In this study, we explored how sterol metabolism altered by the expression of cholesterol-7␣-hydroxylase NADPH:oxygen oxidoreductase (7␣-hydroxylase) affects the ubiquitin-dependent proteasome degradation of translocation-arrested apoB53 in Chinese hamster ovary cells. Stable expression of two different plasmids that encode either rat or human 7␣-hydroxylase inhibited the ubiquitin conjugation of apoB and its subsequent degradation by the proteasome. Oxysterols (25-hydroxycholesterol and 7-ketocholesterol) reversed the inhibition of apoB degradation caused by 7␣-hydroxylase. The combined results suggest that the normally rapid proteasome degradation of translocation-arrested apoB can be regulated by a sterol-sensitive polyubiquitin conjugation step in the endoplasmic reticulum. Blocked ubiquitin-dependent proteasome degradation caused translocation-arrested apoB to become sequestered in segregated membrane domains. Our results described for the first time a novel mechanism through which the "quality control" proteasome endoplasmic reticulum degradative pathway of translocation-arrested apoB is linked to sterol metabolism. Sterol-sensitive blocked ubiquitin conjugation appears to selectively inhibit the proteasome degradation of apoB, but not 7␣-hydroxylase protein, with no impairment of cell vitality or function. Our findings may help to explain why the hepatic production of lipoproteins is increased when familial hypertriglyceridemic patients are treated with drugs that activate 7␣-hydroxylase (e.g. bile acid-binding resins).ApoB is the major structural protein responsible for the assembly of lipoproteins by the liver and intestine. Multiple forms of apoB, designated as the percentage of the N terminus of the largest secretory product apoB100 (4536 amino acids), are produced from a single gene transcript by mRNA editing and proteolytic cleavage (reviewed in Refs. 1-3). Overproduction of apoB-containing lipoproteins by the liver is responsible for familial combined hyperlipidemia (4). In addition, overproduction of triglyceride-rich lipoproteins is responsible for the human disease familial hypertriglyceridemia (5). In these patients, the secretion of triglyceride-rich lipoproteins varies in parallel with the rate of bile acid synthesis (6 -8). These findings suggest that the secretion of very low density lipoprotein triglyceride is linked to hepatic sterol metabolism via an as yet undefined mechanism that is dependent upon genes that contribute to hypertriglyceridemia.The rate of hepatic secretion of apoB is regulated post-transcriptionally. Only a portion of de novo synthesized apoB is secreted; the remaining portion is degraded intracellularly (9). Interruption of apoB translocation is one of several criteria that lead to increased intracellular degradation (reviewed in Ref. 10). Both translocation and lipid addition require the presence of microsomal triglyceride transfer protein (MTP) 1 in the ER (11-13). MTP exists in the ER lumen as a heterodimer with protein-disulfide isomerase (reviewed in Ref. 14). I...

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoBcontaining lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 M), but resistant to N-acetyl-leucylleucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7␣-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7␣-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.The assembly and secretion of lipoproteins containing apoB occur in several tissues (liver, intestine, yolk sac, and heart) and are essential for the transport and delivery of fat during development and in the adult (reviewed in Refs. 1-4). There are at least three distinct human familial disorders associated with developmental and nutritional abnormalities caused by the inability of the liver and/or intestine to assemble and secrete apoB-containing lipoproteins (5). Hypobetalipoproteinemia is caused by mutations of the apoB gene leading to forms that are incapable of forming lipoprotein particles and/or to impaired synthesis of apoB by the liver and intestine (1, 6). The expression of human hypobetalipoproteinemic apoB genes in mice recapitulates many of the phenotypic abnormalities observed in human (7,8). Abetalipoproteinemia is caused by mutations in the MTP 1 gene (9 -12). The absence of MTP lipid transfer activity in the lumen of the endoplasmic reticulum (ER) causes apoB to be degraded rather than secreted as a lipoprotein particle by the liver and intestine (12, 13). The recent demonstration that inactivation of a single allele of the MTP gene in mice causes signifi...

CHO cells expressing the liver-specific gene product cholesterol-7 ␣ -hydroxylase showed a 6-fold increase in the biosynthesis of [ 14 C]cholesterol from [ 14 C]acetate, as well as increased enzymatic activities of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase and squalene synthase. Cells expressing cholesterol-7 ␣ -hydroxylase contained less sterol response element-binding protein 1 (SREBP1) precursor, whereas the cellular content of mature SREBP1, as well as the mRNAs of cholesterol biosynthetic genes (HMG-CoA reductase and squalene synthase), were all increased ϳ 3-fold. Cells expressing cholesterol-7 ␣ -hydroxylase displayed greater activities of luciferase reporters containing the SREBP-dependent promoter elements derived from HMG-CoA reductase and farnesyl diphosphate synthase, in spite of accumulating significantly more free and esterified cholesterol and 7 ␣ -hydroxycholesterol. While cells expressing cholesterol-7 ␣ -hydroxylase displayed increased SREBPdependent transcription, sterol-mediated repression of SREBP-dependent transcription by LDL-cholesterol and exogenous oxysterols was similar in both cell types. Cells expressing cholesterol-7 ␣ -hydroxylase displayed greater rates of secretion of cholesterol as well as increased expression of the ABC1 cassette protein mRNA. Adding 25hydroxycholesterol to the culture medium of both cell types increased the expression of ABC1 cassette protein mRNA. The combined data suggest that in nonhepatic CHO cells multiple regulatory processes sensitive to cellular sterols act independently to coordinately maintain cellular cholesterol homeostasis. -Spitsen, G.