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Squalene synthase (SQS) catalyzes the first reaction of the branch of the isoprenoid metabolic pathway committed specifically to sterol biosynthesis. Regulation of SQS is thought to direct proximal intermediates in the pathway into either sterol or nonsterol branches in response to changing cellular requirements. The importance of SQS in cholesterol metabolism has stimulated research on the mechanism, structure, and regulation of the enzyme. SQS produces squalene, a C30 isoprenoid, in a two-step reaction in which two molecules of farnesyl diphosphate are condensed head to head. Site-directed mutagenesis of rat SQS has identified conserved Tyr, Phe, and Asp residues that are essential for function. The aromatic rings of Tyr and Phe are postulated to stabilize carbocation intermediates of the first and second half-reactions, respectively; the acidic Asp residues may be required for substrate binding. SQS activity, protein level, and gene transcription are strictly and coordinately regulated by cholesterol status, decreasing with cholesterol surfeit and increasing with cholesterol deficit. The human SQS (hSQS) gene has an unusually complex promoter with multiple binding sites for the sterol regulatory element binding proteins SREBP-1a and SREBP-2, and for accessory transcription factors known to be involved in the control of other sterol-responsive genes. SREBP-1a and SREBP-2 require different subsets of hSQS regulatory DNA elements to achieve maximal promoter activation. Current research is directed at elucidating the precise contribution made by individual SREBPs and accessory transcription factors to hSQS transcriptional control.
Mammalian squalene synthase (SQS) catalyzes the first reaction of the branch of the isoprenoid metabolic pathway committed specifically to sterol biosynthesis. SQS produces squalene in an unusual two-step reaction in which two molecules of farnesyl diphosphate are condensed head-to-head. Recent studies have advanced understanding of the reaction mechanism, the functional domains of the enzyme, and transcriptional regulation of the gene. Site-directed mutagenesis has identified conserved Asp, Tyr, and Phe residues that are essential for SQS activity. The Asp residues are hypothesized to be required for substrate binding; the Tyr and Phe residues may stabilize carbocation reaction intermediates. The elucidation of SQS crystal structure will most likely direct future research on the relationship between enzyme structure and function. SQS activity, protein, and mRNA levels are regulated by cholesterol status and by the cytokines TNF-alpha and IL-1beta. Activation of the SQS promoter in response to cholesterol deficit is mediated by sterol regulatory element binding proteins SREBP-1a and SREBP-2. The precise contributions made by individual SREBPs and accessory transcription factors to SQS transcriptional control, and the mechanisms underlying cytokine regulation of SQS are major foci of current research.
Changes in farnesyl protein transferase (FPTase) activity and FPTase beta-subunit protein levels were determined in IgE-sensitized RBL-2H3 mast cells in response to polyvalent antigen administration. Ten minutes after the addition of DNP modified BSA to mast cells, whose high affinity receptor for IgE (FcvarepsilonRI) contained bound anti-DNP IgE, FPTase specific activity increased by 54 +/- 28%. Time course studies showed FPTase specific activity doubled during a 20- to 30-min period after antigen-induced cell aggregation. Also, an increase in FPTase beta-subunit protein during this time ( approximately 30%) was observed; this protein increase was not accompanied by a similar increase in FPTase beta-subunit m-RNA levels. The FcvarepsilonRI aggregation had no significant effect on the activities of other enzymes involved with farnesyl diphosphate (FPP) metabolism: FPP synthase, isopentenyl diphosphate isomerase, geranylgeranyl protein transferase, and squalene synthase. Specific inhibition of FPTase activity by manumycin was studied to determine what role FPTase plays in mast cell activation. Manumycin profoundly inhibited hexosaminidase release in activated cells, indicating FPTase is required for signal transduction involved with protein exocytosis from RBL-2H3 mast cells.
