Publications

• 3-aminopyrrolidinone farnesyltransferase inhibitors: design of macrocyclic compounds with improved pharmacokinetics and excellent cell potency.

  Bell I.M., Gallicchio S.N., Abrams M., Beese L.S., Beshore D.C., Bhimnathwala H., Bogusky M.J., Buser C.A., Culberson J.C., Davide J., Ellis-Hutchings M., Fernandes C., Gibbs J.B., Graham S.L., Hamilton K.A., Hartman G.D., Heimbrook D.C., Homnick C.F., Huber H.E., Huff J.R., Kassahun K., Koblan K.S., Kohl N.E., Lobell R.B., Lynch J.J. Jr., Robinson R., Rodrigues A.D., Taylor J.S., Walsh E.S., Williams T.M., Zartman C.B.

  A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side eff ... >> More

  A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date. << Less

  J. Med. Chem. 45:2388-2409(2002) [PubMed] [EuropePMC]

• Plant farnesyltransferase can restore yeast Ras signaling and mating.

  Yalovsky S., Trueblood C.E., Callan K.L., Narita J.O., Jenkins S.M., Rine J., Gruissem W.

  Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ra ... >> More

  Farnesyltransferase (FTase) is a heterodimeric enzyme that modifies a group of proteins, including Ras, in mammals and yeasts. Plant FTase alpha and beta subunits were cloned from tomato and expressed in the yeast Saccharomyces cerevisiae to assess their functional conservation in farnesylating Ras and a-factor proteins, which are important for cell growth and mating. The tomato FTase beta subunit (LeFTB) alone was unable to complement the growth defect of ram1 delta mutant yeast strains in which the chromosomal FTase beta subunit gene was deleted, but coexpression of LeFTB with the plant alpha subunit gene (LeFTA) restored normal growth, Ras membrane association, and mating. LeFTB contains a novel 66-amino-acid sequence domain whose deletion reduces the efficiency of tomato FTase to restore normal growth to yeast ram1 delta strains. Coexpression of LeFTA and LeFTB in either yeast or insect cells yielded a functional enzyme that correctly farnesylated CaaX-motif-containing peptides. Despite their low degree of sequence homology, yeast and plant FTases shared similar in vivo and in vitro substrate specificities, demonstrating that this enzymatic modification of proteins with intermediates from the isoprenoid biosynthesis pathway is conserved in evolutionarily divergent eukaryotes. << Less

  Mol. Cell. Biol. 17:1986-1994(1997) [PubMed] [EuropePMC]

• Design, synthesis, and characterization of peptide-based rab geranylgeranyl transferase inhibitors.

  Tan K.T., Guiu-Rozas E., Bon R.S., Guo Z., Delon C., Wetzel S., Arndt S., Alexandrov K., Waldmann H., Goody R.S., Wu Y.W., Blankenfeldt W.

  Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various can ... >> More

  Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various cancers, RabGGTase may be a target for novel therapeutics. The development of selective inhibitors is, however, difficult because two related enzymes involved in other cellular processes exist in eukaryotes and because RabGGTase recognizes protein substrates indirectly, resulting in relaxed specificity. We report the synthesis of a peptidic library based on the farnesyl transferase inhibitor pepticinnamin E. Of 469 compounds investigated, several were identified as selective for RabGGTase with low micromolar IC(50) values. The compounds were not generally cytotoxic and inhibited Rab isoprenylation in COS-7 cells. Crystal structure analysis revealed that selective inhibitors interact with a tunnel unique to RabGGTase, implying that this structural motif is an attractive target for improved RabGGTase inhibitors. << Less

  J. Med. Chem. 52:8025-8037(2009) [PubMed] [EuropePMC]

• cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I.

  Zhang F.L., Diehl R.E., Kohl N.E., Gibbs J.B., Giros B., Casey P.J., Omer C.A.

  Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48 ... >> More

  Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I. << Less

  J. Biol. Chem. 269:3175-3180(1994) [PubMed] [EuropePMC]

• Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution.

  Zhang H., Seabra M.C., Deisenhofer J.

  <h4>Background</h4>Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesy ... >> More

  <h4>Background</h4>Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP).<h4>Results</h4>The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT.<h4>Conclusions</h4>LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes. << Less

  Structure 8:241-251(2000) [PubMed] [EuropePMC]

• Aryl tetrahydropyridine inhibitors of farnesyltransferase: glycine, phenylalanine and histidine derivatives.

