RESUMEN
Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.
Asunto(s)
Farnesiltransferasa , Oryza , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Farnesiltransferasa/química , Oryza/genética , Oryza/enzimología , Cinética , Ingeniería de Proteínas , BiocatálisisRESUMEN
Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.
Asunto(s)
Biología Computacional , Biblioteca de Péptidos , Humanos , Biología Computacional/métodos , Especificidad por Sustrato , Farnesiltransferasa/metabolismo , Farnesiltransferasa/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Unión ProteicaRESUMEN
Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.
Asunto(s)
Inhibidores Enzimáticos , Neoplasias , Humanos , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/química , Terpenos/química , Terpenos/farmacología , Prenilación de Proteína , Neoplasias/tratamiento farmacológicoRESUMEN
Farnesyltransferase (FTase) is a heterodimeric enzyme, which catalyzes covalent attachment of the farnesyl group to target proteins, thus coordinating their trafficking in the cell. FTase has been demonstrated to be highly expressed in cancer and neurological diseases; hence considered as a hot target for therapeutic purposes. However, due to the nonspecific inhibition, there has been only one inhibitor that could be translated into the clinic. Importantly, it has been shown that phosphorylation of the α-subunit of FTase increases the activity of the enzyme in certain diseases. As such, understanding the impact of phosphorylation on dynamics of FTase provides a basis for targeting a specific state of the enzyme that emerges under pathological conditions. To this end, we performed 18 µs molecular dynamics (MD) simulations using complexes of (non)-phosphorylated FTase that are representatives of the farnesylation reaction. We demonstrated that phosphorylation modulated the catalytic site by rearranging interactions between farnesyl pyrophosphate (FPP)/peptide substrate, catalytic Zn2+ ion/coordinating residues and hot-spot residues at the interface of the subunits, all of which led to the stabilization of the substrate and facilitation of the release of the product, thus collectively expediting the reaction rate. Importantly, we also identified a likely allosteric pocket on the phosphorylated FTase, which might be used for specific targeting of the enzyme. To the best of our knowledge, this is the first study that systematically examines the impact of phosphorylation on the enzymatic reaction steps, hence opens up new avenues for drug discovery studies that focus on targeting phosphorylated FTase.
Asunto(s)
Transferasas Alquil y Aril , Transferasas Alquil y Aril/metabolismo , Catálisis , Dominio Catalítico , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Péptidos/química , FosforilaciónRESUMEN
In the incessant search for innovative cancer control strategies, this study was devoted to the design, synthesis and pharmacological evaluation of dual inhibitors of farnesyltransferase and tubulin polymerization (FTI/MTIs). A series of indolizine-phenothiazine hybrids 16 (amides) and 17 (ketones) has been obtained in a 4-step procedure. The combination of the two heterocycles provided potent tubulin polymerization inhibitors with similar efficiency as the reference phenstatin and (-)-desoxypodophyllotoxin. Ketones 17 were also able to inhibit human farnesyltransferase (FTase) in vitro. Interestingly, three molecules 17c, 17d and 17f were very effective against both considered biological targets. Next, nine indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b were evaluated for their cell growth inhibition potential on the NCI-60 cancer cell lines panel. Ketones 17a-f were the most active and displayed promising cellular activities. Not only they arrested the cell growth of almost all tested cancer cells, but they displayed cytotoxicity potential with GI50 values in the low nanomolar range. The most sensitive cell lines upon treatment with indolizine-phenothiazine hybrids were NCI-H522 (lung cancer), COLO-205 and HT29 (colon cancer), SF-539 (human glioblastoma), OVCAR-3 (ovarian cancer), A498 (renal cancer) and especially MDA-MB-435 (melanoma). Demonstrating the preclinical effectiveness of these dual inhibitors can be crucial. A single dual molecule could induce a synergy of antitumor activity, while increasing the effectiveness and reducing the toxicity of the classical combo treatments currently used in chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Indolizinas/farmacología , Fenotiazinas/farmacología , Moduladores de Tubulina/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Humanos , Indolizinas/síntesis química , Indolizinas/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Fenotiazinas/síntesis química , Fenotiazinas/metabolismo , Unión Proteica , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/metabolismoRESUMEN
