Title: Insig-dependent Ubiquitination and Degradation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Stimulated by δ- and γ-Tocotrienols
Abstract: Sterol-regulated ubiquitination marks 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-determining enzyme in cholesterol synthesis, for endoplasmic reticulum (ER)-associated degradation by 26 S proteasomes. This degradation, which results from sterol-induced binding of reductase to ER membrane proteins called Insigs, contributes to the complex, multivalent feedback regulation of the enzyme. Degradation of HMG-CoA reductase is also stimulated by various forms of vitamin E, a generic term for α-, β-, δ-, and γ-tocopherols and tocotrienols, which are primarily recognized for their potent antioxidant activity. Here, we show that δ-tocotrienol stimulates ubiquitination and degradation of reductase and blocks processing of sterol regulatory element-binding proteins (SREBPs), another sterol-mediated action of Insigs. The γ-tocotrienol analog is more selective in enhancing reductase ubiquitination and degradation than blocking SREBP processing. Other forms of vitamin E neither accelerate reductase degradation nor block SREBP processing. In vitro assays indicate that γ- and δ-tocotrienol trigger reductase ubiquitination directly and do not require further metabolism for activity. Taken together, these results provide a biochemical mechanism for the hypocholesterolemic effects of vitamin E that have been observed in animals and humans. Sterol-regulated ubiquitination marks 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-determining enzyme in cholesterol synthesis, for endoplasmic reticulum (ER)-associated degradation by 26 S proteasomes. This degradation, which results from sterol-induced binding of reductase to ER membrane proteins called Insigs, contributes to the complex, multivalent feedback regulation of the enzyme. Degradation of HMG-CoA reductase is also stimulated by various forms of vitamin E, a generic term for α-, β-, δ-, and γ-tocopherols and tocotrienols, which are primarily recognized for their potent antioxidant activity. Here, we show that δ-tocotrienol stimulates ubiquitination and degradation of reductase and blocks processing of sterol regulatory element-binding proteins (SREBPs), another sterol-mediated action of Insigs. The γ-tocotrienol analog is more selective in enhancing reductase ubiquitination and degradation than blocking SREBP processing. Other forms of vitamin E neither accelerate reductase degradation nor block SREBP processing. In vitro assays indicate that γ- and δ-tocotrienol trigger reductase ubiquitination directly and do not require further metabolism for activity. Taken together, these results provide a biochemical mechanism for the hypocholesterolemic effects of vitamin E that have been observed in animals and humans. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 3The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein; LDL, low density lipoprotein; CMV, cytomegalovirus. reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids, which include ubiquinone, dolichol, heme, and the farnesyl and geranylgeranyl groups covalently attached to many cellular proteins (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4549) Google Scholar). HMG-CoA reductase, a resident glycoprotein of the endoplasmic reticulum (ER), consists of two contiguous domains (2Liscum L. Finer-Moore J. Stroud R.M. Luskey K.L. Brown M.S. Goldstein J.L. J. Biol. Chem. 1985; 260: 522-530Abstract Full Text PDF PubMed Google Scholar): a hydrophobic N-terminal domain consisting of eight membrane-spanning segments that anchors the protein to ER membranes (3Roitelman J. Olender E.H. Bar-Nun S. Dunn Jr., W.A. Simoni R.D. J. Cell Biol. 1992; 117: 959-973Crossref PubMed Scopus (153) Google Scholar); and a large C-terminal domain located in the cytosol contains the enzyme catalytic activity (4Gil G. Faust J.R. Chin D.J. Goldstein J.L. Brown M.S. Cell. 1985; 41: 249-258Abstract Full Text PDF PubMed Scopus (258) Google Scholar). The reductase is one of the most highly regulated enzymes in biology, as evidenced by its tight control through a multivalent regulatory system mediated by mevalonate-derived products (5Brown M.S. Goldstein J.L. J. Lipid Res. 1980; 21: 505-517Abstract Full Text PDF PubMed Google Scholar). Part of this regulatory system involves sterol-regulated ubiquitination (6Ravid T. Doolman R. Avner