Translocation-arrested Apolipoprotein B Evades Proteasome Degradation via a Sterol-sensitive Block in Ubiquitin Conjugation
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1856
ISSN1083-351X
AutoresEmma Du, James F. Fleming, Shui-Long Wang, Gary M. Spitsen, Roger J. Davis,
Tópico(s)Lipid metabolism and biosynthesis
ResumoIn this study, we explored how sterol metabolism altered by the expression of cholesterol-7α-hydroxylase NADPH:oxygen oxidoreductase (7α-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 7α-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 7α-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 7α-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 7α-hydroxylase (e.g. bile acid-binding resins). In this study, we explored how sterol metabolism altered by the expression of cholesterol-7α-hydroxylase NADPH:oxygen oxidoreductase (7α-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 7α-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 7α-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 7α-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 7α-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. 1Davis R.A. Vance J. New Compr. Biochem. 1996; 31: 473-494Crossref Scopus (46) Google Scholar, 2Innerarity T.L. Boren J. Yamanaka S. Olofsson S.-O. J. Biol. Chem. 1996; 271: 2353-2356Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Yao Z. Tran K. McLeod R.S. J. Lipid Res. 1997; 38: 1937-1953Abstract Full Text PDF PubMed Google Scholar). Overproduction of apoB-containing lipoproteins by the liver is responsible for familial combined hyperlipidemia (4Venkatesan S. Cullen P. Pacy P. Halliday D. Scott J. Arterioscler. Thromb. 1993; 13: 1110-1118Crossref PubMed Google Scholar). In addition, overproduction of triglyceride-rich lipoproteins is responsible for the human disease familial hypertriglyceridemia (5Chait A. Albers J.J. Brunzell J.D. Eur. J. Clin. Invest. 1980; 10: 17-22Crossref PubMed Scopus (200) Google Scholar). In these patients, the secretion of triglyceride-rich lipoproteins varies in parallel with the rate of bile acid synthesis (6Angelin B. Einarsson K. Hellstrom K. Leijd B. J. Lipid Res. 1978; 19: 1004-1010Abstract Full Text PDF PubMed Google Scholar, 7Angelin B. Hershon K.S. Brunzell J.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5434-5438Crossref PubMed Scopus (81) Google Scholar, 8Duane W.C. J. Lipid Res. 1997; 38: 183-188Abstract Full Text PDF PubMed Google Scholar). 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 novosynthesized apoB is secreted; the remaining portion is degraded intracellularly (9Borchardt R.A. Davis R.A. J. Biol. Chem. 1987; 262: 16394-16402Abstract Full Text PDF PubMed Google Scholar). Interruption of apoB translocation is one of several criteria that lead to increased intracellular degradation (reviewed in Ref. 10Davis R.A. Subcellular Biochem. 1993; 21: 169-183Crossref PubMed Scopus (9) Google Scholar). Both translocation and lipid addition require the presence of microsomal triglyceride transfer protein (MTP) 1The abbreviations used are: MTP, microsomal triglyceride transfer protein; 7α-hydroxylase, cholesterol-7α-hydroxylase NADPH:oxygen oxidoreductase (EC1.14.13.17); ALLN, N-acetyl-leucyl-leucyl-norleucinal; apoB, apolipoprotein B; CMV, cytomegalovirus; MEM, modified Eagle's medium; ER, endoplasmic reticulum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Ub, ubiquitin; CHO, Chinese hamster ovary. in the ER (11Wang S. McLeod R.S. Gordon D.A. Yao Z. J. Biol. Chem. 1996; 271: 14124-14133Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Gordon D.A. Jamil H. Gregg R.E. Olofsson S.O. Boren J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). MTP exists in the ER lumen as a heterodimer with protein-disulfide isomerase (reviewed in Ref. 14Wetterau J.R. Lin M.C. Jamil H. Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (286) Google Scholar). In the absence of either sufficient lipid (15Sakata N. Wu X. Dixon J.L. Ginsberg H.N. J. Biol. Chem. 1993; 268: 22967-22970Abstract Full Text PDF PubMed Google Scholar, 16Bonnardel J.A. Davis R.A. J. Biol. Chem. 1995; 270: 28892-28896Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 17Macri J. Adeli K. J. Biol. Chem. 1997; 272: 7328-7337Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) or MTP lipid transfer activity (11Wang S. McLeod R.S. Gordon D.A. Yao Z. J. Biol. Chem. 1996; 271: 14124-14133Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Gordon D.A. Jamil H. Gregg R.E. Olofsson S.O. Boren J. J. Biol. Chem. 1996; 271: 33047-33053Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S.O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), apoB translocation and lipoprotein assembly are blocked. The C terminus of resulting translocation-arrested apoB, which resides in the cytoplasm (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar), is rapidly degraded by a ubiquitin-dependent proteasome process (19Yeung S.J. Chen S.H. Chan L. Biochemistry. 1996; 35: 13843-13848Crossref PubMed Scopus (170) Google Scholar, 20Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 21Benoist F. Grand-Perret T. J. Biol. Chem. 1997; 272: 20435-20442Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The essential requirement of MTP for apoB translocation and lipoprotein assembly is exemplified by the finding that genetic loss of the expression of the MTP gene is responsible for the human recessive disorder abetalipoproteinemia (22Wetterau J.R. Aggerbeck L.P. Bouma M.-E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (644) Google Scholar, 23Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager S.K. Gil C.M. Turck C.W. Bouma M.E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Nature. 1993; 365: 65-69Crossref PubMed Scopus (408) Google Scholar). Similar to the CHO cells used in the studies reported here, the liver of abetalipoproteinemics lacks the ability to fully translocate apoB into the ER (24Du E.Z. Wang S.-L. Kayden H.J. Sokol R. Curtiss L.K. Davis R.A. J. Lipid Res. 1996; 37: 1309-1315Abstract Full Text PDF PubMed Google Scholar) and to secrete apoB-containing lipoproteins (25Gregg R.E. Wetterau J.R. Curr. Opin. Lipidol. 1994; 5: 81-86Crossref PubMed Scopus (119) Google Scholar, 26Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M.E. Jaruz A. Grantham T.T. Leoni P.R.D. Bhattacharya S. Pease R.J. Cullen P.M. Levi S. Byfield P.G.H. Purkiss P. Scott J. Hum. Mol. Genet. 1993; 2: 2109-2116Crossref PubMed Scopus (228) Google Scholar). Recent studies in mice having one MTP allele inactivated show significant reductions in MTP-lipid transfer activity and the ability to secrete apoB-containing lipoproteins by the liver (27Raabe M. Flynn L.M. Zlot C.H. Wong J.S. Veniant M.M. Hamilton R.L. Young S.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8686-8691Crossref PubMed Scopus (223) Google Scholar). These findings raise the possibility that MTP expression may contribute to the rate-limiting step in the lipoprotein assembly/secretion pathway. Cholesterol-7α-hydroxylase NADPH:oxygen oxidoreductase (7α-hydroxylase) is a liver-specific gene product that controls bile acid synthesis, the major pathway responsible for eliminating cholesterol from the body (reviewed in Ref. 28Edwards P.A. Davis R.A. New Compr. Biochem. 1996; 31: 341-362Crossref Scopus (20) Google Scholar). The level of expression of 7α-hydroxylase, which is highly variable in response to diet, metabolic state, and diurnal cycle, has a marked influence on the assembly and secretion of apoB-containing lipoproteins by cultured rat hepatoma cells (29Wang S.-L. Du E.Z. Martin T.D. Davis R.A. J. Biol. Chem. 1997; 272: 19351-19358Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Oxysterols, which are hydroxylated sterol derivatives, antagonize 7α-hydroxylase-induced changes in the assembly and secretion of apoB-containing lipoproteins (29Wang S.-L. Du E.Z. Martin T.D. Davis R.A. J. Biol. Chem. 1997; 272: 19351-19358Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In this study, we examined how expression of 7α-hydroxylase affected the ER degradation of translocation-arrested apoB in CHO cells that lack MTP. Due to a lack of MTP, CHO cells can not fully translocate apoB across the ER, resulting in its rapid degradation (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar, 30Thrift R.N. Drisko J. Dueland S. Trawick J.D. Davis R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9161-9165Crossref PubMed Scopus (81) Google Scholar). Our results show that the normally rapid proteasome degradation of translocation-arrested apoB can be reversibly blocked by 7α-hydroxylase via a sterol-sensitive ubiquitin conjugation step in the ER. An expression plasmid encoding rat 7α-hydroxylase driven by the CMV promoter was constructed and transfected into CHO cells, as described (29Wang S.