Farnesyl diphosphate synthase (FPPS: EC2.5.1.10), a key enzyme in isoprenoid metabolic pathways, catalyzes the synthesis of farnesyl diphosphate (FPP) an intermediate in the biosynthesis of both sterol and non-sterol isoprenoid end products. The localization of FPPS to peroxisomes has been reported (Krisans, S. K., J. Ericsson, P. A. Edwards, and G. A. Keller. 1994. J. Biol. Chem. 269: 14165;-14169). Using indirect immunofluorescence and immunoelectron microscopic techniques we show here that FPPS is localized predominantly in the peroxisomes of rat hepatoma H35 cells. However, the partial release of 60;-70% of cellular FPPS activity is observed by selective permeabilization of these cells with digitonin. Under these conditions, lactate dehydrogenase, a cytosolic enzyme, is completely released whereas catalase, a known peroxisomal enzyme, is fully retained. Digitonin treatment of H35 cells differentially affects the release of other peroxisomal enzymes involved in isoprenoid metabolism. For instance, mevalonate kinase and phosphomevalonate kinase are almost totally released (95% and 91%, respectively), whereas 3-hydroxy-3-methylglutaryl-CoA reductase is fully retained. Indirect immunoflourescence studies indicate that FPPS is localized in peroxisomes of Chinese hamster ovary (CHO)-K1 cells but is dispersed in the cytosol of ZR-82 cells, a mutant that lacks peroxisomes. Unlike in H35 cells, FPPS is completely released upon digitonin permeabilization of CHO-K1 and ZR-82 cells. In contrast, under the same permeabilization conditions, catalase is fully retained in CHO-K1 cells but completely released from ZR-82 cells. These studies indicate that FPPS and other enzymes in the isoprenoid biosynthetic pathways, involved in the formation of FPP, are differentially associated with peroxisomes and may easily diffuse to the cytosol. Based on these observations, the significance and a possible regulatory model in the formation of isoprenoid end-products are discussed.
Rat hepatic squalene synthase (RSS, EC 2.5.1.21) contains three conserved sections, A, B, and C, that were proposed to be involved in catalysis (McKenzie, T. L., Jiang, G., Straubhaar, J. R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374). Here we use the high expression vector pTrxRSS and site-directed mutagenesis to determine the specific residues in these sections that are essential for the two reactions catalyzed by RSS. Section C mutants F288Y, F288L, F286Y, F286W, F286L, Q293N, and Q283E accumulate presqualene diphosphate (PSPP) from trans-farnesyl diphosphate (FPP) with reduced production of squalene. F288L, which retains approximately 50% first step activity, displays only residual activity (0.2%) in the production of squalene from either FPP or PSPP. Substitution of either Phe288 or Phe286 with charged residues completely abolishes the enzyme activity. Thus, F288W, F288D, F288R, F286D, and F286R cannot produce squalene from either FPP or PSPP. All single residue mutants in Section A, except Tyr171, retain most of the RSS activity, with no detectable accumulation of PSPP in an assay mixture complete with NADPH. Y171F, Y171S, and Y171W are all inactive. Section B, which binds the diphosphate moieties of the allylic diphosphate subtrates, contains four negatively charged residues: Glu222, Glu226, Asp219, and Asp223. The two Glu residues can be replaced with neutral or with positively charged residues without signficantly affecting enzyme activity. However, replacement of either Asp residues with Asn eliminates all but a residual level of activity, and substitution with Glu abolishes all activity. These results indicate that 1) Section C, in particular Phe288, may be involved in the second step of catalysis, 2) Tyr171 of Section A is essential for catalysis, most likely for the first reaction, 3) the two Asp residues in Section B are essential for the activity and most likely bind the substrate via magnesium salt bridges. Based on these results, a mechanism for the first reaction is proposed.