R. Harats D. Roitelman J. J. Biol. Chem. 2000; 275: 35840-35847Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), which is mediated by the reductase membrane domain and leads to ER-associated degradation of the enzyme (7Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Degradation of reductase requires the action of at least one of two ER membrane proteins called Insig-1 and Insig-2 (7Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 8Sever N. Lee P.C.W. Song B.L. Rawson R.B. DeBose-Boyd R.A. J. Biol. Chem. 2004; 279: 43136-43147Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Lee P.C.W. Sever N. DeBose-Boyd R.A. J. Biol. Chem. 2005; 280: 25242-25249Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 10Engelking L.J. Liang G. Hammer R.E. Takaishi K. Kuriyama H. Evers B.M. Li W.P. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. 2005; 115: 2489-2498Crossref PubMed Scopus (178) Google Scholar, 11Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar). The central event in this process is sterol-stimulated binding of the reductase membrane domain to Insigs, which in turn are bound to a ubiquitin ligase called gp78 (12Song B.L. Sever N. DeBose-Boyd R.A. Mol. Cell. 2005; 19: 829-840Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). The membrane-anchored gp78 mediates ubiquitination of reductase, an obligate reaction for accelerated degradation of the enzyme (13Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Nonsterol, mevalonate-derived products cannot stimulate ubiquitination or degradation of reductase in sterol-deprived cells but do augment degradation of the enzyme in cells replete with sterols (14Nakanishi M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1988; 263: 8929-8937Abstract Full Text PDF PubMed Google Scholar, 15Roitelman J. Simoni R.D. J. Biol. Chem. 1992; 267: 25264-25273Abstract Full Text PDF PubMed Google Scholar, 16Correll C.C. Edwards P.A. J. Biol. Chem. 1994; 269: 633-638Abstract Full Text PDF PubMed Google Scholar). Metabolic evidence indicates that the nonsterol component of reductase degradation can be derived from the 20-carbon isoprenoid geranylgeraniol (GGOH), but not the 15-carbon isoprenoid farnesol (13Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). It is presently unclear whether the nonsterol potentiator of reductase degradation is GGOH itself or the pyrophosphate derivative, geranylgeranyl pyrophosphate, which may become attached to a specific protein that mediates the effect. Insigs also play a prominent role in the sterol-dependent retention of SREBP cleavage-activating protein (SCAP) in the ER. SCAP, like reductase, contains a hydrophobic N-terminal domain with eight membrane-spanning regions and a large C-terminal domain located in the cytosol (17Nohturfft A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 17243-17250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 18Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, 19Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (423) Google Scholar). SCAP is a sterol-responsive escort protein that associates with SREBPs and facilitates their translocation to the Golgi for sequential proteolytic processing. Proteolysis releases the N-terminal fragments of SREBPs into the cytosol, from which they enter the nucleus to enhance transcription of genes encoding cholesterol biosynthetic enzymes (including reductase) and the low density lipoprotein (LDL)-receptor, which mediates extracellular uptake of cholesterol-rich LDL particles (20Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). Sterols stimulate binding of Insigs to the sterol-sensing domain of SCAP, a stretch of ∼170 amino acids containing five transmembrane domains that resemble the Insig binding site in reductase (18Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar, 21Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Insig binding does not trigger rapid degradation of SCAP, but rather leads to retention of SCAP in the ER, thereby preventing delivery of bound SREBPs to the Golgi for proteolytic activation (22DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 23Nohturfft A. Yabe D. Goldstein J.L. Brown M.S. Espenshade P.J. Cell. 2000; 102: 315-323Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). In the absence of proteolytic activation of SREBPs, rates of cholesterol synthesis and uptake are decreased. Three classes of sterols are believed to control