Β-subunit Of Farnesyltransferase Research Articles

FNTB Promoter Polymorphisms Are Independent Predictors of Survival in Patients with Triple Negative Breast Cancer.

Simple SummaryIn breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical trials. Therefore, this study focussed on the clinical relevance of polymorphisms in FNTB, the gene encoding the catalytically active β-subunit of farnesyltransferase, in early breast cancer. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms are independent prognostic biomarkers, particularly in patients with early triple negative breast cancer (TNBC), and possibly modulate FNTB transcriptional activity. Taken together, we describe for the first time, a link between FNTB promoter polymorphism and the prognosis of breast cancer patients. We propose that FNTB genotyping, which is easily possible from a single blood drawing, may allow independent prognostic stratification, particularly in TNBC, in order to identify patients with a high risk of recurrence and poor prognosis. Ultimately, our results encourage further prospective evaluations of the role of FNTB promoter polymorphisms in predicting response to FTIs, particularly in TNBC patients.In breast cancer, the promising efficacy of farnesyltransferase inhibitors (FTIs) in preclinical studies is in contrast to only limited effects in clinical Phase II–III trials. The objective of this study was to explore the clinical relevance of farnesyltransferase β-subunit (FNTB) single nucleotide promoter polymorphisms (FNTB-173 6G > 5G (rs3215788), -609 G > C (rs11623866) and -179 T > A (rs192403314)) in early breast cancer. FNTB genotyping was performed by pyrosequencing in 797 patients from a prospective multicentre observational PiA trial (NCT 01592825). In the total cohort, the FNTB-173 6G > 5G polymorphism was an independent predictor of RFI (HR = 0.568; 95% CI = 0.339–0.949, p = 0.031), OS (HR = 0.629; 95% CI = 0.403–0.980, p = 0.040) and BCSS (HR = 0.433; 95% CI = 0.213–0.882; p = 0.021), whereas the FNTB-609 G > C polymorphism was an independent predictor of RFI (HR = 0.453; 95% CI = 0.226–0.910, p = 0.026) and BCSS (HR = 0.227; 95% CI = 0.075–0.687, p = 0.009). Subtype analysis revealed the independent prognostic relevance of FNTB promoter polymorphisms, particularly in TNBC but not in luminal or HER2-positive intrinsic subtypes. Finally, we used electrophoretic mobility shift assays (EMSAs) to confirm in vitro that the polymorphism FNTB-173 6G > 5G resulted in the differential binding of nuclear proteins from five different breast cancer cell lines. This is the first study on breast cancer suggesting that FNTB promoter polymorphisms (i) are independent prognostic biomarkers, particularly in patients with early TNBC, and (ii) could modulate FNTB’s transcriptional activity.

CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice.

Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants. Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the β-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.

The Farnesyltransferase β-Subunit Ram1 Regulates Sporisorium scitamineum Mating, Pathogenicity and Cell Wall Integrity.

The basidiomycetous fungus Sporisorium scitamineum causes a serious sugarcane smut disease in major sugarcane growing areas. Sexual mating is essential for infection to the host; however, its underlying molecular mechanism has not been fully studied. In this study, we identified a conserved farnesyltransferase (FTase) β subunit Ram1 in S. scitamineum. The ram1Δ mutant displayed significantly reduced mating/filamentation, thus of weak pathogenicity to the host cane. The ram1Δ mutant sporidia showed more tolerant toward cell wall stressor Congo red compared to that of the wild-type. Transcriptional profiling showed that Congo red treatment resulted in notable up-regulation of the core genes involving in cell wall integrity pathway in ram1Δ sporidia compared with that of WT, indicating that Ram1 may be involved in cell wall integrity regulation. In yeast the heterodimeric FTase is responsible for post-translational modification of Ras (small G protein) and a-factor (pheromone). We also identified and characterized two conserved Ras proteins, Ras1 and Ras2, respectively, and a MAT-1 pheromone precursor Mfa1. The ras1Δ, ras2Δ and mfa1Δ mutants all displayed reduced mating/filamentation similar as the ram1Δ mutant. However, both ras1Δ and ras2Δ mutants were hypersensitive to Congo red while the mfa1Δ mutant was the same as wild-type. Overall our study displayed that RAM1 plays an essential role in S. scitamineum mating/filamentation, pathogenicity, and cell wall stability.

