Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that increase the synthesis of fatty acids as well as cholesterol in animal cells. All three SREBP isoforms (SREBP-1a, -1c, and -2) are subject to feedback regulation by cholesterol, which blocks their proteolytic release from membranes. Previous data indicate that the SREBPs are also negatively regulated by unsaturated fatty acids, but the mechanism is uncertain. In the current experiments, unsaturated fatty acids decreased the nuclear content of SREBP-1, but not SREBP-2, in cultured human embryonic kidney (HEK)-293 cells. The potency of unsaturated fatty acids increased with increasing chain length and degree of unsaturation. Oleate, linoleate, and arachidonate were all effective, but the saturated fatty acids palmitate and stearate were not effective. Downregulation occurred at two levels. The mRNAs encoding SREBP-1a and SREBP-1c were markedly reduced, and the proteolytic processing of these SREBPs was inhibited. When SREBP-1a was produced by a cDNA expressed from an independent promoter, unsaturated fatty acids reduced nuclear SREBP-1a without affecting the mRNA level. There was no effect when the cDNA encoded a truncated version that was not membranebound. When administered together, sterols and unsaturated fatty acids potentiated each other in reducing nuclear SREBP-1. In the absence of fatty acids, sterols did not cause a sustained reduction of nuclear SREBP-1, but they did reduce nuclear SREBP-2. We conclude that unsaturated fatty acids, as well as sterols, can downregulate nuclear SREBPs and that unsaturated fatty acids have their greatest inhibitory effects on SREBP-1a and SREBP-1c, whereas sterols have their greatest inhibitory effects on SREBP-2.
Through suppressive subtractive hybridization, we identified a new gene whose transcription is induced by sterol regulatory element-binding proteins (SREBPs). The gene encodes acetyl-CoA synthetase (ACS), the cytosolic enzyme that activates acetate so that it can be used for lipid synthesis or for energy generation. ACS genes were isolated previously from yeast, but not from animal cells. Recombinant human ACS was produced by expressing the cloned cDNA transiently in human cells. After purification by nickel chromatography, the 701-amino acid cytosolic enzyme was shown to function as a monomer. The recombinant enzyme produced acetylCoA from acetate in a reaction that required ATP. As expected for a gene controlled by SREBPs, ACS mRNA was induced when cultured cells were deprived of sterols and repressed by sterol addition. The pattern of regulation resembled the regulation of enzymes of fatty acid synthesis. ACS mRNA was also elevated in livers of transgenic mice that express dominant-positive versions of all three isoforms of SREBP. We conclude that ACS mRNA, and hence the ability of cells to activate acetate, is regulated by SREBPs in parallel with fatty acid synthesis in animal cells.Sterol regulatory element-binding proteins (SREBPs) 1 are transcription factors that activate more than 20 genes that produce enzymes required for the synthesis of cholesterol and unsaturated fatty acids in animal cells (1-3). The SREBPs differ from other transcription factors because they are synthesized as membrane-bound proteins whose active fragments must be released by proteolysis in order to enter the nucleus and activate transcription. The proteolytic release of the nuclear fragments is controlled by the cholesterol content of the cell; release is rapid when cells are depleted of cholesterol, and it is blocked when cholesterol overaccumulates (1).In tissue culture cells and in livers of transgenic mice, nuclear SREBPs (nSREBPs) increase the levels of mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway, including 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase), HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, lanosterol demethylase, and others. In the fatty acid biosynthetic pathway, SREBPs increase the mRNAs encoding acetyl-CoA carboxylase, fatty acid synthetase, and stearoyl-CoA desaturase. As a result of these changes, there is a massive increase in the content of unsaturated fatty acids and cholesterol in livers of transgenic mice that overexpress either of two nuclear isoforms of SREBP (nSREBP-1a and nSREBP-2) (4, 5).In addition to increasing enzymes that participate directly in lipid biosynthesis, the SREBPs increase the mRNA encoding ATP-citrate lyase, which is the major source of the acetyl-CoA that is the ultimate building block for lipid synthesis (6, 7). The SREBPs also increase the mRNAs for three enzymes that supply the NADPH that is needed for lipogenesis (malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) (8).In th...
RhoB, a small GTP-binding protein, was shown previously to contain farnesyl (C-15) as well as geranylgeranyl (C-20) groups (Adamson, P., Marshall, C. J., Hall, A., and Tilbrook, P. A. (1992) J. Biol. Chem. 267, 20033-20038). The COOH-terminal sequence of the protein is CCKVL. According to current rules of prenylation, the COOH-terminal leucine should render the protein a substrate for CAAX geranylgeranyl transferase (GGTase-1), but not for CAAX farnesyltransferase (FTase). To determine the mechanism of farnesylation, we prepared recombinant RhoB and incubated it with recombinant preparations of either FTase or GGTase-1. RhoB was neither farnesylated nor geranylgeranylated efficiently by FTase, but it was farnesylated as well as geranylgeranylated by GGTase-1. The enzyme attached farnesyl more efficiently than geranylgeranyl to RhoB. Neither farnesylation nor geranylgeranylation required the cysteine at the fifth position from the COOH terminus. However, replacement of the cysteine at the fourth position abolished attachment of both prenyl groups. We conclude that the previously observed farnesylation of RhoB is attributable to the FTase activity of GGTase-1. These data, and other accumulating data, indicate that GGTase-1 is a highly unusual enzyme that efficiently transfers both farnesyl and geranylgeranyl groups and that the choice of prenyl group is dictated by the nature of the protein acceptor.
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