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Novel Salvage Pathway Utilizing Farnesol and Geranylgeraniol for Protein Isoprenylation
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
237, 483–487 (1997)
RC977145
BREAKTHROUGHS AND VIEWS Novel Salvage Pathway Utilizing Farnesol and Geranylgeraniol for Protein Isoprenylation Dean C. Crick, Douglas A. Andres, and Charles J. Waechter1 Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536
Received July 14, 1997
INTRODUCTION AND BACKGROUND Since Glomset and coworkers first detected the incorporation of a mevalonate-derived intermediate into protein in Swiss 3T3 cells in 1984 (1), the enzymology and function of protein isoprenylation has been an active area of research. In the past seven years, studies from many contributors have described the structures and properties of protein farnesyltransferase (FTaseI), protein geranylgeranyltransferase I (GGTase-I) and the Rab geranylgeranyltransferase type II (GGTaseII), the three major enzymes catalyzing the farnesylation and geranylgeranylation of proteins in mammalian cells and lower eukaryotic organisms. In these post-translational modifications t,t,-farnesyl pyrophosphate (F-P-P) and t,t,t-geranylgeranyl pyrophosphate (GG-P-P) serve as the isoprenyl donors. Several excellent reviews have followed the progress in understanding the structure of the lipophilic modification, the enzymes catalyzing the covalent attachment of the thio ether-linked isoprenyl groups and the function of the post-translational modification (2-6). More recently, metabolic labeling experiments have provided evidence that mammalian cells can utilize free geranylgeraniol (GG-OH) for protein isoprenylation and free farnesol (F-OH) for sterol biosynthesis and protein isoprenylation (7-9). These studies represent an important new development in the field of isoprenoid biosynthesis, and indicate that mammalian cells are capable of synthesizing F-P-P and GG-P-P by a ‘‘salvage pathway’’ in addition to the conventional de novo biosynthetic route via mevalonate (Figure 1, ref. 10). In this article the metabolic steps in the conversion of F-OH and GG-OH to the pyrophosphate intermediates or novel isoprenyl donors is referred to as a ‘‘sal1 To whom correspondence should be addressed. Fax: (606) 3231037. E-mail: [email protected].
vage pathway’’ since it is quite likely that the free isoprenols are derived from dephosphorylation of preformed F-P-P and GG-P-P and possibly by degradation of isoprenylated proteins, and not by de novo biosynthesis of the free isoprenol. The objective of this minireview is to summarize the recent literature documenting the metabolic utilization of the allylic isoprenols, and to speculate about possible mechanism(s) for the conversion of GG-OH and F-OH to the respective allylic pyrophosphate intermediates, or perhaps novel isoprenyl donors, and the physiological significance of the salvage pathway. EVIDENCE FOR THE UTILIZATION OF GERANYLGERANIOL FOR PROTEIN ISOPRENYLATION Metabolic labeling experiments in C6 glioma cells published in 1994 provided the first direct proof for the utilization of GG-OH for the geranylgeranylation of cysteine residues of proteins in the size range of small GTP-binding proteins in animal cells (7). Virtually no radioactivity was incorporated into CoQ, as would be expected if t,t,t-[3H]GG-OH were converted to t,t,t,-[3H]GG-P-P (see Figure 1). It is possible that GG-P-P is produced by the salvage pathway in a compartment that is not accessible to the trans-isoprenyltransferases involved in CoQ biosynthesis or that the GG-P-P pools are are small and the affinity of GGTases I and II for GG-P-P is considerably higher than the trans-isoprenyltransferases in vivo. If a mechanism existed for the conversion of t,t,cGG-OH to t,t,c-GG-P-P, this metabolite would provide a selective isotopic precursor for dolichyl phosphate biosynthesis (Figure 1). However, comparative metabolic labeling experiments with this stereoisomer of GG-OH in C6 glial cells indicated that t,t,c-GG-OH does not serve as a precursor for dolichyl phosphate,
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FIG. 1. Proposed Metabolic Relationship Between Salvage Pathways for F-OH and GG-OH and the Conventional Biosynthetic Routes for Isoprenoid Biosynthesis. F-OH Å farnesol; GG-OH Å geranylgeraniol; F-P-P Å farnesyl pyrophosphate; GG-P-P Å geranylgeranyl pyrophosphate; SQ1 Å squalestatin 1, NTP Å nucleoside triphosphate; white arrows denote proposed enzymatic reactions that have not been extensively characterized.