Insig-mediated reactions (ER retention of SCAP·SREBP and accelerated degradation of reductase): cholesterol, methylated sterols, and oxysterols (11Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar). Cholesterol, the bulk end-product of mevalonate metabolism, directly binds to the membrane domain of SCAP, inducing a conformational change in the protein that triggers Insig binding, thereby preventing escape of SCAP·SREBP complexes from the ER (24Brown A.J. Sun L. Feramisco J.D. Brown M.S. Goldstein J.L. Mol. Cell. 2002; 10: 237-245Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 25Radhakrishnan A. Sun L.P. Kwon H.J. Brown M.S. Goldstein J.L. Mol. Cell. 2004; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 26Adams C.M. Reitz J. De Brabander J.K. Feramisco J.D. Li L. Brown M.S. Goldstein J.L. J. Biol. Chem. 2004; 279: 52772-52780Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Lanosterol, the first sterol intermediate in cholesterol synthesis, has no effect on the ER to Golgi transport of SCAP·SREBP, but this methylated sterol potently stimulates Insig-dependent ubiquitination and degradation of reductase (27Song B.L. Javitt N.B. DeBose-Boyd R.A. Cell Metabolism. 2005; 1: 179-189Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Oxysterols, derived from the conversion of either endogenous or LDL-derived cholesterol, have dual actions in that they stimulate the acceleration of reductase degradation and promote ER retention of SCAP·SREBP. Thus, selective recognition of sterol ligands by reductase and SCAP may contribute to the ability of Insigs to mediate regulation of both proteins through distinct mechanisms. Vitamin E is a generic term for eight naturally occurring forms of lipophilic compounds called tocopherols and tocotrienols. Tocopherols and tocotrienols share a polar chromanol ring that is linked to an isoprenoid-derived hydrocarbon side chain (Fig. 1A). Tocopherols carry a saturated phytyl group that is derived from homogentisic acid and phytyl pyrophosphate, whereas tocotrienols are thought to arise from the condensation of homogentisic acid and geranylgeranyl pyrophosphate (28Sen C.K. Khanna S. Roy S. Life Sci. 2006; 78: 2088-2098Crossref PubMed Scopus (458) Google Scholar). Tocopherols and tocotrienols can be subdivided into four isomers (α, β, γ, and δ) with regards to the numbers and position of methyl groups on their chromanol ring (Fig. 1A). Vitamin E is an essential component of the human diet, and various forms of the vitamin are synthesized exclusively by photosynthetic organisms. For example, tocopherols are generally present in common vegetable oils such as soybean, canola, wheat germ, and sunflower, whereas tocotrienols are concentrated (>70%) in cereal grains (i.e. oat, barley, rye, and rice brans), with the richest source found in palm fruits (29Deleted in proofGoogle Scholar). The nutritive value of tocopherols and tocotrienols in food products emanates from their well-known antioxidant capacity, which helps to prevent oxidative damage to polyunsaturated fatty acids (30Brigelius-Flohe R.E.G. Traber M.G. FASEB J. 1999; 13: 1145-1155Crossref PubMed Scopus (1256) Google Scholar). Tocotrienols, but not tocopherols, have been linked to additional beneficial therapeutic properties that include antithrombotic and neuroprotective activities and the ability to inhibit proliferation of breast cancer cells and lower serum cholesterol when administered in the diet of chickens, swine, rats, and hypercholesterolemic humans (31Qureshi A.A. Pearce B.C. Nor R.M. Gapor A. 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Lipids. 1998; 33: 461-469Crossref PubMed Scopus (200) Google Scholar). Early studies revealed an association between the hypocholesterolemic activity of tocotrienol-rich extracts and decreased levels of hepatic HMG-CoA reductase activity (35Qureshi A.A. Burger W.C. Peterson D.M. Elson C.E. J. Biol. Chem. 1986; 261: 10544-10550Abstract Full Text PDF PubMed Google Scholar). Subsequent structure-activity studies revealed that δ- and γ-tocotrienols were the most potent suppressors of reductase in primary rat hepatocytes and cultured HepG2 cells (39Pearce B.C. Parker R.A. Deason M.E. Qureshi A.A. Wright J.J. J. Med. Chem. 1992; 35: 3595-3606Crossref PubMed Scopus (247) Google Scholar, 40Pearce B.C. Parker R.A. Deason M.E. Dischino D.D. Gillespie E. Qureshi A.A. Volk K. Wright J.J. J. Med. Chem. 1994; 37: 526-541Crossref PubMed