Mutation in barley ERA1 (Enhanced Response to ABA1) gene confers better photosynthesis efficiency in response to drought as revealed by transcriptomic and physiological analysis

Farnesylation is a post-translational modification that promotes the interaction between the modified signaling protein and membrane lipids and/or other proteins. Farnesyltransferase is the crucial enzyme involved in this process. Strikingly, plant mutants in the ERA1 (Enhanced response to ABA 1) gene, encoding β-subunit of farnesyltransferase, exhibited ABA-hypersensitivity during seed germination and drought tolerance in several species including Arabidopsis, wheat and soybean. However, the mechanism of ERA1 action has not been resolved yet. Here, we present the potential regulatory role of ERA1 in the drought signaling network in barley. With the aim of decoding the role of the ERA1 gene, we developed a unique barley mutant using TILLING analysis. Mutation in HvERA1 confers semi-dwarf phenotype, ABA-sensitivity during seed germination and drought tolerance. Our transcriptomic analysis suggested a role of HvERA1 in regulation of the crosstalk between ABA and ethylene at the onset of drought. Furthermore, analysis of hvera1.b response to prolonged drought stress linked HvERA1 to the metabolism of galactolipids, that build the chloroplast membranes. It might results in the protection of hvera1.b photosystem and thus, in its better photosynthesis performance under water stress. Together, these results indicate the possible mechanism of the primary cause of the observed alterations in the hvera1.b mutant.

The Aspergillus fumigatus farnesyltransferase β-subunit, RamA, mediates growth, virulence, and antifungal susceptibility

ABSTRACTPost-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αβ heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The β-subunit is unique to each enzyme. Farnesyltransferase activity is an important mediator of protein localization and subsequent signaling for multiple proteins, including Ras GTPases. Here, we examined the importance of protein farnesylation in the opportunistic fungal pathogen Aspergillus fumigatus through generation of a mutant lacking the farnesyltransferase β-subunit, ramA. Although farnesyltransferase activity was found to be non-essential in A. fumigatus, diminished hyphal outgrowth, delayed polarization kinetics, decreased conidial viability, and irregular distribution of nuclei during polarized growth were noted upon ramA deletion (ΔramA). Although predicted to be a target of the farnesyltransferase enzyme complex, we found that localization of the major A. fumigatus Ras GTPase protein, RasA, was only partially regulated by farnesyltransferase activity. Furthermore, the farnesyltransferase-deficient mutant exhibited attenuated virulence in a murine model of invasive aspergillosis, characterized by decreased tissue invasion and development of large, swollen hyphae in vivo. However, loss of ramA also led to a Cyp51A/B-independent increase in resistance to triazole antifungal drugs. Our findings indicate that protein farnesylation underpins multiple cellular processes in A. fumigatus, likely due to the large body of proteins affected by ramA deletion.

The Role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis Stomatal Responses Is Beyond ABA Signaling.

Proper stomatal responses are essential for plant function in an altered environment. The core signaling pathway for abscisic acid (ABA)-induced stomatal closure involves perception of the hormone that leads to the activation of guard cell anion channels by the protein kinase OPEN STOMATA1. Several other regulators are suggested to modulate the ABA signaling pathway, including the protein ENHANCED RESPONSE TO ABA1 (ERA1), that encodes the farnesyl transferase β-subunit. The era1 mutant is hypersensitive to ABA during seed germination and shows a more closed stomata phenotype. Using a genetics approach with the double mutants era1 abi1-1 and era1 ost1, we show that while era1 suppressed the high stomatal conductance of abi1-1 and ost1, the ERA1 function was not required for stomatal closure in response to ABA and environmental factors. Further experiments indicated a role for ERA1 in blue light-induced stomatal opening. In addition, we show that ERA1 function in disease resistance was independent of its role in stomatal regulation. Our results indicate a function for ERA1 in stomatal opening and pathogen immunity.

Ram1 gene, encoding a subunit of farnesyltransferase, contributes to growth, antifungal susceptibility to amphotericin B of Aspergillus fumigatus.

Farnesylation, which is catalyzed by farnesyltransferase, is an important posttranslational process. The function of farnesyltransferase has been previously explored in Cryptococcus neoformans and Candida albicans. Aspergillus fumigatus is an important human opportunistic fungal pathogen in immunocompromised patients. Here we discover the role of the ram1 gene, encoding the β-subunit of farnesyltransferase in A. fumigatus, in the fungal growth and antifungal susceptibility. In this study the ram1 gene was disrupted using A. tumefaciens-mediated transformation. The morphology and radial growth of Δram1 were observed. Assays of disk diffusion and broth microdilution were used to determine the susceptibility of Δram1 mutant to commonly clinical used antifungals and the farnesyltransferase inhibitor manumycin A. Deletion of ram1 resulted in a reduced radial growth of A. fumigatus but did not affect the microscopic morphology. Δram1 showed increased susceptibility to the antifungal amphotericin B; however, its susceptibility to azoles and caspofungin was the same to that to the parental strain. Our data indicate that farnesyltransferase is a potential target for design new antifungal agents.