and it was not incorporated into protein (D. C. Crick, B. Mayer and C. J. Waechter, unpublished observations). The apparent lack of a salvage mechanism for t,t,c-GGOH suggests that the a-isoprene unit with a transdouble bond, present in t,t,-F-OH and t,t,t-GG-OH, is recognized by the enzyme catalyzing the initial reaction in the ‘‘activation’’ process. It is also unlikely that an isomerase exists that catalyzes the conversion of t,t,c-GG-OH to the all-trans stereoisomer. Another plausible explanation for the failure to detect metabolically labeled CoQ and dolichyl phosphate after incubating C6 cells with t,t,t-[3H]GG-OH or t,t,c[3H]GG-OH is that CoQ and Dol-P biosynthesis is only initiated with F-P-P. Although t,t,t,GG-P-P and t,t,cGG-P-P are formally considered to be intermediates, they may only occur transiently tightly bound to the respective isoprenyltransferase. Nevertheless, a number of additional studies with unlabeled and radiolabeled t,t,t,-GG-OH have been reported in the last three years to corroborate a mecha-
nism for the ‘‘re-activation’’ of GG-OH and its incorporation into protein (see below). EVIDENCE FOR THE UTILIZATION OF FARNESOL FOR STEROL BIOSYNTHESIS AND PROTEIN ISOPRENYLATION IN MAMMALIAN CELLS The first direct evidence for a biosynthetic role for FOH in sterol biosynthesis was obtained from metabolic labeling studies in the microalga, Botryococcus braunii (11). More recently, two studies have documented that F-OH can be used as a precursor for sterol biosynthesis and protein isoprenylation in mammalian cells. Fliesler and Keller showed that when [3H]F-OH is injected into rat eyes, radioactivity is incorporated into cholesterol (8). It appeared that in retinas the free alcohol was initially converted to F-P-P and then metabolized by the conventional pathway (Figure 1, ref. 10) since the labeling of sterol was blocked by the squalene epoxidase inhibitor, NB-598.
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In 1995, Crick et al. (9) also found that radioactivity was incorporated into sterol and cysteine residues of proteins when rat C6 glial cells and African green monkey kidney cells (CV-1) were incubated with [3H]F-OH. A lipid, that was chromatographically similar to CoQ with a side-chain consisting of 9-10 isoprene units, was also recovered, but subsequent analyses indicated that the compound either had a much shorter side-chain or was not CoQ (Crick and Waechter, unpublished observation). Virtually no [3H]GG-cysteine was detected in the C6 and CV-1 protein fractions after incubation with [3H]FOH. Thus, any F-P-P, or another yet to be characterized farnesyl donor (F-X), formed from F-OH is either not converted to GG-P-P (or GG-X) at a significant rate or may be converted to the geranylgeranyl donor in a compartment lacking GGTases I and II and/or the corresponding protein substrates. It is also puzzling that dolichyl phosphate and CoQ were not metabolically labeled when C6 and CV-1 cells were incubated with [3H]F-OH since F-P-P is a common intermediate in these biosynthetic pathways (Figure 1, ref. 10). PHYSIOLOGICAL SIGNIFICANCE FOR A SALVAGE PATHWAY FOR GG-OH AND F-OH Metabolically labeled F-OH was detected a number of years ago when ascites tumor cells (12) and human platelets (13) were incubated with [14C]mevalonic acid, and there is now substantial evidence that the isoprenol plays a physiological role in the regulated proteolysis of HMG-CoA reductase (14-16). Evidence for the physiological relevance of the salvage pathway for FOH and GG-OH has been obtained from studies designed to determine if HMG-CoA reductase inhibitors interfere with cell cycle progression due to the block in F-P-P or GG-P-P biosynthesis, and consequently the loss of protein isoprenylation. The experimental strategy of these experiments, illustrated in Figure 1, is based on the discovery that mammalian cells utilize exogenously supplied F-OH and