Scopus (127) Google Scholar). Importantly, these studies offered evidence for tocotrienols as direct, post-transcriptional suppressors that presumably mimic nonsterol isoprenoids in accelerating reductase degradation (41Parker R.A. Pearce B.C. Clark R.W. Gordon D.A. Wright J.J. J. Biol. Chem. 1993; 268: 11230-11238Abstract Full Text PDF PubMed Google Scholar). In the current study, we report the unexpected finding that δ- and γ-tocotrienols mimic sterols, rather than nonsterol isoprenoids, in promoting Insig-dependent ubiquitination/degradation of reductase. Furthermore, δ-tocotrienol, but not the γ-form, also effectively blocks cleavage of SREBPs. Other forms of vitamin E, which include all of the tocopherols and α-tocotrienol, have no measurable effect on reductase degradation or SREBP processing. In vitro ubiquitination assays indicate that γ- and δ-tocotrienols are directly recognized by the sterol-sensing system that mediates formation of the reductase-Insig complex and thereby initiates accelerated degradation of reductase. Considered together, these results provide a plausible mechanism for the hypocholesterolemic activity of tocotrienols that has been observed in animals and humans. Materials—We obtained MG-132, α-, β-, δ-, γ-tocopherols and tocotrienols from Calbiochem; horseradish peroxidase-conjugated donkey anti-mouse, and anti-rabbit IgG from Jackson ImmunoResearch Laboratories; FLAG-ubiquitin from Sigma; and ubiquitin-aldehyde and ubiquitin-activating enzyme from Boston Biochem (Cambridge, MA). Other reagents were obtained from previously described sources (22DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Lipoprotein-deficient serum (LPDS) (d > 1.215 g/ml) was prepared from newborn calf serum by ultracentrifugation (42Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1284) Google Scholar). Expression Plasmids—The following expression plasmids were described in the indicated reference: pCMV-Insig-1-Myc, which encodes amino acids 1–277 of human Insig-1 followed by six tandem copies of a c-Myc epitope tag under control of the cytomegalovirus promoter (CMV) (18Yang T. Espenshade P.J. Wright M.E. Yabe D. Gong Y. Aebersold R. Goldstein J.L. Brown M.S. Cell. 2002; 110: 489-500Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar); and wild-type, K89R/K248R, and YIYF to AAAA versions of pCMV-HMG-Red-T7, which encodes the full-length hamster HMG CoA reductase (amino acids 1–887) followed by three tandem copies of the T7-epitope tag under control of a CMV promoter (13Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Cell Culture—SV589 cells, an immortalized line of human fibroblasts expressing the SV-40 large T-antigen (43Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (979) Google Scholar), were maintained in monolayer at 37 °C in 5% CO2. Stock cultures of SV-589 cells were grown in medium A (Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate) supplemented with 10% (v/v) fetal calf serum. Stock cultures of Chinese hamster ovary-K1 (CHO-K1) cells were grown in monolayer at 37 °C in 8–9% CO2 in medium B (1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 μg/ml streptomycin sulfate) supplemented with 5% fetal calf serum. Ubiquitination of HMG-CoA Reductase in Intact Cells—The conditions of incubations prior to harvesting of cells are described in the figure legends. At the end of the incubations, cells were harvested, lysed in detergent-containing buffer, and immunoprecipitations were carried out with polyclonal antibodies directed against the C-terminal domain of human HMG-CoA reductase as previously described (13Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 44Sato R. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9261-9265Crossref PubMed Scopus (141) Google Scholar). Aliquots of the immunoprecipitates were subjected to 6% SDS-PAGE, transferred to nylon membranes, and subjected to immunoblot analysis. Transient Transfection, Cell Fractionation, and Immunoblot Analysis—Transfection of CHO-K1 cells with FuGENE-6 transfection reagent (Roche Diagnostics) was performed as described previously (7Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Conditions of the incubations are described in the figure legends. At the end of the incubations, triplicate dishes of cells for each variable were harvested and pooled for analysis. Pooled cell pellets were used to isolate nuclear extracts, 2 × 105 g membrane fractions, or whole