Terpene synthases (TPSs) and trans-isoprenyl diphosphate synthases (IDSs) are among the core enzymes for creating the enormous diversity of terpenoids. Despite having no sequence homology, TPSs and IDSs share a conserved "α terpenoid synthase fold" and a trinuclear metal cluster for catalysis, implying a common ancestry with TPSs hypothesized to evolve from IDSs anciently. Here we report on the identification and functional characterization of novel IDS-like TPSs (ILTPSs) in fungi that evolved from IDS relatively recently, indicating recurrent evolution of TPSs from IDSs. Through large-scale bioinformatic analyses of fungal IDSs, putative ILTPSs that belong to the geranylgeranyl diphosphate synthase (GGDPS) family of IDSs were identified in three species of Melampsora. Among the GGDPS family of the two Melampsora species experimentally characterized, one enzyme was verified to be bona fide GGDPS and all others were demonstrated to function as TPSs. Melampsora ILTPSs displayed kinetic parameters similar to those of classic TPSs. Key residues underlying the determination of GGDPS versus ILTPS activity and functional divergence of ILTPSs were identified. Phylogenetic analysis implies a recent origination of these ILTPSs from a GGDPS progenitor in fungi, after the split of Melampsora from other genera within the class of Pucciniomycetes. For the poplar leaf rust fungus Melampsora larici-populina, the transcripts of its ILTPS genes were detected in infected poplar leaves, suggesting possible involvement of these recently evolved ILTPS genes in the infection process. This study reveals the recurrent evolution of TPSs from IDSs since their ancient occurrence and points to the possibility of a wide distribution of ILTPS genes in three domains of life.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Basidiomycota/enzimología , Difosfatos/metabolismo , Evolución Molecular , Farnesiltransferasa/química , Populus/microbiología , Terpenos/metabolismo , Transferasas Alquil y Aril/genética , Basidiomycota/genética , Basidiomycota/crecimiento & desarrollo , Mutación , FilogeniaRESUMEN
Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid "Cxx" C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.
Asunto(s)
Farnesiltransferasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencias de Aminoácidos , Farnesiltransferasa/química , Farnesiltransferasa/genética , Cinética , Prenilación , Prenilación de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Difosfonatos/química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/química , Mieloma Múltiple/tratamiento farmacológico , Terpenos/química , Antineoplásicos/química , Apoptosis , Proliferación Celular , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Mieloma Múltiple/enzimología , Mieloma Múltiple/patología , Prenilación de Proteína , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Geranylgeranyl diphosphate synthase (GGPPS) is a central metalloenzyme in the mevalonate pathway, crucial for the prenylation of small GTPases. As small GTPases are pivotal for cellular survival, GGPPS was highlighted as a potential target for treating human diseases, including solid and hematologic malignancies and parasitic infections. Most available GGPPS inhibitors are bisphosphonates, but the clinically available compounds demonstrate poor pharmacokinetic properties. Although the design of novel bisphosphonates with improved physicochemical properties is highly desirable, the structure of wild-type human GGPPS (hGGPPS) bound to a bisphosphonate has not been resolved. Moreover, various metal-bisphosphonate-binding stoichiometries were previously reported in structures of yeast GGPPS (yGGPPS), hampering computational drug design with metal-binding pharmacophores (MBP). In this study, we report the 2.2 Å crystal structure of hGGPPS in complex with ibandronate, clearly depicting the involvement of three Mg2+ ions in bisphosphonate-protein interactions. Using drug-binding assays and computational docking, we show that the assignment of three Mg2+ ions to the binding site of both hGGPPS and yGGPPS greatly improves the correlation between calculated binding energies and experimentally measured affinities. This work provides a structural basis for future rational design of additional MBP-harboring drugs targeting hGGPPS. SIGNIFICANCE STATEMENT: Bisphosphonates are inhibitors of geranylgeranyl diphosphate synthase (GGPPS), a metalloenzyme crucial for cell survival. Bisphosphonate binding depends on coordination by Mg2+ ions, but various Mg2+-bisphosphonate-binding stoichiometries were previously reported. In this study, we show that three Mg2+ ions are vital for drug binding and provide a structural basis for future computational design of GGPPS inhibitors.