GG-OH for protein isoprenylation, coupled with the observation that little if any F-P-P formed from FOH is converted to GG-P-P (see above). Therefore these alternate routes for the biosynthesis of the isoprenyl donors provide a simple means of restoring either protein farnesylation or geranylgeranylation selectively by supplementing the culture medium with the appropriate isoprenol. The utilization of GG-OH for protein geranylgeranylation has provided insight into the intriguing question of which mevalonate-derived product(s) is responsible for the inhibitory effects of HMG-CoA reductase inhibitors on DNA replication and cell proliferation (17, 18). In early reports, GG-OH, as well as mevalonate, restored cell growth in simvastatin-
treated human arterial (19) and bronchial smooth muscle cells (20). In two later related studies, GGOH and mevalonate, but not F-OH, were shown to restore DNA synthesis in lovastatin-treated NIH 3T3 cells (21) and C6 glioma cells (22). Convincing evidence for the role of geranylgeranylated proteins in the control of cell proliferation was also obtained with the CHO mutant clone, UT-2, which has a genetic defect in HMG-CoA reductase (23). UT2 cells, previously shown to be ‘‘auxotrophic’’ for mevalonate, were stimulated to re-enter the cell cycle when cultured in the presence of 5-10 mM GG-OH, but not similar concentrations of F-OH (24). When 10 mM GGOH was supplemented in the culture medium, C6 glial (22) and UT-2 cells (24) divided at least 3 times before cell division was arrested. Failure to continue to divide could be due to the conversion of GG-OH to inhibitory metabolites or the depletion of another essential mevalonate-derived compound. Under these conditions radiolabeled GG-OH was shown to be incorporated into a set of polypeptides in the size range (19-27 kDa) of small GTP-binding proteins. All of these studies are consistent with the idea that the effects of HMG-CoA reductase inhibitors on DNA replication and cell growth are due to a block in GG-P-P synthesis and the inability to geranylgeranylate key regulatory proteins. Different experimental approaches have also been used to implicate the geranylgeranylated proteins, Rho, Rac and CDC42, in the regulation of cell cycle progression (25-27). In another study documenting the physiological relevance of the salvage pathway, exogenous GG-OH and not F-OH, overcame the block on the tyrosine-phosphorylation of the PDGF receptor imposed by lovastatin (28), presumably by restoring protein geranylgeranylation. Similarly, Finder et al. (29) restored protein geranylgeranylation to lovastatin-treated pulmonary artery smooth muscle cells by GG-OH supplementation, implicating one or more geranylgeranylated proteins in the regulation of nitric-oxide synthase expression by IL-1b. The results of the experiments summarized above indicate that the salvage pathway provides an alternate route for the biosynthesis of physiological levels of GG-P-P, and probably F-P-P. Moreover, since F-OH was unable to substitute effectively for GG-OH in these cell studies, the mevalonate requirement is not satisfied by only restoring protein farnesylation. SPECULATION ON THE MECHANISM FOR THE ‘‘ACTIVATION’’ OF F-OH AND GG-OH As noted above, the formation of free F-OH in mammalian cells was reported over thirty years ago (12). FOH and GG-OH could be produced in mammalian cells by the action of rat liver microsomal phosphatases (30)