Role of protein farnesylation events in the ABA-mediated regulation of the Pinoresinol–Lariciresinol Reductase 1 (LuPLR1) gene expression and lignan biosynthesis in flax (Linum usitatissimum L.)

A Linum usitatissimum LuERA1 gene encoding a putative ortholog of the ERA1 (Enhanced Response to ABA 1) gene of Arabidopsis thaliana (encoding the beta subunit of a farnesyltransferase) was analyzed in silico and for its expression in flax. The gene and the protein sequences are highly similar to other sequences already characterized in plants and all the features of a farnesyltransferase were detected. Molecular modeling of LuERA1 protein confirmed its farnesyltransferase nature. LuERA1 is expressed in the vegetative organs and also in the outer seedcoat of the flaxseed, where it could modulate the previously observed regulation operated by ABA on lignan synthesis. This effect could be mediated by the regulation of the transcription of a key gene for lignan synthesis in flax, the LuPLR1 gene, encoding a pinoresinol lariciresinol reductase. The positive effect of manumycin A, a specific inhibitor of farnesyltransferase, on lignan biosynthesis in flax cell suspension systems supports the hypothesis of the involvement of such an enzyme in the negative regulation of ABA action. In Arabidopsis, ERA1 is able to negatively regulate the ABA effects and the mutant era1 has an enhanced sensitivity to ABA. When expressed in an Arabidopsis cell suspension (heterologous system) LuERA1 is able to reverse the effect of the era1 mutation. RNAi experiments in flax targeting the farnesyltransferase β-subunit encoded by the LuERA1 gene led to an increase LuPLR1 expression level associated with an increased content of lignan in transgenic calli. Altogether these results strongly suggest a role of the product of this LuERA1 gene in the ABA-mediated upregulation of lignan biosynthesis in flax cells through the activation of LuPLR1 promoter. This ABA signaling pathway involving ERA1 probably acts through the ABRE box found in the promoter sequence of LuPLR1, a key gene for lignan synthesis in flax, as demonstrated by LuPLR1 gene promoter-reporter experiments in flax cells using wild type and mutated promoter sequences.

A farnesylated G-protein suppresses Akt phosphorylation in INS 832/13 cells and normal rat islets: Regulation by pertussis toxin and PGE 2

Protein isoprenylation constitutes incorporation of either 15-carbon farnesyl or 20-carbon geranylgeranyl derivative of mevalonic acid onto the C-terminal cysteine, culminating in increased hydrophobicity of the modified proteins for optimal membrane anchoring and interaction with their respective effectors. Emerging evidence confirms the participatory role of prenylated proteins in pancreatic β-cell function including insulin secretion. Herein, we investigated the putative regulatory roles of protein farnesylation in cell survival signaling pathways in insulin-secreting INS 832/13 cells and normal rodent islets, specifically at the level of protein kinase-B/Akt phosphorylation induced by insulin-like growth factor [IGF-1]. Selective inhibitors of farnesylation [e.g., FTI-277 or FTI-2628] or knockdown of the β-subunit of farnesyl transferase by siRNA significantly increased Akt activation under basal and IGF-1-stimulated conditions. Consequentially, the relative abundance of phosphorylated FoxO1 and Bad were increased implicating inactivation of critical components of the cell death machinery. In addition, FTI-induced Akt activation was attenuated by the PI3-kinase inhibitor, LY294002. Exposure of INS 832/13 cells to pertussis toxin [PTx] markedly potentiated Akt phosphorylation suggesting involvement of a PTx-sensitive G-protein in this signaling axis. Furthermore, prostaglandin E 2, a known agonist of inhibitory G-proteins, significantly attenuated FTI-induced Akt phosphorylation. Taken together, our findings suggest expression of a farnesylated G-protein in INS 832/13 cells and normal rat islets, which appear to suppress Akt activation and subsequent cell survival signaling steps. Potential regulatory roles of the islet endogenous protein kinase-B inhibitory protein [Probin] in islet function are discussed.

Functional Analysis of Arabidopsis Postprenylation CaaX Processing Enzymes and Their Function in Subcellular Protein Targeting

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase beta-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins.

