Dean C. Crick

Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells

Abstract: It is well documented that 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate‐derived product. Recently, it has been shown that free geranylgeraniol (GG‐OH) and farnesol (F‐OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG‐OH and F‐OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post‐translational modification of key regulatory proteins. In this study we report that GG‐OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [3H]thymidine incorporation. The increase in cell number and [3H]thymidine incorporation were significantly lower when F‐OH was added. Under these conditions [3H]mevalonate and [3H]GG‐OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP‐binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase. Analysis of the proteins metabolically labeled by [3H]mevalonate and [3H]GG‐OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG‐OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG‐OH was added to lovastatin‐treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.

Long‐Chain os‐Isoprenyltransferase Activity Is Induced Early in the Developmental Program for Protein N‐Glycosylation in Embryonic Rat Brain Cells

A large developmental increase in Glc3Man9‐ GlcNAc2‐P‐P‐dolichol (Oligo‐P‐P‐Dol) synthesis and protein W‐glycosylation in primary cultures of embryonic rat brain cells has been reported previously. In vitro enzyme studies and metabolic labeling experiments now show that there is a coordinate induction of long‐chain c/s‐iso‐ prenyltransferase (IPTase) activity, an activity required for the chain‐elongation stage of dolichyl monophosphate (Dol‐P) biosynthesis de novo, and Oligo‐P‐P‐Dol biosynthesis in embryonic rat brain. Different developmental patterns were observed for IPTase and |8‐hydroxy‐/3‐methyl‐ glutaryl‐CoA (HMG‐CoA) reductase activity as well as Dol‐ P and cholesterol biosynthesis, indicating that these pathways are regulated independently in rat brain. Three separate experimental approaches provide evidence that the amount of Dol‐P available in the rough endoplasmic reticulum (RER) is a rate‐limiting factor in the expression of the lipid intermediate pathway. First, metabolic labeling experiments show that the biosynthesis of Dol‐P is induced at the same time or just prior to the induction of Oligo‐P‐P‐Dol biosynthesis. Second, the time of induction and rate of Oligo‐P‐P‐Dol synthesis are accelerated when Dol‐P is supplemented in the culture medium. Third, in vitro assays of mannosylphosphoryldolichol synthase and A/‐acetylglucosaminylpyrophosphoryldolichol synthase indicate that there are only minor increases in the levels of these enzymes during development, but the amount of endogenous Dol‐P in the RER that is accessible to the glycosyltransferases increases when IPTase activity is induced. In summary, the current studies with embryonic rat brain cells document the coordinate induction of IPTase activity and Oligo‐P‐P‐Dol synthesis, support the hypothesis that the availability of Dol‐P in the RER is one rate‐limiting factor in Oligo‐P‐P‐Dol synthesis, and strongly suggest that increases in IPTase activity and the rate of de novo Dol‐P biosynthesis enhance the capacity of embryonic rat brain cells for lipid intermediate synthesis early in the developmental program for N‐linked glycoprotein biosynthesis.

Selective Inhibition of Cholesterol Biosynthesis in Brain Cells by Squalestatin 1

Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F‐P‐P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 nM), pig brain (IC50 = 21 nM), and C6 glioma cells (IC50 = 35 nM) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long‐chain cis‐isoprenyltransferase catalyzing the conversion of F‐P‐P to polyprenyl pyrophosphate (Poly‐P‐P), the precursor of dolichyl phosphate (Dol‐P). SQ also had no effect on F‐P‐P synthase; the conversion of [3H]F‐P‐P to geranylgeranyl pyrophosphate (GG‐P‐P) catalyzed by partially purified GG‐P‐P synthase from bovine brain; the enzymatic farnesylation of recombinant H‐p21ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [3H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3–5 µM) but either had no effect or slightly stimulated the labeling of Dol‐P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase. Thus, SQ provides a useful tool for evaluating the obligatory requirement for de novo cholesterol biosynthesis in neurobiological processes without interfering with other critical reactions involving F‐P‐P.

Rapid Identification of Cysteine-Linked Isoprenyl Groups by Metabolic Labeling with [ 3 H]Farnesol and [ 3 H]Geranylgeraniol

The posttranslational modification of proteins by the covalent attachment of farnesyl and geranylgeranyl groups to cysteine residues at or near the C-terminus via a thioether bond is now well established in mammalian cells (1–6). Most isoprenylated proteins are thought to serve as regulators of cell signaling and membrane trafficking. Farnesylation and geranylgeranylation of the cysteinyl residues have been shown to promote both protein–protein and protein–membrane interactions (6–8). Isoprenylation, and, in some cases, the subsequent palmitoylation, provide a mechanism for the membrane association of polypeptides, which lack a transmembrane domain, and appear to be prerequisite for their in vivo activity (6,9,10). Three distinct protein prenyltransferases catalyzing these modifications have been identified (1–5). Two geranylgeranyltransferases (GGTases) have been characterized, and are known to modify distinct protein substrates. The CaaX GGTase (also known as GGTase-1) geranylgeranylates proteins that end in a CaaL(F) sequence, where C is cysteine, a is usually an aliphatic amino acid, and the C-terminal amino acyl group is leucine (L) or phenylalanine (F). Rab GGTase (also known as GGTase-2) catalyzes the attachment of two geranylgeranyl groups to paired carboxyl-terminal cysteines in most members of the Rab family of GTP-binding proteins (11). These proteins terminate in a Cys-Cys, Cys-X-Cys or Cys-Cys-X-X motif, where X is a small hydrophobic amino acid. Another set