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and possibly the turnover of isoprenylated proteins. With respect to the latter possibility pig liver microsomes catalyze the S-oxidation of farnesyl-cysteine (31). The observation that the incorporation of [3H]farnesol into sterol in C6 glioma cells was effectively blocked by squalestatin 1 (Figure 1), a potent inhibitor of squalene synthetase (32-36), suggests that the free isoprenol is converted to F-P-P prior to being utilized for sterol biosynthesis, and probably protein isoprenylation. The allylic isoprenols, F-OH and GG-OH, could plausibly be ‘‘re-activated’’ by their conversion to the respective pyrophosphate intermediates by a (pyro)phosphorylation system. This could occur mechanistically as two successive phosphoryl transfer reactions, similar to the formation of mevalonate pyrophosphate (10), or a single pyrophosphoryl transfer. The CTP-mediated phosphorylation of the long-chain polyisoprenol, dolichol, has been thoroughly characterized in microsomal fractions from several animal tissues (37-39). A CTP-mediated F-OH kinase activity has been reported for a 100,0001g pellet from the microalga, Botryococcus braunii (40). Under these in vitro conditions a very small amount of F-P-P was also detected. Membrane fractions from the archaebacterium, Sulfolobus acidocaldarius, catalyzed the monophosphorylation of GG-OH in the presence of ATP (41). UTP, CTP and GTP could substitute for ATP as phosphoryl donors in this system. The conversion of GG-P to GG-P-P was catalyzed by cytosolic fractions, but not crude microsomes. A preliminary report of the CTP-mediated conversion of F-OH to F-P-P by membrane fractions from rat liver has appeared recently (42). The possibility that t,t,-F-OH and t,t,t-GG-OH are incorporated into proteins via novel, previously uncharacterized ‘‘activated’’ isoprenyl donors can not yet be excluded. Based on their failure to detect metabolically labeled GG-P-P when NIH 3T3 cells were incubated with [3H]GG-OH, McGuire and Sebti (43) concluded that GG-OH could be converted to GG-X, an unidentified geranylgeranyl donor. However, since it was not demonstrated that metabolically labeled GGP-P could be detected with the same protocol when the cells were incubated with radiolabeled mevalonate, the formation of a novel isoprenyl donor remains to be established. GG-OH may also be converted to metabolites that are responsible for arresting cell division after extended periods of time in culture (24). Ohizumi et al. (44) have reported that relatively high concentrations (50 mM) of GG-OH induce apoptosis in human leukemia HL-60 cells, and oxidative pathways for F-OH metabolism are well documented (45-47). PROSPECTUS There is now convincing evidence for a quantitatively and physiologically significant salvage pathway that
provides a mechanism for the utilization of F-OH and GG-OH for sterol biosynthesis and protein isoprenylation. It will now be important to learn more about the enzymatic details of how the free isoprenols are converted to the pyrophosphate intermediates or novel isoprenyl donors, the subcellular locations of these enzymes and the relative contribution of enzymatic dephosphorylation of the allylic pyrophosphates, extracellular sources and perhaps the turnover of isoprenylated proteins to the intracellular F-OH and GG-OH pools. Also considering the ability of F-OH and GG-OH to serve as precursors for protein isoprenylation and the role of F-OH in the regulated proteolysis of HMGCoA reductase, more information is needed on the factors controlling the size of the intracellular pools of these free isoprenols. In view of the limited solubility of the isoprenols, particularly GG-OH, in aqueous solutions, carrier proteins may exist and significant amounts may be imbedded in cellular membranes. Finally, the observation that F-OH can be utilized for sterol biosynthesis and protein farnesylation, but is apparently not converted to GG-P-P, dolichyl phosphate or CoQ, raises interesting questions regarding the compartmentalization of the various branches of isoprenoid metabolism. ACKNOWLEDGMENTS The work cited by the authors was supported by NIH Grant GM 36065 (C.J.W.) and EY11231 (D.A.A.).