Transcription of the human squalene synthase (HSS) gene is regulated by variations in the level of cellular cholesterol. Three regulatory elements in the HSS promoter region are known to be involved in the regulation: 1) a modified sterol regulatory element (SRE) 1 (HSS-SRE-1), 2) an inverted SRE-3 (Inv-SRE-3), 3) an inverted Y box (Inv-Y-Box). We report here the regulatory role of distinct cis-elements in the HSS promoter by using mutants of an HSS-luciferase promoter reporter. The activity of a wild-type promoter reporter transiently transfected into HepG-2 cells is increased by sterol depletion of the cells or by coexpression of mature forms of the SRE-binding proteins (SREBP) 1a and SREBP-2. Differential activation by SREBP-1a and SREBP-2 of the reporter gene mutated at various regions of the promoter is observed. Mutation of either the HSS-SRE-1 or the Inv-SRE-3 sequence diminished the activation by SREBP-1a and by sterol depletion but did not affect the activation by SREBP-2. Simultaneous mutations of both of these sequences almost completely abolished activation of the promoter by SREBP-1a or by sterol depletion, but activation by SREBP-2 was retained at 70%. Mutation of the Inv-Y-Box sequence element decreased the activity of the promoter by 50% or more, and if mutated together with both SREs, the activation was almost completely abolished. Mutation of any single GC box of the two located at -40 to -57 did not affect activity, whereas simultaneous mutation of the two decreased activation by SREBP-2 by 60%, by lipid depletion by 20%, and had no effect on the activation by SREBP-1a. A Y box motif at -159 to -166 and an SRE-like sequence element (SRE-1(8/10)) at position -101 to -108 are also involved in the sterol regulation. These results indicate that the complex sterol-mediated transcriptional regulation of the HSS gene is due to the presence of multiple copies of diverse cis elements in the HSS promoter. The differential activation of the HSS promoter may point to specific role of the SREBPs in cholesterogenesis.
The expression of human squalene synthase (HSS) gene is transcriptionally regulated in HepG-2 cells, up to 10-fold, by variations in cellular cholesterol homeostasis. An earlier deletion analysis of the 5'-flanking region of the HSS gene demonstrated that most of the HSS promoter activity is detected within a 69-base pair sequence located between nucleotides -131 and -200. ADD1/SREBP-1c, a rat homologue of sterol regulatory element-binding protein (SREBP)-1c binds to sterol regulatory element (SRE)-1-like sequence (HSS-SRE-1) present in this region (Guan, G., Jiang, G., Koch, R. L. and Shechter, I. (1995) J. Biol. Chem. 270, 21958-21965). In our present study, we demonstrate that mutation of this HSS-SRE-1 element significantly reduced, but did not abolish, the response of HSS promoter to change in sterol concentration. Mutation scanning indicates that two additional DNA promoter sequences are involved in sterol-mediated regulation. The first sequence contains an inverted SRE-3 element (Inv-SRE-3) and the second contains an inverted Y-box (Inv-Y-box) sequence. A single mutation in any of these sequences reduced, but did not completely remove, the response to sterols. Combination mutation studies showed that the HSS promoter activity was abolished only when all three elements were mutated simultaneously. Co-expression of SRE-1- or SRE-2-binding proteins (SREBP-1 or SREBP-2) with HSS promoter-luciferase reporter resulted in a dramatic increase of HSS promoter activity. Gel mobility shift studies indicate differential binding of the SREBPs to regulatory sequences in the HSS promoter. These results indicate that the transcription of the HSS gene is regulated by multiple regulatory elements in the promoter.
Recent studies have shown that endotoxin (LPS) administration to Syrian hamsters markedly increased hepatic HMG-CoA reductase activity, protein mass, and mRNA levels, but only produced a modest increase in hepatic cholesterol synthesis, suggesting that LPS may also influence other key enzymes involved in the regulation of cholesterol metabolism. In the present study, we have examined the effect of LPS and cytokines on the activity, protein mass, and mRNA level of squalene synthase, which is the first committed enzyme in cholesterol biosynthesis and is located at a branch point in the mevalonate pathway. Our results demonstrate that LPS administration produces a marked decrease in the mRNA levels of squalene synthase. This decrease in squalene synthase mRNA occurred very rapidly (90 min after LPS) and required relatively small doses of LPS (1 microg/100 gm body weight). LPS also significantly decreased squalene synthase activity and protein mass. Finally, LPS produced a marked decrease in squalene synthase mRNA, activity, and protein levels when the basal levels of squalene synthase expression were increased 4-fold by prior treatment with bile acid binding resin, colestipol. Tumor necrosis factor and interleukin-1, which mediate many of the metabolic effects of LPS, also decreased hepatic squalene synthase activity and mRNA levels. Taken together, our results suggest that the discordant regulation of HMG-CoA reductase and squalene synthase during the host response to infection and inflammation may have substantial effects on the regulation of substrate flux into the non-sterol pathways of mevalonate metabolism.