  Gwaltney S.L., O'Connor S.J., Nelson L.T., Sullivan G.M., Imade H., Wang W., Hasvold L., Li Q., Cohen J., Gu W.Z., Tahir S.K., Bauch J., Marsh K., Ng S.C., Frost D.J., Zhang H., Muchmore S., Jakob C.G., Stoll V., Hutchins C., Rosenberg S.H., Sham H.L.

  Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered a series of aryl tetrahydropyridines that incorpora ... >> More

  Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered a series of aryl tetrahydropyridines that incorporate substituted glycine, phenylalanine and histidine residues. The design, synthesis, SAR and biological properties of these compounds will be discussed. << Less

  Bioorg. Med. Chem. Lett. 13:1359-1362(2003) [PubMed] [EuropePMC]

• Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation.

  Guo Z., Wu Y.W., Das D., Delon C., Cramer J., Yu S., Thuns S., Lupilova N., Waldmann H., Brunsveld L., Goody R.S., Alexandrov K., Blankenfeldt W.

  Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation event ... >> More

  Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP. << Less

  EMBO J. 27:2444-2456(2008) [PubMed] [EuropePMC]

• Rab geranylgeranyl transferase catalyzes the geranylgeranylation of adjacent cysteines in the small GTPases Rab1A, Rab3A, and Rab5A.

  Farnsworth C.C., Seabra M.C., Ericsson L.H., Gelb M.H., Glomset J.A.

  Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the foll ... >> More

  Rab proteins are Ras-related small GTPases that are geranylgeranylated on cysteine residues located at or near their C termini. They differ from other geranylgeranylated small GTPases in several important respects. (i) Most Rab proteins contain two adjacent cysteine residues within one of the following C-terminal sequence motifs: -XXCC, -XCXC, or -CCXX; (ii) a Rab protein that ends in a -XCXC motif has been shown to be geranylgeranylated on both adjacent cysteine residues; and (iii) Rab proteins are substrates of a unique Rab-specific geranylgeranyltransferase. Whether this enzyme catalyzes the geranylgeranylation of both cysteines is unknown. We addressed this question by direct structural analysis of in vitro prenylated proteins. We incubated recombinant Rab geranylgeranyltransferase, Rab escort protein, and [1-3H]geranylgeranyl pyrophosphate with recombinant wild-type Rab1A (-XXCC), Rab3A (-XCXC), or Rab5A (-CCXX) and treated each labeled protein with trypsin. We then analyzed the resulting peptides by HPLC and electrospray mass spectrometry and found that for each protein both C-terminal adjacent cysteines were geranylgeranylated. These results indicate that Rab geranylgeranyltransferase/Rab escort protein catalyzes the geranylgeranylation of both cysteines in Rab proteins with three distinct C-terminal motifs and suggest that other Rab proteins with these motifs may be similarly modified. << Less

  Proc. Natl. Acad. Sci. U.S.A. 91:11963-11967(1994) [PubMed] [EuropePMC]

• Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases.

  Wilson A.L., Erdman R.A., Castellano F., Maltese W.A.

  Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab s ... >> More

  Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 prenylation in vivo, the incorporation of [3H]mevalonate into Rab8 was measured in human embryonal kidney 293 cells under conditions where the activity of GGTaseI, but not GGTaseII, was blocked by the peptidomimetic inhibitor GGTI-298. The GGTaseI inhibitor did not prevent prenylation of either overexpressed Myc-tagged Rab8 or endogenous Rab8, whereas prenylation of a known GGTaseI substrate with the same carboxyl-terminal motif, Cdc42Hs, was completely blocked. To rule out the possibility that the apparent prenylation of Rab8 by GGTaseII occurs only when GGTaseI activity is eliminated, metabolic labelling studies were carried out in the absence of the GGTaseI inhibitor, using a REP-binding-deficient Rab8 construct (Y78D) that cannot serve as a substrate for GGTaseII, but is indistinguishable from wild-type Rab8 as a substrate for GGTaseI. Prenylation of the Y78D mutant was reduced by 60-70% in intact cells, consistent with the conclusion that the majority of Rab8 is prenylated by the REP/GGTaseII system in vivo. << Less

  Biochem J 333:497-504(1998) [PubMed] [EuropePMC]

• Caged protein prenyltransferase substrates: tools for understanding protein prenylation.

  DeGraw A.J., Hast M.A., Xu J., Mullen D., Beese L.S., Barany G., Distefano M.D.

  Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and ... >> More

  Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications. << Less

  Chem. Biol. Drug Des. 72:171-181(2008) [PubMed] [EuropePMC]

• Mutational analysis of alpha-subunit of protein farnesyltransferase. Evidence for a catalytic role.