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15. Bradfute, D. L., and Simoni, R. D. (1994) J. Biol. Chem. 269, 6645–6650. 16. Meigs, T. E., Roseman, D. S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 7916–7922. 17. Quesney-Huneeus, V., Wiley, M. H., and Siperstein, M. D. (1979) Proc. Natl. Acad. Sci. USA 76, 5056–5060. 18. Habenicht, A. J. R., Glomset, J. A., and Ross, R. (1980) J. Biol. Chem. 255, 5134–5140. 19. Corsini, A., Mazzotti, M., Raiteri, M., Soma, M. R., Gabbiani, G., Fumagalli, R., and Paolettii, R. (1993) Atherosclerosis 101, 117– 125. 20. Vigano, T., Hernandez, A., Corsini, A., Granata, A., Belloni, P., Fumagalli, R., Paoletti, R., and Folco, G. (1995) Eur. J. Pharm. 291, 201–203. 21. Vogt, A., Qian, Y., McGuire, T. F., Hamilton, A. D., and Sebti, S. M. (1996) Oncogene 13, 1991–1999. 22. Crick, D. C., Waechter, C. J., and Andres, D. A. (1996) SAAS Bull. Biochem. Biotech. 9, 37–42. 23. Mosley, S. T., Brown, M. S., Anderson, R. G. W., and Goldstein, J. L. (1983) J. Biol. Chem. 258, 13875–13881. 24. Crick, D. C., Andres, D. A., and Waechter, C. J. (1997) Exp. Cell Res. 231, 302–307. 25. Olson, M. F., Ashworth, A., and Hall, A. (1995) Science 269, 1270–1272. 26. Symons, M. (1996) TIBS 21, 178–181. 27. Hirai, A., Nakamura, S., Noguchi, Y., Yasuda, T., Kitagawa, M., Tatsuno, I., Oeda, T., Tahara, K., Terano, T., Narumiya, S., Kohn, L. D., and Saito, Y. (1997) J. Biol. Chem. 272, 13–16. 28. McGuire, T. F., Qian,Y., Vogt, A., Hamilton, A. D., and Sebti, S. (1996) J. Biol. Chem. 271, 27402–27407. 29. Finder, J. D., Litz, J. L., Blaskovich, M. A., McQuire, T. F., Qian, Y., Hamilton, A. D., Davies, and Sebti, S. (1997) J. Biol. Chem. 272, 13484–13486. 30. Bansal, V., and Vaidya, S. (1994) Arch. Biochem. Biophys. 315, 393–399. 31. Park, S. B., Howald, W. N., and Cashman, J. R. (1994) Chem. Res. Toxicol. 7, 191–198. 32. Baxter, A., Fitzgerald, B. J., Hutson, J. L., McCarthy, A. D., Mot-
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RC977145
BREAKTHROUGHS AND VIEWS Novel Salvage Pathway Utilizing Farnesol and Geranylgeraniol for Protein Isoprenylation Dean C. Crick, Douglas A. Andres, and Charles J. Waechter1 Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536
Received July 14, 1997
INTRODUCTION AND BACKGROUND Since Glomset and coworkers first detected the incorporation of a mevalonate-derived intermediate into protein in Swiss 3T3 cells in 1984 (1), the enzymology and function of protein isoprenylation has been an active area of research. In the past seven years, studies from many contributors have described the structures and properties of protein farnesyltransferase (FTaseI), protein geranylgeranyltransferase I (GGTase-I) and the Rab geranylgeranyltransferase type II (GGTaseII), the three major enzymes catalyzing the farnesylation and geranylgeranylation of proteins in mammalian cells and lower eukaryotic organisms. In these post-translational modifications t,t,-farnesyl pyrophosphate (F-P-P) and t,t,t-geranylgeranyl pyrophosphate (GG-P-P) serve as the isoprenyl donors. Several excellent reviews have followed the progress in understanding the structure of the lipophilic modification, the enzymes catalyzing the covalent attachment of the thio ether-linked isoprenyl groups and the function of the post-translational modification (2-6). More recently, metabolic labeling experiments have provided evidence that mammalian cells can utilize free geranylgeraniol (GG-OH) for protein isoprenylation and free farnesol (F-OH) for sterol biosynthesis and protein isoprenylation (7-9). These studies represent an important new development in the field of isoprenoid biosynthesis, and indicate that mammalian cells are capable of synthesizing F-P-P and GG-P-P by a ‘‘salvage pathway’’ in addition to the conventional de novo biosynthetic route via mevalonate (Figure 1, ref. 10). In this article the metabolic steps in the conversion of F-OH and GG-OH to the pyrophosphate intermediates or novel isoprenyl donors is referred to as a ‘‘sal1 To whom correspondence should be addressed. Fax: (606) 3231037. E-mail: [email protected].