As part of an effort to dissect the genetic factors involved in cholesterol homeostasis in the mouse model, we report the mapping of 12 new candidate genes using linkage analysis. The genes include: cytoplasmic HMG-CoA synthase (Hmgcs 1, Chr 13), mitochondrial synthase (Hmgcs 2, Chr 3), a synthase-related sequence (Hmgcs 1-rs, Chr 12), mevalonate kinase (Mvk, Chr 5), farnesyl diphosphate synthase (Fdps, Chr 3), squalene synthase (Fdft 1, Chr 14), acyl-CoA:cholesterol acyltransferase (Acact, Chr 1), sterol regulatory element binding protein-1 (Srebf1, Chr 8) and -2 (Srebf2, Chr 15), apolipoprotein A-I regulatory protein (Tcfcoup2, Chr 7), low density receptor-related protein-related sequence (Lrp-rs, Chr 10), and Lrp-associated protein (Lrpap 1, Chr 5). In addition, the map positions for several lipoprotein receptor genes were refined. These genes include: low density lipoprotein receptor (Ldlr, Chr 9), very low density lipoprotein receptor (Vldlr, Chr 19), and glycoprotein 330 (Gp330, Chr 2). Some of these candidate genes are located within previously defined chromosomal regions (quantitative trait loci, QTLs) contributing to plasma lipoprotein levels, and Acact maps near a mouse mutation, ald, resulting in depletion of cholesteryl esters in the adrenals. The combined use of QTL and candidate gene mapping provides a powerful means of dissecting complex traits such as cholesterol homeostasis.
We have cloned and characterized the 5'-flanking region of the gene encoding human squalene synthase. We report here the promoter activity of successively 5'-truncated sections of a 1 kilobase of this region by fusing it to the coding region of a luciferase reporter gene. DNA segments of 200 base pairs (bp) 5' to the transcription start site, as determined by primer extension analysis, show a strong promoter effect on the expression of the luciferase chimeric gene and a high response to the presence of sterols when transiently transfected into the human hepatoma cell line HepG2 or to the hamster-derived CHO-K1 cells. An approximately 50-fold induction of luciferase activity, in the absence of sterols, was observed in transiently transfected HepG2 cells for fusion constructs containing sections of 200, 459, and 934 bp of the putative human squalene synthase promoter. Loss of promoter activity and response to sterols was localized to a 69-bp section located 131 nucleotides 5' to the transcription start site. Sequence analysis of this region showed that it contained a sterol regulatory element 1 (SRE-1) previously identified in other sterol regulated genes (Smith, J. R., Osborne, T. F., Brown, M. S., Goldstein, J. L., and Gil, G. (1988). J. Biol. Chem. 263, 18480-18487) and two potential NF-1 binding sites. Additional CCAAT box, SRE-1 element, and two Sp1 sites were identified 3' to this section. Sequences within this 69-bp DNA, including the SRE-1 cis-acting element, show strong binding to the purified nuclear transcription factor ADD1 (Tonzonoz, P., Kim, J. B., Graves, R. A., and Spiegelman B. M. (1993) Mol. Cell Biol. 13, 4753-4759) by mobility shift assay and footprinting analyses.