  Andres D.A., Goldstein J.L., Ho Y.K., Brown M.S.

  Protein farnesyltransferase from rat brain is composed of tightly associated alpha- and beta-subunits of 377 and 437 amino acids that migrate on SDS-polyacrylamide gels with apparent molecular masses of 49 and 46 kDa, respectively. The enzyme attaches farnesyl groups to cysteines in p21ras and oth ... >> More

  Protein farnesyltransferase from rat brain is composed of tightly associated alpha- and beta-subunits of 377 and 437 amino acids that migrate on SDS-polyacrylamide gels with apparent molecular masses of 49 and 46 kDa, respectively. The enzyme attaches farnesyl groups to cysteines in p21ras and other proteins that contain cysteine residues at the fourth position from the COOH terminus. Production of stable enzyme in animal cells requires the simultaneous synthesis of both subunits, and all activity is lost when the subunits are dissociated chemically. The beta-subunit functions in the Zn(2+)-dependent binding of the protein substrate. The role of the alpha-subunit is unknown. In the current studies we used in vitro mutagenesis and transfection of cloned cDNAs to define the parts of the alpha-subunit that are necessary to stabilize the beta-subunit and to support farnesyl transfer. Deletion of 51 amino acids at the NH2 terminus of the alpha-subunit allowed normal stabilization of the beta-subunit and production of normal enzyme activity, but deletion of 106 amino acids abolished both of these properties. A proline-rich region at residues 12-34 of the alpha-subunit is not required for activity, but its presence explains the anomalously slow migration of the polypeptide on SDS-polyacrylamide gels. Deletion of only 5 amino acids at the COOH terminus of the alpha-subunit reduced activity appreciably. Substitution of asparagine for a conserved lysine at position 164 produced an alpha-subunit that stabilized the beta-subunit normally and permitted normal binding of the two substrates, farnesyl pyrophosphate and p21H-ras. Nevertheless, the rate of transfer of the bound farnesyl group to p21H-ras was markedly reduced. The latter finding suggests that the alpha-subunit plays a direct role in the catalytic reaction in addition to its role in the stabilization of the beta-subunit. << Less

  J. Biol. Chem. 268:1383-1390(1993) [PubMed] [EuropePMC]

• Psoromic acid is a selective and covalent Rab-prenylation inhibitor targeting autoinhibited RabGGTase.

  Deraeve C., Guo Z., Bon R.S., Blankenfeldt W., DiLucrezia R., Wolf A., Menninger S., Stigter E.A., Wetzel S., Choidas A., Alexandrov K., Waldmann H., Goody R.S., Wu Y.W.

  Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been ... >> More

  Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC(50) of 1.3 μM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal (α)His2 coordination with the catalytic zinc ion. Mutation of (α)His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors. << Less

  J. Am. Chem. Soc. 134:7384-7391(2012) [PubMed] [EuropePMC]

• cDNA cloning and expression of the alpha and beta subunits of rat Rab geranylgeranyl transferase.

  Armstrong S.A., Seabra M.C., Suedhof T.C., Goldstein J.L., Brown M.S.

  Rab geranylgeranyl transferase (Rab GG transferase) attaches 20-carbon geranylgeranyl groups to cysteine residues in Rab proteins that contain the COOH-terminal sequence Cys-X-Cys or Cys-Cys. Rab GG transferase consists of two components that are separable in high salt solutions. Component A is a ... >> More

  Rab geranylgeranyl transferase (Rab GG transferase) attaches 20-carbon geranylgeranyl groups to cysteine residues in Rab proteins that contain the COOH-terminal sequence Cys-X-Cys or Cys-Cys. Rab GG transferase consists of two components that are separable in high salt solutions. Component A is a 95-kDa protein, and Component B is a heterodimer consisting of a 60-kDa alpha subunit and a 38-kDa beta subunit. In the current paper, we have cloned cDNAs for the alpha and beta subunits of Component B. The cDNAs for the rat alpha and beta subunits predict proteins of 567 and 331 amino acids, respectively. The mRNAs for both subunits are expressed in many tissues. When transfected together in embryonic kidney 293 cells, the alpha and beta subunit cDNAs produced Rab GG transferase activity that was stimulated in vitro by the addition of purified Component A. Comparisons of the amino acid sequences suggest that the proteins encoded by the Saccharomyces cerevisiae genes MAD2 and BET2 are the yeast counterparts of the mammalian Rab GG transferase alpha and beta subunits, respectively. << Less

  J. Biol. Chem. 268:12221-12229(1993) [PubMed] [EuropePMC]

• Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate.