vage pathway’’ since it is quite likely that the free isoprenols are derived from dephosphorylation of preformed F-P-P and GG-P-P and possibly by degradation of isoprenylated proteins, and not by de novo biosynthesis of the free isoprenol. The objective of this minireview is to summarize the recent literature documenting the metabolic utilization of the allylic isoprenols, and to speculate about possible mechanism(s) for the conversion of GG-OH and F-OH to the respective allylic pyrophosphate intermediates, or perhaps novel isoprenyl donors, and the physiological significance of the salvage pathway. EVIDENCE FOR THE UTILIZATION OF GERANYLGERANIOL FOR PROTEIN ISOPRENYLATION Metabolic labeling experiments in C6 glioma cells published in 1994 provided the first direct proof for the utilization of GG-OH for the geranylgeranylation of cysteine residues of proteins in the size range of small GTP-binding proteins in animal cells (7). Virtually no radioactivity was incorporated into CoQ, as would be expected if t,t,t-[3H]GG-OH were converted to t,t,t,-[3H]GG-P-P (see Figure 1). It is possible that GG-P-P is produced by the salvage pathway in a compartment that is not accessible to the trans-isoprenyltransferases involved in CoQ biosynthesis or that the GG-P-P pools are are small and the affinity of GGTases I and II for GG-P-P is considerably higher than the trans-isoprenyltransferases in vivo. If a mechanism existed for the conversion of t,t,cGG-OH to t,t,c-GG-P-P, this metabolite would provide a selective isotopic precursor for dolichyl phosphate biosynthesis (Figure 1). However, comparative metabolic labeling experiments with this stereoisomer of GG-OH in C6 glial cells indicated that t,t,c-GG-OH does not serve as a precursor for dolichyl phosphate,
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FIG. 1. Proposed Metabolic Relationship Between Salvage Pathways for F-OH and GG-OH and the Conventional Biosynthetic Routes for Isoprenoid Biosynthesis. F-OH Å farnesol; GG-OH Å geranylgeraniol; F-P-P Å farnesyl pyrophosphate; GG-P-P Å geranylgeranyl pyrophosphate; SQ1 Å squalestatin 1, NTP Å nucleoside triphosphate; white arrows denote proposed enzymatic reactions that have not been extensively characterized.
and it was not incorporated into protein (D. C. Crick, B. Mayer and C. J. Waechter, unpublished observations). The apparent lack of a salvage mechanism for t,t,c-GGOH suggests that the a-isoprene unit with a transdouble bond, present in t,t,-F-OH and t,t,t-GG-OH, is recognized by the enzyme catalyzing the initial reaction in the ‘‘activation’’ process. It is also unlikely that an isomerase exists that catalyzes the conversion of t,t,c-GG-OH to the all-trans stereoisomer. Another plausible explanation for the failure to detect metabolically labeled CoQ and dolichyl phosphate after incubating C6 cells with t,t,t-[3H]GG-OH or t,t,c[3H]GG-OH is that CoQ and Dol-P biosynthesis is only initiated with F-P-P. Although t,t,t,GG-P-P and t,t,cGG-P-P are formally considered to be intermediates, they may only occur transiently tightly bound to the respective isoprenyltransferase. Nevertheless, a number of additional studies with unlabeled and radiolabeled t,t,t,-GG-OH have been reported in the last three years to corroborate a mecha-
nism for the ‘‘re-activation’’ of GG-OH and its incorporation into protein (see below). EVIDENCE FOR THE UTILIZATION OF FARNESOL FOR STEROL BIOSYNTHESIS AND PROTEIN ISOPRENYLATION IN MAMMALIAN CELLS The first direct evidence for a biosynthetic role for FOH in sterol biosynthesis was obtained from metabolic labeling studies in the microalga, Botryococcus braunii (11). More recently, two studies have documented that F-OH can be used as a precursor for sterol biosynthesis and protein isoprenylation in mammalian cells. Fliesler and Keller showed that when [3H]F-OH is injected into rat eyes, radioactivity is incorporated into cholesterol (8). It appeared that in retinas the free alcohol was initially converted to F-P-P and then metabolized by the conventional pathway (Figure 1, ref. 10) since the labeling of sterol was blocked by the squalene epoxidase inhibitor, NB-598.