Recently, we reported the isolation of a cDNA encoding the human enzyme squalene synthase, the first step of sterol biosynthesis uniquely committed to synthesis of cholesterol (6). As such, it is likely that this enzyme occupies a critical regulatory position in the synthesis of cholesterol. As part of continuing studies of the role of this gene in cellular metabolism, we undertook the mapping of this gene on the human chromosomes. To localize the gene, we have first isolated a yeast artificial chromosome (YAC) containing the squalene synthase gene. We then used fluorescence in situ hybridization (FISH) with yeast DNA containing the YAC to localize the gene to chromosome 8. Assignment to human chromosome 8 was confirmed by polymerase chain reaction analysis of a somatic cell hybrid containing human chromosome 8. Use of a somatic cell hybrid regional mapping panel dividing chromosome 8 into several fragments localized the gene to 8p21-pter. Fractional length analysis of the FISH mapping placed the signal generated with this YAC at 8p22-p23.1.
Primers, based on the cDNA nucleotide sequences for rat hepatic squalene synthase (EC 2.5.1.21) (McKenzie, T.L., Jiang, G., Straubhaar, J.R., Conrad, D., and Shechter, I. (1992) J. Biol. Chem. 267, 21368-21374), were synthesized and used for the amplification and sequencing of a 1672-base pair (bp) cDNA for the human hepatic squalene synthase (HSS) from human hepatic RNA. An open reading frame of 1251 bp encoding 417 amino acids (M(r) = 48,200) was detected for HSS. We have constructed a pHSS 1286 expression vector by molecular cloning of a 1286-bp cDNA, that includes sequences of the entire coding region for HSS, into pBluescript. Expression in Escherichia coli of a functional, full-length HSS was confirmed by immunoblot analysis and enzymatic activity. Northern blot analyses of poly(A+) RNA obtained from the human hepatoma cell line HepG2 show three distinct size classes of mRNA for HSS. 1.4-, 1.6- and 2.1-kilobase mRNA were observed. The relative abundance is in the order 1.6 > 1.4 > 2.1 and did not change when the cells were grown in the presence of 25-hydroxycholesterol or lovastatin. The ratio between the level of HSS mRNA in cells grown in the absence and presence of 5 micrograms/ml 25-hydroxycholesterol varies between 8- and 16-fold. This lowering of the mRNA level was observed when the cells were grown in 10% of either full serum or lipid-depleted serum. A 2.7- and 4.0-fold increase of HSS mRNA was observed when HepG2 cells were grown in the presence of 5 micrograms/ml lovastatin in lipid-depleted or full serum, respectively. These studies show that HSS exhibit a relatively high level of transcriptional regulation in response to 25-hydroxycholesterol regardless of the presence of cholesterol in the growth media.
In the present study we investigated the subcellular localization of squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21). Squalene synthase catalyzes the formation of squalene from trans-farnesyl diphosphate in two distinct steps and is the first committed enzyme for the biosynthesis of cholesterol. Recently, a truncated form of the enzyme from rat hepatocytes was purified, and monospecific antibodies for squalene synthase were produced. This enabled the subcellular localization of squalene synthase by three different methods: (i) analytical subcellular fractionation and measurements of enzyme activities; (ii) immunodeterminations of squalene synthase in the isolated subcellular fractions with a monospecific antibody; and (iii) immunoelectron microscopy. All three methods gave consistent results. The data clearly illustrate that squalene synthase enzymatic activity and squalene synthase are exclusively localized in the endoplasmic reticulum. In rat hepatic peroxisomes we were not able to detect any squalene synthase. In addition, we also demonstrated that squalene synthase in the microsomal fraction is dramatically regulated by a number of hypolipidemic drugs and dietary treatments.