-L. Du E.Z. Martin T.D. Davis R.A. J. Biol. Chem. 1997; 272: 19351-19358Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Another pcDNA3 plasmid containing the coding region of human 7α-hydroxylase driven by the CMV promoter was a gift from Alan McClelland (Genetic Therapy, Inc.). Plasmid pCW8 encoding dominant negative ubiquitin, Ub (K48R), was generously provided by Ron Kopito. An affinity-purified rabbit antibody against ubiquitin was generously provided by Arthur Haas. Lactacystin was a generous gift from Ardythe McCracken. All cells were cultured in modified Eagle's medium (MEM; Life Technologies) containing 5% fetal bovine serum (Gemni) and antibiotics (100 units/ml penicillin, 100 units/ml streptomycin, and 500 μg/ml fungizone) and other antibiotics for selection as indicated below. Within each cell type, there was no observed difference in cell viability or growth rate between individual clones used for the experiments. For most experiments, cells were grown to 80% confluence in the absence of antibiotics unless indicated. B53 cells (CHO cells expressing apoB53 (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar, 30Thrift R.N. Drisko J. Dueland S. Trawick J.D. Davis R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9161-9165Crossref PubMed Scopus (81) Google Scholar) were plated at 106 cells/100-mm plate and were co-transfected with an expression plasmid encoding rat 7α-hydroxylase driven by the CMV promoter and a plasmid conferring resistance to hygromycin (31Blochlinger K. Diggelmann H. Cell. Mol. Biol. 1984; 4: 2929-2931Crossref Scopus (187) Google Scholar) at an 18:1 molar ratio. Cells were selected in 500 μg/ml hygromycin (for 7α-hydroxylase) and 400 μg/ml G418 (for apoB). Resistant cells were characterized as described below. B53 cells were co-transfected with a pcDNA3 plasmid containing the coding region of human 7α-hydroxylase driven by the CMV promoter and a plasmid conferring resistance to hygromycin, as described above. Resistant cells were characterized as described below. The coding sequence for the dominant negative form of ubiquitin Ub (K48R) (with a Lys → Arg mutation at amino acid 48 (32Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1133) Google Scholar) was released from plasmid pCW8 using restriction enzymeBamHI and cloned into expression vector pIND at theBamHI site (Ecdysone-inducible expression kit, Invitrogen). The pIND-K48R plasmid is then co-transfected with the pVgRXR plasmid at a 1:19 molar ratio into B53 cells. Cells were selected in 500 μg/ml Zeocin for Ub (K48R) and then maintained in 250 μg/ml Zeocin. Single cell clones were obtained. Poly(A+) RNA was isolated from cells using a modification of the guanidinium isothiocyanate method, as described (33Trawick J.D. Lewis K.D. Dueland S. Moore G.L. Simon F.R. Davis R.A. J. Lipid Res. 1996; 37: 24169-24176Abstract Full Text PDF Google Scholar). The resulting mRNA (2–5 μg) was separated on a 0.8% agarose gel, transferred to a nylon membrane, and probed with nick-translated 32P-cDNA probes for 7α-hydroxylase, and β-actin was prepared from gel-purified inserts as described (33Trawick J.D. Lewis K.D. Dueland S. Moore G.L. Simon F.R. Davis R.A. J. Lipid Res. 1996; 37: 24169-24176Abstract Full Text PDF Google Scholar). Cells were harvested in phosphate-buffered saline (PBS) containing a mixture of protease inhibitors (100 μg/ml aprotinin, 100 μg/ml leupeptin, and 2 mmphenylmethylsulfonyl fluoride). Western blotting was performed as described (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar). Following SDS-PAGE, the gels were electroblotted onto nitrocellulose membranes. The nonspecific binding sites of the membranes were blocked using 10% defatted dry milk, followed by the addition of primary antibody. The relative amount of primary antibody bound to the proteins in the nitrocellulose membranes was detected with species-specific horseradish peroxidase-conjugated IgG. Blots were developed using the ECL detection kit (Amersham Pharmacia Biotech). B53 and JF7 cells were grown to 80% confluence on 60-mm plates, after which the culture medium was changed to methionine-free MEM (Sigma). One hour later, cells were pulsed with [35S]methionine (100 