Farnesyltransferase Inhibitors Induce DNA Damage via Reactive Oxygen Species in Human Cancer Cells

Farnesyltransferase inhibitors (FTIs) possess antitumor activity. Based on recent findings, we hypothesized that FTIs induce reactive oxygen species (ROS) that damage DNA, leading to DNA damage responses. To test this hypothesis, we investigated the effects of FTIs on the generation of ROS, DNA double-strand breaks (DSB), DNA damage responses, and RhoB, and the effects of quenching ROS on these FTI effects. We evaluated four FTIs in human cancer cell lines of different tissue origins. We found that FTIs induced ROS and DSBs. Suppressing expression of the beta-subunit of farnesyltransferase with siRNA did not induce ROS, but slightly attenuated the ROS induced by FTIs. N-acetyl-L-cysteine (NAC), but not caspase inhibitors, blocked FTI-induced DSBs, suggesting that the DSBs were caused by ROS and did not result from apoptosis. The DSBs led to DNA damage responses. H2AX became phosphorylated and formed nuclear foci. The DNA-damage-sensing molecules involved were probably ataxia-telangiectasia mutated protein (ATM) and DNA-dependent protein kinase (DNA-PK) but not ATM- and Rad3-related protein (ATR). Key components of the homologous recombination and nonhomologous end joining repair pathways (DNA-PK, BRCA1, and NBS1) underwent phosphorylation and formed nuclear foci. RhoB, a mediator of the antineoplastic effect of FTIs and a protein inducible by DNA damage, was increased by FTIs. This increase was blocked by NAC. We concluded that FTIs induced oxidative DNA damage by inducing ROS and initiated DNA damage responses, including RhoB induction, and there was a complex relationship among FTIs, farnesyltransferase, ROS, and RhoB. Our data also imply that inhibitors of DNA repair may accentuate the clinical efficacy of FTIs.

Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases.

Meristems require a myriad of intercellular signaling pathways for coordination of cell division within and between functional zones and clonal cell layers. This control of cell division ensures a constant availability of stem cells throughout the life span of the meristem while limiting overproliferation of meristematic cells and maintaining the meristem structure. We have undertaken a genetic screen to identify additional components of meristem signaling pathways. We identified pluripetala (plp) mutants based on their dramatically larger meristems and increased floral organ number. PLURIPETALA encodes the alpha-subunit shared between protein farnesyltransferase and protein geranylgeranyltransferase-I. plp mutants also have altered abscisic acid responses and overall much slower growth rate. plp is epistatic to mutations in the beta-subunit of farnesyltransferase and shows a synergistic interaction with clavata3 mutants. plp mutants lead to insights into the mechanism of meristem homeostasis and provide a unique in vivo system for studying the functional role of prenylation in eukaryotes.

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase β-subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three beta-subunit C-terminal truncation mutants, Delta5, Delta10 and Delta14, which were designed to eliminate a lattice interaction between the beta-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2-3-fold. A new crystalline form of FPT was obtained for the Delta10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 A resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.

Substrate specificity determinants in the farnesyltransferase beta-subunit.

Protein prenyltransferases catalyze the covalent attachment of isoprenoid lipids (farnesyl or geranylgeranyl) to a cysteine near the C terminus of their substrates. This study explored the specificity determinants for interactions between the farnesyltransferase of Saccharomyces cerevisiae and its protein substrates. A series of substitutions at amino acid 149 of the farnesyltransferase beta-subunit were tested in combination with a series of substitutions at the C-terminal amino acid of CaaX protein substrates Ras2p and a-factor. Efficient prenylation was observed when oppositely charged amino acids were present at amino acid 149 of the yeast farnesyltransferase beta-subunit and the C-terminal amino acid of the CaaX protein substrate, but not when like charges were present at these positions. This evidence for electrostatic interaction between amino acid 149 and the C-terminal amino acid of CaaX protein substrates leads to the prediction that the C-terminal amino acid of the protein substrate binds near amino acid 149 of the yeast farnesyltransferase beta-subunit.

Expression of the Farnesyltransferase β-Subunit Gene in Human Ovarian Carcinoma: Correlation to K-rasMutation

Therassignaling protein requires a posttranslational modification to localize it to the inner surface of plasma membrane. In this state it can behave as a signal transduction mediator. Farnesyltransferase plays an important role in this posttranslational processing ofrasby attaching a farnesyl group to the cysteine of therasC-terminal tetrapeptide. In this study, we investigated the relationship of K-rasexpression and mutation with farnesyltransferase β-subunit expression in 20 ovarian tumors (17 carcinomas and 3 low malignant potential tumors) and 4 normal ovaries. The expression level of mRNA was determined by using quantitative PCR and mutation analysis was performed by direct cDNA sequencing. K-rasmutations were found in 1 of 3 low malignant potential tumors and in 4 of 17 carcinoma cases. K-rasmRNA overexpression was found in 1 of 3 low malignant potential tumors (one with mutatedras) and in only 1 of 17 carcinoma cases. Farnesyltransferase β-subunit mRNA overexpression was found in 2 of 3 low malignant potential tumors and in 7 of 17 carcinoma cases. Interestingly, all K-rasmutation cases showed farnesyltransferase β-subunit overexpression. These findings suggest that there may be a direct relationship between K-ras(rasdysfunction) mutation and expression of farnesyltransferase β-subunit gene.