Definition
Prenylated proteins are modified by formation of cysteine thioethers with the isoprenoid lipids, farnesol (C-15) or geranylgeranyl (C-20), at the carboxy terminus. Geranylgeranylation and farnesylation are catalyzed by the enzymes geranylgeranyltransferase (GGTase) and farnesyltransferase (FTase), respectively. The cysteines from CAAX motif of their substrates are modified by these enzymes.
Discovery
Dursina B et al., in 2006 developed two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1} pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. They demonstrated that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, they developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, they found that, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase 1. Numerous effective and specific inhibitors of FTase and GGTase I are also known 2,3.
Structural Characteristics
FTase and GGTase-I are α, β heterodimeric enzymes, share the same α subunit with a molecular mass of 48 kDa. Beese and co-workers have extensively investigated the CAAX requirements on the substrate by FTase and GGTase-I enzyme. The FTase specificity pocket is more polar and accepts Met (hydrophobic AA), Ser (small AA), and Glu (polar AA) and to a lesser extent Ala, Thr, and Cys (small AA) through a specific network of electrostatic interactions. Interestingly, Phe is too bulky for the specificity pocket, but is still tolerated by binding in an adjacent hydrophobic cavity. In GGTase-I, the crystal structure revealed only one X-binding site that discriminates against polar, charged, and small amino acids. X is occupied by hydrophobic amino acids such as Leu or Phe and occasionally by Ile or Val. Both FPP and GGPP display distinct lipid substrate specificities in vivo. This can be explained by the amino acids that occupy the bottom of their lipid binding site. In GGTase-I and RabGGTase, residues 49β and 48β, respectively, are always small amino acids like Thr or Ser, whereas the corresponding residue is a Trp in FTase (W102β) 4,5. In GGTase-I, the crystal structure revealed only one X-binding site that discriminates against polar, charged, and small amino acids. X is occupied by hydrophobic amino acids such as Leu or Phe and occasionally by Ile or Val. The studies by Beese and co-workers suggest that the CAAX peptide is anchored to FTase/GGTase-I through the coordination of the cysteine thiol by Zn2+ and through water-mediated hydrogen bondings of the C-terminal carboxyl group. As a result, the enzymes are capable of discriminating against peptides that are too long or too short, or that lack a cysteine residue 6.
Mode of Action
Prenylation of protein is an important posttranslational modification, which is required for cellular localization and biological function of small G-proteins. The addition of isoprenoid lipid farnesyl or geranylgeranyl is mediated by the enzymes farnesyltransferase (FTase) and geranylgeranyl transferase (GGTase), respectively. Lovastatin and related drugs are inhibitors of HMG-CoA reductase, an early and rate-limiting enzyme in the sterol synthesis pathway. The inhibitors reduce the level of isoprenoids including geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) by depleting cellular pools of the precursors, which are substrates for GGTase and FTase, respectively. Several studies have shown that lovastatin as well as other HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) inhibitors inhibit both geranylgeranylation and farnesylation in various cell types. Both enzymes modify cysteines of proteins that end with the motif CAAX (C is Cys, A is an aliphatic amino acid, X is any amino acid) at their carboxyl terminal. GGTase prefers leucine or isoleucine in the X position, whereas FTase RhoA/RhoA kinase pathway promotes endothelial cell survival 2 serine or methionine. GGTI-298 and FTI-277 (the isoprenoid inhibitors) are CAAX peptidomimetics that potently and selectively inhibit GGTase I and FTase, respectively. Prenylation is required for proper subcellular localization and biological function of these proteins 7.
The two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively display a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases 1.
Functions
GGTase-I inhibitors (GTIs) have demonstrated efficacy in pre-clinical models of tumor progression and show promise in the treatment of smooth muscle hyperplasia 7.
Countering parasitic infections, GGTase-I has also been proposed as a target for countering parasitic infections such as malaria by selective inhibition of the parasite enzyme 7.
Biological functions, protein prenylation has been shown to be involved in cell adhesion, cell proliferation, malignant transformation, and cell survival 2.
Prenylation of small G-proteins with farnesyl or geranylgeranyl groups is essential for their localization to cell membranes and hence for their biological functions. RhoA is geranylgeranylated, whereas H-Ras is selectively farnesylated 6.
References
1. Dursina B, Reents R, Delon C, Wu Y, Kulharia M, Thutewohl M, Veligodsky A, Kalinin A, Evstifeev V, Ciobanu D, Szedlacsek SE, Waldmann H, Goody RS, Alexandrov K (2006). Identification and Specificity Profiling of Protein Prenyltransferase Inhibitors Using New Fluorescent Phosphoisoprenoids . Am. Chem. Soc., 128(9):2822-2835.
2. Hamilton AD, Sebti SM (2000). Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies. Oncogene, 19(56):6584-6593.
3. El Oualid F, Cohen LH, van der Marel GA, Overhand M (2006). Inhibitors of protein: geranylgeranyl transferases. Curr. Med. Chem., 13(20):2385-2427.
4. Andres DA, Goldstein JL, Ho YK, Brown MS (1993). Mutational analysis of alpha-subunit of protein farnesyltransferase. Evidence for a catalytic role. J Biol Chem., 268 (2):1383-1390.
5. Dunten P, Kammlott U, Crowther R, Weber D, Palermo R, Birktoft J (1998). Protein farnesyltransferase: structure and implications for substrate binding. Biochemistry, 37 (37):13042.
6. Reid TS, Terry KL, Casey PJ, Beese LS (2004). Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J Mol Biol., 343 (2):417-433.
7. Efuet ET and Keyomarsi K (2006). Farnesyl and Geranylgeranyl Transferase Inhibitors Induce G1 Arrest by Targeting the Proteasome. Cancer Res., 66(2):1040-1051.