μCi/ml; DuPont) for 10 min, after which cells were chased with culture medium containing a 1000-fold excess of unlabeled methionine. At indicated chase time points, cells were lysed in 1 ml of TETN buffer (25 mm Tris, pH 7.5, 5 mm EDTA, 250 mm NaCl, and 1% Triton X-100) containing a mixture of protease inhibitors (100 μg/ml aprotinin, 100 μg/ml leupeptin, and 2 mm phenylmethylsulfonyl fluoride). Immunoprecipitation was carried out as described (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar). Solubilized proteins were precleared with Sepharose CL-4B. 5 μl of a rabbit antiserum specific for human apoB was preincubated with 20 μl of protein A-Sepharose beads (dry volume) in 1 ml of TETN buffer at 4 °C overnight. The beads were then washed with TETN buffer three times. The antibody-bound protein A-Sepharose conjugates were incubated overnight at 4 °C with the cellular protein samples in an amount determined empirically to completely bind the apoB present in each sample. Beads were recovered by centrifugation in a microcentrifuge and were washed three times with the TETN buffer. The immunoprecipitates were dissolved in sample buffer containing SDS and β-mercaptoethanol and separated on a 1–20% gradient SDS-PAGE. The protein content of the cell lysate was determined by the Bradford assay (Bio-Rad). JF7 cells cultured to 85% confluence were disrupted by nitrogen cavitation, and microsomes were isolated by subsequent ultracentrifugation (9Borchardt R.A. Davis R.A. J. Biol. Chem. 1987; 262: 16394-16402Abstract Full Text PDF PubMed Google Scholar). Protein content was analyzed by the Bradford protein assay (Bio-Rad). Trypsin digestion was carried out as described (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar). Each microsomal sample (containing 50 μg of protein) was incubated with 15 μg/ml trypsin in ST buffer (0.25 m sucrose, 10 mm Tris-HCl buffer, pH 7.4) for 30 min on ice. The digestion was stopped by adding protease inhibitors (300 μg/ml soybean trypsin inhibitor, 2 mm phenylmethylsulfonyl fluoride, 100 μg/ml aprotinin, and 100 μg/ml leupeptin each). Microsomes were recovered by centrifugation at 45,000 rpm in a Beckman TLA 45 rotor for 2 h at 4 °C. The microsomal pellet was resuspended in ST buffer and solubilized in sample buffer containing β-mercaptoethanol. The samples were separated on a linear 1–20% polyacrylamide gradient SDS-PAGE, Western blotted, and detected by using anti-human apoB monoclonal Ab 1D1 and a rabbit antiserum against protein-disulfide isomerase (a generous gift from Steve Fuller). Cells were grown on coverslips in 100-mm plates to 60% confluence. Media were removed from the plates, and the cells were washed three times with PBS before they were fixed with 3% paraformaldehyde for 15 min. Cells were then permeabilized with 1% Triton X-100 for 7 min. Nonspecific binding sites in the cells were blocked by incubation with 3% bovine serum albumin in PBS for 30 min. The cells were then incubated for 45 min with 20 μl of affinity-purified antibodies. At the end of the incubation, cells were washed four times with PBST (PBS containing 0.1% Tween 20) and then incubated with the appropriate species-specific Texas Red-conjugated IgG. After washing with PBST, the coverslips were examined using a Nikon microscope with a camera attached. Cells were washed once with ice-cold PBS. Cellular proteins were then solubilized in 1 ml of TETN buffer containing a mixture of protease inhibitors (see "Pulse-Chase Analysis and Immunoprecipitation"). ApoB was immunoprecipitated using 5 μl of a rabbit antiserum specific for human apoB preincubated with 20 μl of protein A-Sepharose beads. The immunoprecipitates were separated on a 1–20% gradient SDS-PAGE and then electroblotted onto nitrocellulose membranes. The ubiquitin conjugated on apoB was detected by an affinity-purified rabbit antibody against ubiquitin, followed by chemiluminescence (Amersham Pharmacia Biotech). Western blots were scanned by a densitometer and analyzed by the ImageQuant program. Previous results showed that when apoB53 is stably expressed in CHO cells (B53 cells), it is rapidly degraded, producing an N-terminal fragment that is secreted without a lipid core (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar). The proteolytic inhibitor ALLN blocks the formation of the N-terminal apoB fragment