  Thoma N.H., Niculae A., Goody R.S., Alexandrov K.

  Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing ... >> More

  Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction. << Less

  J Biol Chem 276:48631-48636(2001) [PubMed] [EuropePMC]

• Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control.

  Qian D., Zhou D., Ju R., Cramer C.L., Yang Z.

  Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesylt ... >> More

  Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes. In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants. A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy. A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit. The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll. RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division. The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells. A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth. Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells. Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase. These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase. << Less

  Plant Cell 8:2381-2394(1996) [PubMed] [EuropePMC]

• Structure-guided development of selective RabGGTase inhibitors.

  Bon R.S., Guo Z., Stigter E.A., Wetzel S., Menninger S., Wolf A., Choidas A., Alexandrov K., Blankenfeldt W., Goody R.S., Waldmann H.

  Angew. Chem. Int. Ed. Engl. 50:4957-4961(2011) [PubMed] [EuropePMC]

• Efficient prenylation by a plant geranylgeranyltransferase-I requires a functional CaaL box motif and a proximal polybasic domain.

  Caldelari D., Sternberg H., Rodriguez-Concepcion M., Gruissem W., Yalovsky S.

  Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common alpha-subunit with farnesyltransferase (FTase) and has a distinct beta-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I beta-subunit (AtGGT- ... >> More

  Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common alpha-subunit with farnesyltransferase (FTase) and has a distinct beta-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I beta-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase alpha-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a(2) position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24 degrees C and 42 degrees C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase beta-subunit. << Less

  Plant Physiol. 126:1416-1429(2001) [PubMed] [EuropePMC]

• Protein prenylation: genes, enzymes, targets, and functions.

  Schafer W.R., Rine J.

  Annu Rev Genet 26:209-237(1992) [PubMed] [EuropePMC]

• Development of selective RabGGTase inhibitors and crystal structure of a RabGGTase-inhibitor complex.

  Guo Z., Wu Y.W., Tan K.T., Bon R.S., Guiu-Rozas E., Delon C., Nguyen T.U., Wetzel S., Arndt S., Goody R.S., Blankenfeldt W., Alexandrov K., Waldmann H.

  Angew. Chem. Int. Ed. Engl. 47:3747-3750(2008) [PubMed] [EuropePMC]

• Influence of metal ions on substrate binding and catalytic activity of mammalian protein geranylgeranyltransferase type-I.

  Zhang F.L., Casey P.J.

  Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group from the prenyl donor geranylgeranyl diphosphate (GGPP) to the cysteine residue of substrate proteins containing a C-terminal CaaX-motif (a sequence motif of proteins consisting of an invariant Cys residue fourth ... >> More

  Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group from the prenyl donor geranylgeranyl diphosphate (GGPP) to the cysteine residue of substrate proteins containing a C-terminal CaaX-motif (a sequence motif of proteins consisting of an invariant Cys residue fourth from the C-terminus). The GGTase-I heterodimer contains one atom of zinc, and this metal is required for enzyme activity. In this regard, GGTase-I is similar to the related enzyme protein farnesyltransferase (FTase); the latter enzyme also requires Mg2+ for activity. The current studies were undertaken in an attempt to explore further the role of bivalent metal ions in the activity of GGTase-I. Surprisingly, we found that GGTase-I and FTase have different metal requirements. Specifically, in marked contrast to FTase, GGTase-I does not require Mg2+ for activity. Direct binding assays, including a novel fluorescence-based technique, were employed to obtain quantitative information on the interaction of substrates with GGTase-I. Using these assays, we demonstrate that the Zn2+ in GGTase-I is required for peptide, but not for isoprenoid, substrate binding. Moreover, binding of GGPP protects GGTase-I from inactivation by zinc-chelating reagents; this protective effect is not seen with binding of peptide substrates. Metal substitution studies show that the Zn2+ in GGTase-I can be replaced by Cd2+, and that the Cd form of GGTase-I has altered specificity with regard to utilization of both peptide and isoprenoid substrates. The significance of these findings in relation to proposed mechanisms for the GGTase-I reaction is discussed. << Less

  Biochem J 320:925-932(1996) [PubMed] [EuropePMC]

• Development of selective, potent RabGGTase inhibitors.

  Stigter E.A., Guo Z., Bon R.S., Wu Y.W., Choidas A., Wolf A., Menninger S., Waldmann H., Blankenfeldt W., Goody R.S.

