract

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...

Nascent Lipidated Apolipoprotein B Is Transported to the Golgi as an Incompletely Folded Intermediate as Probed by Its Association with Network of Endoplasmic Reticulum Molecular Chaperones, GRP94, ERp72, BiP, Calreticulin, and Cyclophilin B

“…NIS, nonimmune serum. quality control mechanism, it was demonstrated that apoB can be targeted for degradation at different stages of its maturation beyond the ER (15,40,55,68,69). This, however, is an unlikely scenario occurring in HepG2 cells under the conditions used in this study, because the cells were incubated under conditions that support optimal folding of apoB resulting in a very efficient secretion.…”

Identification of the Lipoprotein Initiating Domain of Apolipoprotein B

“…The radioactivity recovered in the four lipid classes associated with each purified apoB-containing lipoprotein was used to calculate the percent radiolabeled lipid composition (Table I). assembly process. Hence, in the absence of MTP, forms of apoB containing a sufficient length of the ␤ 1 domain may undergo aggregation and/or non-native interactions with intracellular membranes, ultimately resulting in intracellular retention and turnover (28,29).…”

Microsomal Triglyceride Transfer Protein Promotes the Secretion of Xenopus laevis Vitellogenin A1

“…In the absence of MTP, apoB fails to acquire lipid during translation, causing misfolding and intracellular retention and degradation (25,26). In contrast, coexpression of apoB with MTP promotes nascent lipoprotein formation, which stabilizes apoB and promotes anterograde transport and secretion (23,27).…”