and causes intact apoB53 to accumulate as a transmembrane protein in isolated microsomes. These cells, stably transfected with a plasmid expressing rat 7α-hydroxylase, are hereafter referred to as JF7 cells. The rate of growth of JF7 cells was indistinguishable from B53 and wild-type CHO-K1 cells. In JF7 cells, a single band for 7α-hydroxylase mRNA was detected by Northern blot, whereas none was detected in B53 cells (data not shown). Analysis of the content of 7α-hydroxylase protein by Western blot showed a single protein band, identical in size to the native protein (data shown below). The enzyme activity of 7α-hydroxylase in CHO cells transfected with the CMV-driven plasmid containing the rat cDNA was ∼10 pmol/mg protein/min. In the absence of proteolytic inhibitors, intact apoB53 was nearly absent in B53 cells (Fig. 1, lane 1). Treating B53 cells with ALLN caused intact apoB53 to accumulate (Fig. 1, lane 2). Remarkably, 7α-hydroxylase expression caused intact apoB53 to accumulate (JF7 cells; Fig. 1, lane 3). The amount of apoB53 in JF7 and B53 cells treated with ALLN was similar (Fig. 1, comparelane 3 with lane 2). Moreover, treating JF7 cells with ALLN did not cause further accumulation of apoB53 (Fig. 1, lane 4). This experiment was performed at least five times (using different cell preparations), and there were no significant differences in the content of apoB53 in JF7 cells treated with or without ALLN. Additional studies using [35S]methionine pulse labeling and immunoprecipitation also showed that ALLN did not cause the accumulation of intact apoB53 in JF7 cells (data not shown). Interestingly, treating JF7 cells with ALLN did increase the amount of smaller apoB fragments (Fig. 1, lane 4). These data suggest that, in contrast to apoB53, the degradation of small apoB peptides is blocked by ALLN in JF7 cells. Thus, JF7 cells are sensitive to proteolytic block by ALLN. These combined data suggest that the expression of 7α-hydroxylase caused intact apoB53 to accumulate by blocking a proteolytic process that is blocked by ALLN in cells that do not express 7α-hydroxylase (i.e. B53 cells; Fig. 1,lane 2). To exclude the possibility that the phenotype we observed was due to an artifact of the individual plasmid, the transfection procedure, or the site of genomic integration, we isolated three individual single cell clones of B53 cells expressing rat 7α-hydroxylase. Each clone showed a comparable level of intact apoB53 in the absence of ALLN. Moreover, to rule out the possibility that the JF7 phenotype was due to a phenomenon unique to the transfected plasmid, we used an entirely different plasmid encoding human 7α-hydroxylase (B53–7αh cells). Cells stably expressing human 7α-hydroxylase displayed an accumulation of apoB53 that was similar to that of JF7 cells (Fig. 1,lane 5). Additional experiments showed that B53 cells transfected with different vectors not expressing 7α-hydroxylase (e.g. luciferase and MTP) together with a hygromycin resistance plasmid did not cause the JF7 phenotype (i.e. the accumulation of intact apoB53; data not shown). These data indicate that the accumulation of apoB53 in JF7 cells is due to the expression of 7α-hydroxylase (rat or human) and cannot be ascribed to a phenomenon caused by the transfection procedure, metabolic selection, or a single type of expression plasmid. The finding that inhibition of proteolysis by ALLN caused equivalent amounts of intact apoB53 to accumulate in B53 cells and JF7 cells (Fig.1, compare lanes 2 and 4) suggests that both cells types synthesize similar amounts of apoB53. The turnover of newly synthesized apoB53 was determined in JF7 and B53 cells using pulse-chase analysis. In both groups of cells, maximal accumulation of [35S]methionine-labeled apoB53 was detected after 30 min of chase (Fig.2). Similar pulse-chase labeling of apoB has been observed using rat hepatocytes (9Borchardt R.A. Davis R.A. J. Biol. Chem. 1987; 262: 16394-16402Abstract Full Text PDF PubMed Google Scholar) and human hepatoma cells (16Bonnardel J.A. Davis R.A. J. Biol. Chem. 1995; 270: 28892-28896Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The amount of maximally labeled apoB53 in JF7 cells at the 30-min chase time was 14-fold higher than that in B53 cells. These data are consistent with the proposal that apoB53 was degraded in a manner