cell lysates; all fractions were subjected to 8% SDS-PAGE and immunoblot analysis was carried out as previously described (7Sever N. Yang T. Brown M.S. Goldstein J.L. DeBose-Boyd R.A. Mol. Cell. 2003; 11: 25-33Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Primary antibodies used for immunoblotting were as follows: mouse monoclonal anti-T7 (IgG2b, Novagen); mouse monoclonal anti-Myc (IgG fraction) from the culture medium of hybridoma clone 9E10 (American Type Culture Collection); IgG-A9, a mouse monoclonal antibody against the catalytic domain of hamster HMG-CoA reductase (45Liscum L. Luskey K.L. Chin D.J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1983; 258: 8450-8455Abstract Full Text PDF PubMed Google Scholar); IgG-P4D1, a mouse monoclonal antibody against bovine ubiquitin (Santa Cruz Biotechnology); IgG-1D2, a mouse monoclonal antibody against the N terminus of human SREBP-2 (amino acids 48–403) (19Yabe D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12753-12758Crossref PubMed Scopus (423) Google Scholar); and IgG-M2, a mouse monoclonal antibody against the FLAG epitope (Sigma). RNA Interference—Duplexes of small interfering RNA (siRNA) targeting human Insig-1, human Insig-2, and an irrelevant control gene vesicular stomatitis virus glycoprotein (VSV-G), were synthesized by Dharmacon Research (Lafayette, CO). The sequences of the siRNAs and the procedure for transfection have been described (13Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Ubiquitination of HMG-CoA Reductase in Permeabilized Cells and Isolated Membranes—The conditions of incubations prior to harvesting of cells are described in the figure legends. SV-589 cells were harvested into the medium by scraping and collected by centrifugation, after which pooled cell pellets from triplicate dishes were washed with phosphate-buffered saline and either permeabilized with 0.025% (w/v) digitonin (46Song B.L. DeBose-Boyd R.A. J. Biol. Chem. 2004; 279: 28798-28806Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) or subjected to cell fractionation for the isolation of membrane fractions as described above. Permeabilized cells and isolated membrane fractions were resuspended in 300 μl and 150 μl, respectively, of permeabilization buffer (25 mm Hepes-KOH at pH 7.3, 115 mm potassium acetate, 5 mm sodium acetate, 2.5 mm magnesium chloride, 0.5 mm sodium EGTA) containing protease inhibitors (20 μm leupeptin, 10 μm MG-132, 5 μg/ml pepstatin A, and 2 μg/ml aprotinin), an ATP-regenerating system (2 mm Hepes-KOH at pH 7.3, 1 mm magnesium acetate, 1 mm sodium ATP, 30 mm creatine phosphate, and 0.05 mg/ml creatine kinase), 0.1 mg/ml FLAG-ubiquitin, and 0.01 mg/ml ubiquitin-aldehyde. Reactions were supplemented with either 3 mg/ml rat liver cytosol or 5 μg/ml purified ubiquitin-activating enzyme (E1) to facilitate activation of FLAG-ubiquitin (27Song B.L. Javitt N.B. DeBose-Boyd R.A. Cell Metabolism. 2005; 1: 179-189Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). 25-Hydroxycholesterol, α-, β-, δ-, or γ-tocopherol and tocotrienol were added to reactions in a final concentration of 1% (v/v) ethanol. Reactions were carried out at 37 °C for 30 min and terminated by centrifugation at 4,000 rpm (permeabilized cells) or 2 × 105 g (isolated membranes) at 4 °C. The resulting pellets were lysed, clarified, and subjected to immunoprecipitation with polyclonal anti-HMG-CoA reductase and immunoblotted as described above. Fig. 1B shows the results of an experiment that compare the effects of various tocopherols and tocotrienols on the degradation of reductase and the processing of SREBP-2 in SV589 cells (43Yamamoto T. Davis C.G. Brown M.S. Schneider W.J. Casey M.L. Goldstein J.L. Russell D.W. Cell. 1984; 39: 27-38Abstract Full Text PDF PubMed Scopus (979) Google Scholar). The cells were first depleted of sterols by incubation for 16 h in medium containing lipoprotein-deficient serum, the reductase inhibitor compactin (47Brown M.S. Faust J.R. Goldstein J.L. J. Biol. Chem. 1978; 253: 1121-1128Abstract Full Text PDF PubMed Google Scholar), and a low level of mevalonate (50 μm). The sterol-depleted cells were then treated for an additional 5 h with 2.5 μm 25-hydroxycholesterol or different concentrations of α-, γ-, or δ-tocopherol and tocotrienol plus 10 mm mevalonate, which allows for production of nonsterol isoprenoids that enhance reductase degradation (13Sever N. Song B.L. Yabe D. Goldstein