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In 1995, Crick et al. (9) also found that radioactivity was incorporated into sterol and cysteine residues of proteins when rat C6 glial cells and African green monkey kidney cells (CV-1) were incubated with [3H]F-OH. A lipid, that was chromatographically similar to CoQ with a side-chain consisting of 9-10 isoprene units, was also recovered, but subsequent analyses indicated that the compound either had a much shorter side-chain or was not CoQ (Crick and Waechter, unpublished observation). Virtually no [3H]GG-cysteine was detected in the C6 and CV-1 protein fractions after incubation with [3H]FOH. Thus, any F-P-P, or another yet to be characterized farnesyl donor (F-X), formed from F-OH is either not converted to GG-P-P (or GG-X) at a significant rate or may be converted to the geranylgeranyl donor in a compartment lacking GGTases I and II and/or the corresponding protein substrates. It is also puzzling that dolichyl phosphate and CoQ were not metabolically labeled when C6 and CV-1 cells were incubated with [3H]F-OH since F-P-P is a common intermediate in these biosynthetic pathways (Figure 1, ref. 10). PHYSIOLOGICAL SIGNIFICANCE FOR A SALVAGE PATHWAY FOR GG-OH AND F-OH Metabolically labeled F-OH was detected a number of years ago when ascites tumor cells (12) and human platelets (13) were incubated with [14C]mevalonic acid, and there is now substantial evidence that the isoprenol plays a physiological role in the regulated proteolysis of HMG-CoA reductase (14-16). Evidence for the physiological relevance of the salvage pathway for FOH and GG-OH has been obtained from studies designed to determine if HMG-CoA reductase inhibitors interfere with cell cycle progression due to the block in F-P-P or GG-P-P biosynthesis, and consequently the loss of protein isoprenylation. The experimental strategy of these experiments, illustrated in Figure 1, is based on the discovery that mammalian cells utilize exogenously supplied F-OH and GG-OH for protein isoprenylation, coupled with the observation that little if any F-P-P formed from FOH is converted to GG-P-P (see above). Therefore these alternate routes for the biosynthesis of the isoprenyl donors provide a simple means of restoring either protein farnesylation or geranylgeranylation selectively by supplementing the culture medium with the appropriate isoprenol. The utilization of GG-OH for protein geranylgeranylation has provided insight into the intriguing question of which mevalonate-derived product(s) is responsible for the inhibitory effects of HMG-CoA reductase inhibitors on DNA replication and cell proliferation (17, 18). In early reports, GG-OH, as well as mevalonate, restored cell growth in simvastatin-
treated human arterial (19) and bronchial smooth muscle cells (20). In two later related studies, GGOH and mevalonate, but not F-OH, were shown to restore DNA synthesis in lovastatin-treated NIH 3T3 cells (21) and C6 glioma cells (22). Convincing evidence for the role of geranylgeranylated proteins in the control of cell proliferation was also obtained with the CHO mutant clone, UT-2, which has a genetic defect in HMG-CoA reductase (23). UT2 cells, previously shown to be ‘‘auxotrophic’’ for mevalonate, were stimulated to re-enter the cell cycle when cultured in the presence of 5-10 mM GG-OH, but not similar concentrations of F-OH (24). When 10 mM GGOH was supplemented in the culture medium, C6 glial (22) and UT-2 cells (24) divided at least 3 times before cell division was arrested. Failure to continue to divide could be due to the conversion of GG-OH to inhibitory metabolites or the depletion of another essential mevalonate-derived compound. Under these conditions radiolabeled GG-OH was shown to be incorporated into a set of polypeptides in the size range (19-27 kDa) of small GTP-binding proteins. All of these studies are consistent with the idea that the effects of HMG-CoA reductase inhibitors on DNA replication and cell growth are due to a block in GG-P-P synthesis and the inability to geranylgeranylate key regulatory proteins. Different experimental approaches have also been used to implicate the geranylgeranylated proteins, Rho, Rac and CDC42, in the regulation of cell cycle progression (25-27). In another study documenting the physiological relevance of the salvage pathway, exogenous GG-OH and not F-OH, overcame the block on the tyrosine-phosphorylation of the PDGF receptor imposed by lovastatin (28), presumably by restoring protein geranylgeranylation. Similarly, Finder et al. (29) restored protein geranylgeranylation to lovastatin-treated pulmonary artery smooth muscle cells by GG-OH supplementation, implicating one or more geranylgeranylated proteins in the regulation of nitric-oxide synthase expression by IL-1b. The results of the experiments summarized above indicate that the salvage pathway provides an alternate route for the biosynthesis of physiological levels of GG-P-P, and probably F-P-P. Moreover, since F-OH was unable to substitute effectively for GG-OH in these cell studies, the mevalonate requirement is not satisfied by only restoring protein farnesylation. SPECULATION ON THE MECHANISM FOR THE ‘‘ACTIVATION’’ OF F-OH AND GG-OH As noted above, the formation of free F-OH in mammalian cells was reported over thirty years ago (12). FOH and GG-OH could be produced in mammalian cells by the action of rat liver microsomal phosphatases (30)
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and possibly the turnover of isoprenylated proteins. With respect to the latter possibility pig liver microsomes catalyze the S-oxidation of farnesyl-cysteine (31). The observation that the incorporation of [3H]farnesol into sterol in C6 glioma cells was effectively blocked by squalestatin 1 (Figure 1), a potent inhibitor of squalene synthetase (32-36), suggests that the free isoprenol is converted to F-P-P prior to being utilized for sterol biosynthesis, and probably protein isoprenylation. The allylic isoprenols, F-OH and GG-OH, could plausibly be ‘‘re-activated’’ by their conversion to the respective pyrophosphate intermediates by a (pyro)phosphorylation system. This could occur mechanistically as two successive phosphoryl transfer reactions, similar to the formation of mevalonate pyrophosphate (10), or a single pyrophosphoryl transfer. The CTP-mediated phosphorylation of the long-chain polyisoprenol, dolichol, has been thoroughly characterized in microsomal fractions from several animal tissues (37-39). A CTP-mediated F-OH kinase activity has been reported for a 100,0001g pellet from the microalga, Botryococcus braunii (40). Under these in vitro conditions a very small amount of F-P-P was also detected. Membrane fractions from the archaebacterium, Sulfolobus acidocaldarius, catalyzed the monophosphorylation of GG-OH in the presence of ATP (41). UTP, CTP and GTP could substitute for ATP as phosphoryl donors in this system. The conversion of GG-P to GG-P-P was catalyzed by cytosolic fractions, but not crude microsomes. A preliminary report of the CTP-mediated conversion of F-OH to F-P-P by membrane fractions from rat liver has appeared recently (42). The possibility that t,t,-F-OH and t,t,t-GG-OH are incorporated into proteins via novel, previously uncharacterized ‘‘activated’’ isoprenyl donors can not yet be excluded. Based on their failure to detect metabolically labeled GG-P-P when NIH 3T3 cells were incubated with [3H]GG-OH, McGuire and Sebti (43) concluded that GG-OH could be converted to GG-X, an unidentified geranylgeranyl donor. However, since it was not demonstrated that metabolically labeled GGP-P could be detected with the same protocol when the cells were incubated with radiolabeled mevalonate, the formation of a novel isoprenyl donor remains to be established. GG-OH may also be converted to metabolites that are responsible for arresting cell division after extended periods of time in culture (24). Ohizumi et al. (44) have reported that relatively high concentrations (50 mM) of GG-OH induce apoptosis in human leukemia HL-60 cells, and oxidative pathways for F-OH metabolism are well documented (45-47). PROSPECTUS There is now convincing evidence for a quantitatively and physiologically significant salvage pathway that
provides a mechanism for the utilization of F-OH and GG-OH for sterol biosynthesis and protein isoprenylation. It will now be important to learn more about the enzymatic details of how the free isoprenols are converted to the pyrophosphate intermediates or novel isoprenyl donors, the subcellular locations of these enzymes and the relative contribution of enzymatic dephosphorylation of the allylic pyrophosphates, extracellular sources and perhaps the turnover of isoprenylated proteins to the intracellular F-OH and GG-OH pools. Also considering the ability of F-OH and GG-OH to serve as precursors for protein isoprenylation and the role of F-OH in the regulated proteolysis of HMGCoA reductase, more information is needed on the factors controlling the size of the intracellular pools of these free isoprenols. In view of the limited solubility of the isoprenols, particularly GG-OH, in aqueous solutions, carrier proteins may exist and significant amounts may be imbedded in cellular membranes. Finally, the observation that F-OH can be utilized for sterol biosynthesis and protein farnesylation, but is apparently not converted to GG-P-P, dolichyl phosphate or CoQ, raises interesting questions regarding the compartmentalization of the various branches of isoprenoid metabolism. ACKNOWLEDGMENTS The work cited by the authors was supported by NIH Grant GM 36065 (C.J.W.) and EY11231 (D.A.A.).
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