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At least 19 recordsLinked to original sources
PubMed1999

Enzymology and biology of CaaX protein prenylation.

Protein prenylation refers to a type of lipid modification in which either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins. The majority of prenylated proteins belong to a group termed "CaaX proteins" that are defined by a specific C-terminal motif that directs their modification by this process. The ménage of CaaX-type prenylated proteins encompasses a wide variety of molecules that are found primarily at the cytoplasmic face of cellular membranes. These include nuclear lamins, Ras and a multitude of GTP-binding proteins (G proteins), several protein kinases and phosphatases, as well as other important proteins. A tremendous number of cellular signaling processes and regulatory events are under the control of CaaX prenyl proteins. While the attached isoprenoid lipids, in general, support the membrane association of the modified proteins, some proteins also clearly participate directly in protein-protein interactions. This chapter will emphasize 1) the biochemistry of the two enzymes termed farnesyltransferase and geranylgeranyltransferase type I, responsible for CaaX protein prenylation, and 2) biological roles for these modifications. Throughout, we will attempt to highlight the significance of prenylation in specific cellular events. The critical importance of this class of lipid modifications is attested to by the emergence of farnesyltransferase as a target for the development of anti-cancer therapeutics.

Alkyl and Aryl Transferases↗
PubMed1989

Prenylated proteins: demonstration of a thioether linkage to cysteine of proteins.

Prenylated amino acid fragments have been isolated from prenylated proteins of Chinese hamster ovary cells. Gel-exclusion chromatography indicates that these proteins are modified by two different prenyl groups. The prenyl-amino acid fragments are labeled by 35S from cysteine, and this bond is cleaved by Raney-Ni, proving that the prenyl residue is linked to protein via a thioether to cysteine. Hydrazinolysis has been used to demonstrate that the cysteine is carboxy terminal.

Animals↗
PubMed1999

Novel farnesol and geranylgeraniol analogues: A potential new class of anticancer agents directed against protein prenylation.

Protein farnesyltransferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Herein it is shown that certain novel prenyl diphosphate analogues are potent inhibitors of mammalian FTase. Furthermore, the alcohol precursors of two of these compounds are able to block anchorage-independent growth of ras-transformed cells. While 3-allylfarnesol inhibits protein farnesylation, 3-vinylfarnesol instead leads to abnormal prenylation of proteins with the 3-vinylfarnesyl group. In a similar manner, 3-allylgeranylgeraniol acts as a highly specific inhibitor of protein geranylgeranylation, while 3-vinylgeranylgeraniol restores protein geranylgeranylation in cells. This study indicates that certain prenyl alcohol analogues can act as prenyltransferase inhibitors in situ, via a novel prodrug mechanism. These analogues may prove to be valuable tools for investigating the therapeutic consequences of inhibiting geranylgeranylation relative to farnesylation. Furthermore, the 3-vinyl alcohol analogues can inhibit transformed cell growth through a mechanism not involving prenyltransferase inhibition.

Alkyl and Aryl Transferases↗
PubMed1998

Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation: evidence from structure-activity relationships in J774 macrophages.

Recent evidence suggests that bisphosphonates (BPs) may inhibit bone resorption by mechanisms that lead to osteoclast apoptosis. We have previously shown that BPs also reduce cell viability and induce apoptosis in the macrophage-like cell line J774. To determine whether BPs inhibit osteoclast-mediated bone resorption and affect J774 macrophages by the same molecular mechanism, we examined the potency to reduce J774 cell viability of pairs of nitrogen-containing BPs that differ slightly in the structure of the heterocycle-containing side chain but that differ markedly in antiresorptive potency. In all cases, the most potent antiresorptive BP of each pair also caused the greatest loss of J774 viability, while the less potent antiresorptive BPs were also less potent at reducing J774 cell viability. Similarly, the bisphosphinate, phosphonoalkylphosphinate and monophosphonate analogs of BPs (in which one or both phosphonate groups are modified, giving rise to much less potent or inactive antiresorptive agents) were much less potent or inactive at reducing J774 cell viability. Thus, the structure-activity relationships of BPs for inhibiting bone resorption match those for causing loss of cell viability in J774 cells, indicating that BPs inhibit osteoclast-mediated bone resorption and reduce J774 macrophage viability by the same molecular mechanism. Loss of J774 cell viability after treatment with BPs was associated with a parallel increase in apoptotic cell death. We have recently proposed that nitrogen-containing BPs reduce cell viability and cause J774 apoptosis as a consequence of inhibition of enzymes of the mevalonate pathway and hence loss of prenylated proteins. In this study, the BPs that were potent inducers of J774 apoptosis and potent antiresorptive agents were also found to be effective inhibitors of protein prenylation in J774 macrophages, whereas the less potent BP analogs did not inhibit protein prenylation. This provides strong evidence that BPs with a heterocyclic, nitrogen-containing side chain, such as risedronate, inhibit osteoclast-mediated bone resorption and induce J774 apoptosis by preventing protein prenylation.