Asunto(s)
Cristalografía por Rayos X/métodos , Dimetilaliltranstransferasa/metabolismo , Difosfonatos/metabolismo , Farnesiltransferasa/metabolismo , Geraniltranstransferasa/metabolismo , Magnesio/metabolismo , Simulación del Acoplamiento Molecular/métodos , Sitios de Unión/fisiología , Dimetilaliltranstransferasa/química , Difosfonatos/química , Farnesiltransferasa/química , Geraniltranstransferasa/química , Humanos , Magnesio/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Post-translational farnesylation can regulate subcellular localization and protein-protein interaction in eukaryotes. The function of farnesylation is not well identified in plant pathogenic fungi, particularly during the process of fungal infection. Here, through functional analyses of the farnesyltransferase ß-subunit gene, RAM1, we examine the importance of protein farnesylation in the rice blast fungus Magnaporthe oryzae. Targeted disruption of RAM1 resulted in the reduction of hyphal growth and sporulation, and an increase in the sensitivity to various stresses. Importantly, loss of RAM1 also led to the attenuation of virulence on the plant host, characterized by decreased appressorium formation and invasive growth. Interestingly, the defect in appressoria formation of the Δram1 mutant can be recovered by adding exogenous cAMP and IBMX, suggesting that RAM1 functions upstream of the cAMP signalling pathway. We found that two Ras GTPases, RAS1 and RAS2, can interact with Ram1, and their plasma membrane localization was regulated by Ram1 through their C-terminal farnesylation sites. Adding a farnesyltransferase inhibitor Tipifarnib can result in similar defects as in Δram1 mutant, including decreased appressorium formation and invasive growth, as well as mislocalized RAS proteins. Our findings indicate that protein farnesylation regulates the RAS protein-mediated signaling pathways required for appressorium formation and host infection, and suggest that abolishing farnesyltransferase could be an effective strategy for disease control.
Asunto(s)
Farnesiltransferasa/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/crecimiento & desarrollo , Magnaporthe/metabolismo , Proteínas ras/metabolismo , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/fisiología , Magnaporthe/genética , Enfermedades de las Plantas/microbiología , Quinolonas/farmacología , Transducción de Señal/genética , Transducción de Señal/fisiología , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Virulencia , Proteínas ras/genéticaRESUMEN
Pepper ( Capsicum annuum) fruits are a rich source of carotenoids. Geranylgeranyl diphosphate (GGPP) is the precursor for carotenoid biosynthesis and is produced by GGPP synthase (GGPPS), which belongs to the prenyl transferase (PTS) family. In this study, we identified from the pepper genome a total of eight PTS homologues. Our subcellular localization, enzymatic activity, and expression level analyses proved that among these homologues Capana04g000412 is the only functional GGPPS (CaGGPPS1) for carotenoid biosynthesis in pepper fruits. We demonstrated that CaGGPPS1 interacts with a catalytically inactive small subunit homologue protein CaSSUII, and such an interaction promotes CaGGPPS1 enzymatic activity. We also revealed a protein-protein interaction between CaSSUII and a putative phytoene synthase and the repression of carotenoid accumulation by silencing CaSSUII in pepper fruits. Taken together, our results suggest an essential contribution of the CaGGPPS1/CaSSUII interaction to carotenoid biosynthesis in ripening pepper fruits.