  Members of the Ras superfamily of small GTPases are frequently mutated in cancer. Therefore, inhibitors have been developed to address the acitivity of these GTPases by inhibiting their prenylating enzymes FTase, GGTase I, and RabGGTase. In contrast to FTase and GGTase I, only a handful of RabGGTa ... >> More

  Members of the Ras superfamily of small GTPases are frequently mutated in cancer. Therefore, inhibitors have been developed to address the acitivity of these GTPases by inhibiting their prenylating enzymes FTase, GGTase I, and RabGGTase. In contrast to FTase and GGTase I, only a handful of RabGGTase inhibitors have been developed. The most active RabGGTase inhibitor known until recently was an FTase inhibitor which hit RabGGTase as an off-target. We recently reported our efforts to tune the selectivity of these inhibitors toward RabGGTase. Here we describe an extended set of selective inhibitors. The requirements for selective RabGGTase inhibitors are described in detail, guided by multiple crystal structures. In order to relate in vitro and cellular activity, a high-throughput assay system to detect the attachment of [(3)H]geranylgeranyl groups to Rab was used. Selective RabGGTase inhibition allows the establishment of novel drug discovery programs aimed at the development of anticancer therapeutics. << Less

  J. Med. Chem. 55:8330-8340(2012) [PubMed] [EuropePMC]

• In vitro assembly, purification, and crystallization of the rab geranylgeranyl transferase:substrate complex.

  Rak A., Niculae A., Kalinin A., Thoma N.H., Sidorovitch V., Goody R.S., Alexandrov K.

  Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl gr ... >> More

  Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 A on an in-house X-ray source. << Less

  Protein Expr Purif 25:23-30(2002) [PubMed] [EuropePMC]

• Structure of mammalian protein geranylgeranyltransferase type-I.

  Taylor J.S., Reid T.S., Terry K.L., Casey P.J., Beese L.S.

  Protein geranylgeranyltransferase type-I (GGTase-I), one of two CaaX prenyltransferases, is an essential enzyme in eukaryotes. GGTase-I catalyzes C-terminal lipidation of >100 proteins, including many GTP-binding regulatory proteins. We present the first structural information for mammalian GGTase ... >> More

  Protein geranylgeranyltransferase type-I (GGTase-I), one of two CaaX prenyltransferases, is an essential enzyme in eukaryotes. GGTase-I catalyzes C-terminal lipidation of >100 proteins, including many GTP-binding regulatory proteins. We present the first structural information for mammalian GGTase-I, including a series of substrate and product complexes that delineate the path of the chemical reaction. These structures reveal that all protein prenyltransferases share a common reaction mechanism and identify specific residues that play a dominant role in determining prenyl group specificity. This hypothesis was confirmed by converting farnesyltransferase (15-C prenyl substrate) into GGTase-I (20-C prenyl substrate) with a single point mutation. GGTase-I discriminates against farnesyl diphosphate (FPP) at the product turnover step through the inability of a 15-C FPP to displace the 20-C prenyl-peptide product. Understanding these key features of specificity is expected to contribute to optimization of anti-cancer and anti-parasite drugs. << Less

  EMBO J. 22:5963-5974(2003) [PubMed] [EuropePMC]

• Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I.

  Lane K.T., Beese L.S.

  More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and t ... >> More

  More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and the gamma subunit of many small heterotrimeric G proteins. This modification is catalyzed by the protein prenyltransferases: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I), and GGTase-II (or RabGGTase). In this review, we examine the structural biology of FTase and GGTase-I (the CaaX prenyltransferases) to establish a framework for understanding the molecular basis of substrate specificity and mechanism. These enzymes have been identified in a number of species, including mammals, fungi, plants, and protists. Prenyltransferase structures include complexes that represent the major steps along the reaction path, as well as a number of complexes with clinically relevant inhibitors. Such complexes may assist in the design of inhibitors that could lead to treatments for cancer, viral infection, and a number of deadly parasitic diseases. << Less

  J Lipid Res 47:681-699(2006) [PubMed] [EuropePMC]

• Analysis of the eukaryotic prenylome by isoprenoid affinity tagging.

  Nguyen U.T., Guo Z., Delon C., Wu Y., Deraeve C., Franzel B., Bon R.S., Blankenfeldt W., Goody R.S., Waldmann H., Wolters D., Alexandrov K.

  Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to d ... >> More

  Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents. << Less

  Nat. Chem. Biol. 5:227-235(2009) [PubMed] [EuropePMC]