Amino acid sequence information was obtained for the NH2 terminus, and for endogenous peptides generated by trypsin digestion, of a purified, truncated form of rat hepatic squalene synthase (RSS, EC 2.5.1.21) (Shechter, I., Klinger, E., Rucker, M. L., Engstrom, R. G., Spirito, J. A., Islam, M. A., Boettcher, B. R., and Weinstein, D. B. (1992) J. Biol. Chem. 267, 8628-8635). Degenerate primers, based on the amino acid sequences, were synthesized and used for the amplification and sequencing of a 1708-base pair (bp) cDNA for RSS from the rat hepatoma cell line H35. An open reading frame of 1248 bp encoding 416 amino acids (M(r) = 48,103) was detected for RSS. We have constructed a pRSS1327 expression vector by molecular cloning of a 1327-bp cDNA, which includes sequences of the entire coding region for RSS, into pBluescript. Expression in Escherichia coli of a functional, full-length RSS was confirmed by immunoblot analysis and enzymatic activity. We present and evaluate a model for the secondary structure of RSS and its possible membrane orientation. The model predicts a 315-residue domain at the center of the protein that contains the catalytic site and is released in a soluble form by partial proteolysis. The 33-residue NH2-terminal and 98-residue COOH-terminal sections are not involved in catalysis. Sequence analysis of the catalytic domain of RSS indicate three regions with high homology to sequences in a number of functionally distinct proteins that utilize polyprenyl diphosphate substrates.
Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.
Apolipoprotein (apo) B-48 is generated by a unique physiological process. Cytidine 6,666 of the apo B primary transcript is posttranscriptionally converted to a uridine by an RNA editing mechanism that transforms the codon for glutamine 2,153 to a termination codon. The editing reaction can be duplicated in a cell-free extract. In this study, the apo B-48 mRNA editing activity derived from partially purified extracts of rabbit enterocytes was characterized. The optimum conditions for the editing reaction were determined to be a salt concentration of 0.125-0.150 M NaCl or KCl, a pH of 8-8.5, and a temperature of 30 degrees C. The reaction rate was linear up to 45 minutes and was proportional to the editing extract concentration. No metal ion cofactors, DNA or RNA cofactors, or energy requirements were identified. At optimum conditions, the reaction followed Michaelis-Menten kinetics, with a Km of 0.4 nM for the rabbit RNA substrate. In addition, the reaction rate was enhanced by the addition of 25 micrograms/ml heparin or 40% glycerol. The characteristics of the editing reaction suggest that it is catalyzed by a nucleotide sequence-specific cytidine deaminase that is either a single enzyme or a multimeric protein.
The activity of rat liver microsomal squalene epoxidase is inhibited effectively by digitonin. Concentrations of 0.8 to 1.2 mg/ml of digitonin cause total inhibition of microsomal (0.75 mg protein/ml) squalene epoxidase either in microsomes that were pretreated with digitonin and subsequently washed and subjected to epoxidase assay or when digitonin was added directly to the assay. The inhibition of squalene epoxidase by digitonin is concentration-dependent and takes place rapidly within 5 min of exposure of the microsomes to digitonin. Octylglucoside, dimethylsulfoxide, CHAPS, as well as cholesterol or total microsomal lipid extract were ineffective in restoring the digitonin-inhibited squalene epoxidase activity. Epoxidase activity in digitonin-treated microsomes was fully restored by Triton X-100. The reactivation by Triton X-100 displays a concentration optimum with maximal reactivation of the epoxidase (0.7 mg protein/ml) occurring at 0.2% Triton X-100. Microsomal 2,3-oxidosqualene-lanosterol cyclase is also inhibited by digitonin. Higher concentrations of digitonin are required to obtain full inhibition of the cyclase activity and only 40% inhibition of cyclase activity is observed at 1 mg/ml of digitonin. Solubilized (subunit size 55 to 66 kDa) and microsomal (subunit size 97 kDa) 3-hydroxy-3-methylglutaryl CoA reductase are totally unaffected by the same concentration of digitonin. Squalene synthetase, another microsomal enzyme in the biosynthetic pathway of cholesterol, is activated by digitonin. A 2.2-fold activation of squalene synthetase is observed at 0.8 mg/ml of digitonin. The results agree with a model in which squalene, and to a lesser degree 2,3-oxidosqualene, are segregated by digitonin into separate intramembranal pools.(ABSTRACT TRUNCATED AT 250 WORDS)