Asunto(s)
Capsicum/enzimología , Carotenoides/biosíntesis , Farnesiltransferasa/metabolismo , Frutas/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Capsicum/genética , Capsicum/crecimiento & desarrollo , Capsicum/metabolismo , Cromatografía Líquida de Alta Presión , Dimerización , Farnesiltransferasa/química , Farnesiltransferasa/genética , Frutas/enzimología , Frutas/genética , Frutas/metabolismo , Genoma de Planta , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMEN
Farnesyl Transferase is a hetero-dimer transferase that targets Ras proteins and attaches a farnesyl group to it. This Ras protein, on localization to the cell membrane, has the ability to induce activation of various growth and proliferation pathways of the cell. Over-activation of mutated Ras may lead to the development of cancer. Farnesyl Transferase catalyses the initial step in the posttranslational modification of normal as well as mutated Ras gene, thus facilitating its tethering to the cell membrane. Inhibition of Farnesyl Transferase is the main step in restricting the activity of mutant Ras protein. Thus the above enzyme has emerged as a novel target for anti-cancer agents. Here we review the role of Farnesyl Transferase in tumorigenesis and various compounds of synthetic and natural origin acting as Farnesyl Transferase inhibitors as potential anti-cancer agents.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Animales , Antineoplásicos/química , Inhibidores Enzimáticos/química , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Genes ras , Humanos , Sistema de Señalización de MAP Quinasas , Neoplasias/enzimología , Neoplasias/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Relación Estructura-ActividadRESUMEN
Malaria, mainly caused by Plasmodium falciparum and Plasmodium vivax, has been a growing cause of morbidity and mortality. P. falciparum is more lethal than is P. vivax, but there is a vital need for effective drugs against both species. Geranylgeranyl diphosphate synthase (GGPPS) is an enzyme involved in the biosynthesis of quinones and in protein prenylation and has been proposed to be a malaria drug target. However, the structure of P. falciparumGGPPS (PfGGPPS) has not been determined, due to difficulties in crystallization. Here, we created a PfGGPPS model using the homologous P.vivaxGGPPS X-ray structure as a template. We simulated the modeled PfGGPPS as well as PvGGPPS using conventional and Gaussian accelerated molecular dynamics in both apo- and GGPP-bound states. The MD simulations revealed a striking similarity in the dynamics of both enzymes with loop 9-10 controlling access to the active site. We also found that GGPP stabilizes PfGGPPS and PvGGPPS into closed conformations and via similar mechanisms. Shape-based analysis of the binding sites throughout the simulations suggests that the two enzymes will be readily targeted by the same inhibitors. Finally, we produced three MD-validated conformations of PfGGPPS to be used in future virtual screenings for potential new antimalarial drugs acting on both PvGGPPS and PfGGPPS.
Asunto(s)
Antimaláricos/química , Diseño de Fármacos , Farnesiltransferasa/química , Plasmodium falciparum/enzimología , Plasmodium vivax/enzimología , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Antimaláricos/metabolismo , Sitios de Unión , Farnesiltransferasa/metabolismo , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Alineación de SecuenciaRESUMEN
The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.
Asunto(s)
Afidicolina/química , Aspergillus oryzae/genética , Retículo Endoplásmico/química , Farnesiltransferasa/química , Citoplasma/química , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Farnesiltransferasa/genética , Fosfatos de Poliisoprenilo/químicaRESUMEN
Farnesylation is an important post-translational protein modification in eukaryotes. Farnesylation is performed by protein farnesyltransferase, a heterodimer composed of an α- (FTα) and a ß-subunit. Recently, homodimerization of truncated rat and yeast FTα has been detected, suggesting a new role for FTα homodimers in signal transduction. We investigated the putative dimerization behaviour of human and rat FTα. Different in vitro and in vivo approaches revealed no self-dimerization and a presumably artificial formation of homotrimers and higher homo-oligomers in vitro. Our study contributes to the clarification of the physiological features of FTase in different species and may be important for the ongoing development of FTase inhibitors.