J.L. Brown M.S. DeBose-Boyd R.A. J. Biol. Chem. 2003; 278: 52479-52490Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Following treatments, cells were harvested, subjected to fractionation, and aliquots of the resulting membrane and nuclear extracts were subjected to SDS-PAGE. Immunoblot analysis was subsequently carried out with antibodies against reductase (top panel) and SREBP-2 (bottom panel). As expected, 25-hydroxycholesterol accelerated degradation of reductase, as indicated by the disappearance of the reductase protein, and blocked SREBP-2 processing, as demonstrated by the disappearance of nuclear SREBP-2 (top and bottom panels, compare lanes a and b). In contrast, none of the tocopherols accelerated reductase degradation or blocked SREBP-2 processing (top and bottom panels, lanes c–h); similar results were obtained with α-tocotrienol (lanes i–j) and the β-forms of tocopherol and tocotrienol. 4R. A. DeBose-Boyd and B.-L. Song, unpublished observations. Both γ- and δ-tocotrienols accelerated reductase degradation at 10 μm (top panel, lanes l and n), but only δ-tocotrienol completely blocked SREBP-2 processing (bottom panel, lane n). To determine whether δ- and γ-tocotrienols fulfill the sterol or nonsterol requirement for reductase degradation, sterol-depleted cells were treated with these molecules individually or in combination with 10 mm mevalonate (Fig. 2A). When added to cells individually, 25-hydroxycholesterol, γ-tocotrienol, and δ-tocotrienol increased reductase degradation (lanes b–d, f, and g, respectively), whereas 10 mm mevalonate alone had no effect (lane h). Degradation of reductase was further accelerated when 10 mm mevalonate was added together with 25-hydroxycholesterol or δ- and γ-tocotrienols (compare lanes b–d, f, and g with i–k, m, and n). In contrast, α-tocotrienol had no measurable effect on reductase degradation, regardless of the absence or presence of 10 mm mevalonate (lanes e and l). The ubiquitination state of reductase was determined in sterol-deprived cells treated with 25-hydroxycholesterol or the various forms of tocopherols and tocotrienols in the presence of MG-132, which blocks proteasomal degradation of ubiquitinated proteins (Fig. 2B). Following treatments, detergent lysates were prepared and subjected to immunoprecipitation with polyclonal antibodies against reductase. The resulting immunoprecipitates were then subjected to SDS-PAGE and immunoblotted with anti-ubiquitin (top panel) and anti-reductase (bottom panel). In the presence of 25-hydroxycholesterol, γ-, or δ-tocotrienol, reductase became ubiquitinated as indicated by the high molecular weight smears of reactivity in the anti-ubiquitin immunoblot (top panel, lanes 2, 7, and 8). In contrast, ubiquitination of reductase was not stimulated by α-, γ-, and δ-tocopherols or α-tocotrienol to an appreciable extent (top panel, lane 3–6). The Insig requirement for tocotrienol-stimulated degradation and ubiquitination of reductase is demonstrated in the RNA interference (RNAi) experiments of Fig. 3. SV-589 cells were transfected with duplexes of small interfering RNA (siRNA) targeting the control gene vesicular stomatitis virus glycoprotein or the combination of Insig-1 and Insig-2 and depleted of sterols for 16 h. For degradation experiments, cells were subjected to treatments with 25-hydroxycholesterol, α-, γ-, or δ-tocotrienol in the presence of 10 mm mevalonate (Fig. 3A). The combination of 25-hydroxycholesterol and 10 mm mevalonate led to complete degradation of reductase within 5 h in the control-transfected cells (top panel, lane 2). Similarly, γ- or δ-tocotrienol plus 10 mm mevalonate caused reductase to become fully degraded after 5 h (lanes 4 and 5). When siRNAs targeting Insig-1 and Insig-2 were introduced into the cells, the degradation of reductase mediated by 25-hydroxycholesterol or γ- and δ-tocotrienols were abolished (lanes 7, 9, and 10). Expression of a control protein, the transferrin receptor, did not change under any of the experimental conditions (bottom panel, lanes 1–10). Similar results were obtained for reductase ubiquitination (Fig. 3B). In these experiments, sterol-deprived siRNA-transfected cells were treated with MG-132 plus 25-hydroxycholesterol or tocotrienols for 1 h prior to lysate preparation, which was followed by reductase immunoprecipitation and immunoblot analysis. As expected, 25-hydroxycholesterol or γ- and δ-tocotrienols stim