that appeared to be co-translational and that the expression of 7α-hydroxylase in JF7 cells blocked this process. We examined if oxysterols could reverse the block in apoB53 degradation exhibited by JF7 cells. Adding 25-hydroxycholesterol and 7-ketocholesterol to JF7 cells caused a marked increased in the rate of apoB53 degradation (Fig.3). The oxysterol effect was observed during the 30-min pulse period and the 30-min chase period. After the 30-min chase period, the rate of decay of apoB53 was similar to that of untreated cells (Fig. 3). Additional data show that total protein synthesis was indistinguishable between JF7, B53, and JF7 cells treated with oxysterols (i.e. the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein was similar in all groups; data not shown). The pulse-chase experiments were performed three separate times (using different preparations of B53 and JF7 cells). In all experiments, oxysterols reversed the blocked degradation of intact apoB53. In two additional experiments, JF7 cells were incubated with and without oxysterols for 24 h. Western blot analysis of the cell extracts showed a 70–95% decrease in the cellular content of apoB53 in cells treated with oxysterols (data not shown). The combined data indicate that the rapid degradation of apoB that is blocked by expression of 7α-hydroxylase can be reversed by oxysterols. We examined the localization of apoB53 that accumulated in JF7 cells following subcellular membrane fractionation and isolation. Following cell disruption and ultracentrifugation, >90% of the apoB53 in JF7 cells was isolated in the 100,000 × g pellet (i.e. microsomes). Moreover, in microsomes prepared from JF7 cells essentially all apoB53 was susceptible to digestion with exogenous trypsin (Fig. 4). The major and smallest molecular weight proteolytic fragment of apoB produced by trypsin digestion had a molecular mass of 69 kDa, which is identical to the results obtained using B53 cells treated with ALLN (18Du E. Kurth J. Wang S.-L. Humiston P. Davis R.A. J. Biol. Chem. 1994; 269: 24169-24176Abstract Full Text PDF PubMed Google Scholar). There were two additional immunoreactive bands (of about 128 and 116 kDa) that were detected in JF7 microsomes following trypsin treatment. These immunoreactive bands showed markedly less chemiluminescence compared with the 69-kDa band. The 69-kDa fragment contained a defined apoB N-terminal epitope (residues 474–539) as demonstrated by its recognition by monoclonal antibody 1D1 (34Marcel Y.L. Innerarity T.L. Spilman C. Mahley R.W. Protter A.A. Milne R.W. Arteriosclerosis. 1987; 7: 166-175Crossref PubMed Google Scholar) (Fig. 4). In contrast to the complete degradation of apoB53, the ER luminal protein, protein-disulfide isomerase, was resistant to trypsin digestion (Fig.4), indicating that the microsomes remained intact. The combined data suggest that the majority of apoB53 that accumulates in JF7 cells assumes a transmembrane orientation in which 69 kDa of the N terminus resides within the ER lumen and the remaining C terminus is exposed to the cytoplasm. Indirect immunofluorescence was used to examine the accumulation of apoB in JF7 cells. We used two epitope-specific antibodies: monoclonal antibody 1D1, which recognizes the N-terminal residues 474–539 (34Marcel Y.L. Innerarity T.L. Spilman C. Mahley R.W. Protter A.A. Milne R.W. Arteriosclerosis. 1987; 7: 166-175Crossref PubMed Google Scholar), and a rabbit antiserum, which recognizes a C-terminal epitope of apoB53 at residue 2140 (35Innerarity T.L. Young S.G. Poksay K.S. Mahley R.W. Smith R.S. Milne R.W. Marcel Y.L. Weisgraber K.H. J. Clin. Invest. 1987; 80: 1794-1798Crossref PubMed Scopus (27) Google Scholar). The N-terminal epitope-specific antibody will recognize both intact apoB53 and N-terminal apoB peptides, whereas the C-terminal specific antiserum will recognize only intact apoB53. In B53 cells, the N-terminal specific antibody resulted in a diffuse reticular immunofluorescence pattern (Fig. 5). In JF7 cells, the N-terminal epitope-specific antibody showed a unique punctate immunofluorescence pattern that overlaid a more diffuse reticular pattern (Fig. 5). In contrast, the antiserum recognizing the C-terminal epitope showed no specific immunofluorescence in B53 cells, while there was a d
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