Asunto(s)
Farnesiltransferasa/química , Multimerización de Proteína , Animales , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Humanos , RatasRESUMEN
The isoprenoid donor for protein geranylgeranylation reactions, geranylgeranyl diphosphate (GGDP), is the product of the enzyme GGDP synthase (GGDPS) that condenses farnesyl diphosphate (FDP) and isopentenyl pyrophosphate. GGDPS inhibition is of interest from a therapeutic perspective for multiple myeloma because we have shown that targeting Rab GTPase geranylgeranylation impairs monoclonal protein trafficking, leading to endoplasmic reticulum stress and apoptosis. We reported a series of triazole bisphosphonate GGDPS inhibitors, of which the most potent was a 3:1 mixture of homogeranyl (HG) and homoneryl (HN) isomers. Here we determined the activity of the individual olefin isomers. Enzymatic and cellular assays revealed that although HN is approximately threefold more potent than HG, HN is not more potent than the original mixture. Studies in which cells were treated with varying concentrations of each isomer alone and in different combinations revealed that the two isomers potentiate the induced-inhibition of protein geranylgeranylation when used in a 3:1 HG:HN combination. A synergistic interaction was observed between the two isomers in the GGDPS enzyme assay. These results suggested that the two isomers bind simultaneously to the enzyme but within different domains. Computational modeling studies revealed that HN is preferred at the FDP site, that HG is preferred at the GGDP site, and that both isomers may bind to the enzyme simultaneously. These studies are the first to report a set of olefin isomers that synergistically inhibit GGDPS, thus establishing a new paradigm for the future development of GGDPS inhibitors.
Asunto(s)
Difosfonatos/química , Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Triazoles/química , Triazoles/farmacología , Dominio Catalítico , Línea Celular Tumoral , Sinergismo Farmacológico , Inhibidores Enzimáticos/química , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Humanos , Isomerismo , Lovastatina/farmacología , Modelos Moleculares , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismoRESUMEN
We report a molecular dynamics investigation of the structure, function, and inhibition of geranylgeranyl diphosphate synthase (GGPPS), a potential drug target, from the malaria parasite Plasmodium vivax. We discovered several GGPPS inhibitors, benzoic acids, and determined their structures crystallographically. We then used molecular dynamics simulations to investigate the dynamics of three such inhibitors and two bisphosphonate inhibitors, zoledronate and a lipophilic analogue of zoledronate, as well as the enzyme's product, GGPP. We were able to identify the main motions that govern substrate binding and product release as well as the molecular features required for GGPPS inhibition by both classes of inhibitor. The results are of broad general interest because they represent the first detailed investigation of the mechanism of action, and inhibition, of an important antimalarial drug target, geranylgeranyl diphosphate synthase, and may help guide the development of other, novel inhibitors as new drug leads.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/química , Plasmodium vivax/efectos de los fármacos , Antimaláricos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Análisis de Componente Principal , TermodinámicaRESUMEN
Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.
Asunto(s)
Dimetilaliltranstransferasa/antagonistas & inhibidores , Dimetilaliltranstransferasa/química , Colorantes Fluorescentes/química , Sondas Moleculares/química , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Sustitución de Aminoácidos , Dimetilaliltranstransferasa/genética , Difosfonatos/farmacología , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/química , Farnesiltransferasa/genética , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/química , Geraniltranstransferasa/genética , Humanos , Imidazoles/farmacología , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Especificidad por Sustrato , Ácido ZoledrónicoAsunto(s)
Evolución Molecular , Farnesiltransferasa/genética , Duplicación de Gen , Genoma de Planta , Proteínas de Plantas/genética , Solanaceae/genética , Secuencias de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Farnesiltransferasa/química , Expresión Génica , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Secundaria de Proteína , Selección Genética , Alineación de Secuencia , Solanaceae/clasificaciónRESUMEN
The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the ß subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the ß subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking.