RESUMEN
The monoterpenes limonene and perillyl alcohol are effective therapeutic agents against advanced rat mammary cancer. Limonene is currently undergoing clinical testing in cancer patients. These monoterpenes and their oxygenated metabolites have been previously shown to inhibit protein prenylation in cultured cells. Since farnesylation of ras protein is critical for its ability to cause oncogenic transformation, inhibition of protein prenylation may be the basis of the anti-tumor effects of limonene and perillyl alcohol. In this study we test the ability of limonene and its oxygenated analogs to inhibit protein prenylation enzymes in vitro. Limonene and perillyl alcohol and their major in vivo metabolite, perillic acid, are weak inhibitors of both mammalian and yeast protein farnesyl transferase (PFT) and protein geranylgeranyl transferase (PGGT). In contrast, a minor metabolite of both limonene and perillyl alcohol, perillic acid methyl ester, is a potent inhibitor of both enzymes. Perillic acid methyl ester is a competitive inhibitor of yeast PFT with respect to farnesyl pyrophosphate. These studies suggest that if the inhibition of protein prenylation is a mechanism for limonene's and perillyl alcohol's anti-cancer activities, these monoterpenes may be prodrugs that are converted into pharmacologically-active substances by metabolic modification.
Asunto(s)
Monoterpenos , Prenilación de Proteína/efectos de los fármacos , Terpenos/farmacología , Transferasas/antagonistas & inhibidores , Animales , Encéfalo/enzimología , Bovinos , Ciclohexenos , Inhibidores Enzimáticos , Proteínas Fúngicas , Proteínas de Unión al GTP/metabolismo , Técnicas In Vitro , Cinética , Limoneno , Oxidación-Reducción , Saccharomyces cerevisiae/enzimología , Relación Estructura-ActividadRESUMEN
The protein farnesyltransferase (PFT) beta-subunit gene of Saccharomyces cerevisiae, DPR1, was randomly mutagenized by PCR to construct a mutant DPR1 gene library on a high-copy plasmid. The library was screened for suppression of the temperature sensitivity conferred by a mutation in the protein geranylgeranyltransferase type I (PGGT-I) beta-subunit gene, CAL1. A mutant DPR1 gene was identified whose product contained a single amino acid change of Ser-159 to Asn. This mutant gene also suppressed a cal1 disruption even on a low-copy plasmid, suggesting that the product (designated S159N) can substitute for PGGT-I beta subunit in vivo. Its ability to act as a PFT is not drastically reduced, since the mutant gene still complemented a dpr1 disruption. Results of in vitro assays demonstrate that the mutant enzyme has increased activity to farnesylate, a substrate for PGGT-I. On the other hand, the ability to farnesylate its own substrate is reduced. The increased ability to utilize the PGGT-I substrate is due to its increased affinity for the protein substrate. In addition, the mutant enzyme shows a severalfold increase in the sensitivity to a peptidomimetic inhibitor that acts as a competitor of the protein substrate. These results point to the importance of the beta subunit of PFT for the binding of a protein substrate and demonstrate that Ser-159 of DPR1 product is critical for its substrate specificity.
Asunto(s)
Transferasas Alquil y Aril , Saccharomyces cerevisiae/enzimología , Transferasas/metabolismo , Secuencia de Bases , Prueba de Complementación Genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Prenilación de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.