Animals↗
PubMed1998

Protein prenyl transferase activities of Plasmodium falciparum.

Prenylated proteins have been shown to function in important cellular regulatory processes including signal transduction. The enzymes involved in protein prenylation, farnesyl transferase and geranylgeranyl transferase, have been recent targets for development of cancer chemotherapeutics. We have initiated a systematic study of protein prenyl transferases of the malaria parasite, Plasmodium falciparum, to determine whether these enzymes can be developed as targets for antimalarial chemotherapy. We report here the identification of protein farnesyl transferase and protein geranylgeranyl transferase-I in the malaria parasite, P. falciparum. The farnesyl transferase has been partially purified from the cytosolic fraction through ammonium sulfate precipitation and Mono-Q chromatography. Farnesyl and geranylgeranyl transferase-I activities are present at all stages of P. falciparum intraerythrocytic development with maximum specific activity in the ring stage. Geranylgeranyl transferase-I specific activity is two times that of farnesyl transferase in the ring stage. Peptidomimetics and prenyl analogues of protein farnesyl transferase substrates were tested as in vitro inhibitors of partially purified P. falciparum prenyl transferase and of malaria parasite growth. The peptidomimetics were significantly more potent inhibitors than lipid substrate analogues of both the activity of Mono-Q purified enzyme and parasite growth in intraerythrocytic cultures. Exposure of the parasite to the peptidomimetic L-745,631 also showed significant inhibition of morphological development beyond the trophozoite stage. These studies suggest the potential of designing or identifying differential inhibitors of P. falciparum and mammalian prenyl transferases as an approach to novel malaria therapy.

Alkyl and Aryl Transferases↗
PubMed1997

Chloroplastic prenylated proteins.

By in vivo [3H]mevalonate labelling of spinach combined with biochemical analysis, evidence is provided for the existence of protein prenylation in chloroplasts. Approximately 20 prenylated polypeptides were resolved by SDS-PAGE followed by autoradiography. Thermolysin treatment of intact chloroplasts revealed that about 40% of the prenylated polypeptides were associated with the cytoplasmic surface of the outer envelope membrane. The remaining portion was present in thylakoids and/or the inner envelope membrane. The majority of the prenylated polypeptides were associated with larger membrane protein complexes. A farnesyl protein transferase activity was found to be associated with the thylakoid membrane.

Alkyl and Aryl Transferases↗
PubMed1998

Single prenyl-binding site on protein prenyl transferases.

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.

Alkyl and Aryl Transferases↗
PubMed1992

Isopentenoid synthesis in embryonic Drosophila cells: prenylated protein profile and prenyl group usage.

It has been established that vertebrates and yeasts modified a unique subset of polypeptides with farnesyl and geranylgeranyl residues. This observation has been extended to Drosophila Kc cells. [3H]Mevalonate was incorporated into 54 Kc cell peptides (18-92 kDa). As reported for mammalian cells, most of the labeled peptides had molecular weights between 21 and 27 kDa. C18 radio-HPLC tryptic digest profiles for delipidized, [3H]mevalonate-labeled (a) insect (Drosophila and Spodoptera frugiperda) and mammalian (Chinese hamster ovary met 18-2b) cells, (b) Kc cell nuclear lamin, and (c) a 23.5-kDa purified Kc cell GTP-binding protein were compared and analyzed. [35S]Cysteine-labeled Kc cells yielded a tryptic digest radio-HPLC profile which was congruent with that for [3H]mevalonate-labeled cells. A significant fraction (30-33%) of the doubly labeled tryptic peptides were eluted with greater than or equal to 93% acetonitrile. Kc cell nuclear lamin tryptic digests yielded a single 3H-labeled product which migrated as S-farnesylcysteine. The Kc cell 23.5-kDa GTP-binding protein's 3H-labeled oligopeptide(s)/amino acid(s) was geranylgeranylated and its tryptic digest profile was representative of prenylated proteins whose oligopeptides eluted with greater than or equal to 93% acetonitrile. Moreover, the 3H-labeled oligopeptide/amino acid profiles plus prenyl group patterns for [3H]mevalonate-labeled Kc and mammalian cell total extracts were similar. Collectively, these observations supported a prenylated protein spectrum and prenyl group usage as highly conserved eukaryotic cellular characteristics.

Animals↗
PubMed1997

Differential prenyl pyrophosphate binding to mammalian protein geranylgeranyltransferase-I and protein farnesyltransferase and its consequence on the specificity of protein prenylation.

Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the gamma subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.

Alkyl and Aryl Transferases↗
PubMed1994

Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry.

Modification of proteins at C-terminal cysteine residue(s) by the isoprenoids farnesyl (C15) and geranylgeranyl (C20) is essential for the biological function of a number of eukaryotic proteins including fungal mating factors and the small, GTP-binding proteins of the Ras superfamily. Three distinct enzymes, conserved between yeast and mammals, have been identified that prenylate proteins: farnesyl protein transferase, geranylgeranyl protein transferase type I and geranylgeranyl protein transferase type II. Each prenyl protein transferase has its own protein substrate specificity. Much has been learned about the biology, genetics and biochemistry of protein prenylation and prenyl protein transferases through studies of eukaryotic microorganisms, particularly Saccharomyces cerevisiae. The functional importance of protein prenylation was first demonstrated with fungal mating factors. The initial genetic analysis of prenyl protein transferases was in S. cerevisiae with the isolation and subsequent characterization of mutations in the RAM1, RAM2, CDC43 and BET2 genes, each of which encodes a prenyl protein transferase subunit. We review here these and other studies on protein prenylation in eukaryotic microbes and how they relate to and have contributed to our knowledge about protein prenylation in all eukaryotic cells.

Amino Acid Sequence↗
PubMed1999

Protein prenylation in spinach chloroplasts.

Protein prenylation in plants was studied by in vivo metabolic (3)H-mevalonate labeling in combination with a range of protein synthesis inhibitors. In spinach cotyledons, this posttranslational protein modification was found to be divided into two categories, one representing the conventional prenylation involving farnesyl and geranylgeranyl groups bound to cysteine residues via thioether linkages. This category revealed a similar pattern of prenylated proteins to that observed in mammalian cells and depends on nuclear gene expression. The other category was shown to represent a type of prenylation confined to chloroplasts. It depends on plastid gene expression and does not involve a thioether bond. The modifying isoprenoid could be released from the chloroplastic polypeptides by alkaline treatment and was identified as phytol upon GC-MS analysis. The phytol could readily be derived from all-trans-[(3)H]farnesol, which, like all-trans-[(3)H]geranylgeraniol, was taken up by the cotyledons, resulting in incorporation of radiolabel into proteins.

Animals↗
PubMed1993

Characterization of recombinant human farnesyl-protein transferase: cloning, expression, farnesyl diphosphate binding, and functional homology with yeast prenyl-protein transferases.

We have isolated cDNAs encoding the alpha and beta subunits of human farnesyl-protein transferase (FPTase). The proteins encoded by these two cDNAs are 93-95% identical to the corresponding subunits of bovine and rat FPTase and show regions of homology with proteins encoded by Saccharomyces cerevisiae prenyl-protein transferase genes. Human FPTase expressed in Escherichia coli from a translationally coupled operon had kinetic properties similar to those of FPTase isolated from bovine brain. Examination of farnesyl diphosphate binding indicated that while neither individual subunit was capable of isoprenoid binding, a radiolabeled farnesyl diphosphate analog could be specifically photo-cross-linked to the beta subunit of FPTase holoenzyme. To further analyze subunit structure-function and to detect functional similarities with yeast prenyl-protein transferases (FPTase and two geranylgeranyl-protein transferases), amino acid changes homologous to those found in mutant yeast prenyl-protein transferase subunits were made in the subunits of human FPTase. Substitutions in either the alpha or beta subunits that decrease the activity of yeast prenyl-protein transferases were also observed to impair human FPTase. Kinetic analyses showed that these mutant human FPTases have Km and kcat values that are altered with respect to wild-type human FPTase.

Alkyl and Aryl Transferases↗
PubMed1995

In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure.

Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from those induced with lovastatin. These results implicate a defect in protein prenylation in the lovastatin-induced retinal degeneration, and suggest the presence of a dynamic pathway in the retina that requires isoprenylated proteins to maintain retinal cytoarchitecture.

Acetates↗
PubMed1999

Characterization of prenylated protein methyltransferase in Leishmania.

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-l-[methyl-(3)H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans, trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, but N-acetyl-S-trans, trans-geranyl-l-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5'-[gamma-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-l-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5'-[gamma-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

Animals↗
PubMed2006

Therapeutic intervention based on protein prenylation and associated modifications.

In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.

Animals↗