Farnesyl Diphosphate Synthetase Research Articles

Hydroxypropyl methylcellulose/sodium alginate/hydroxyapatite nano biomaterial enriched with zinc to promote bone tissue augmentation

Bone is a dynamic connective tissue that provides structural support and locomotion to human physiology. Severe bone degeneration can occur in cases of trauma, infections, genetic disorders, tumors, and hormonal imbalances. This leads to a demand for scaffolds and composites to enhance bone tissue regeneration. The present study aimed to develop a composite material using hydroxypropyl methylcellulose and sodium alginate in combination with hydroxyapatite. The composite was doped with zinc and developed into a nanocomposite using ionic gelation and solvent precipitation methods. The prepared nanocomposite was characterized by scanning electron microscopy, atomic force microscopy, dynamic light scattering, FTIR, and XRD studies. In vivo pharmacological studies were conducted to establish the bone targeting and bone tissue regeneration efficacy of the developed nanocomposite. The nanocomposite was checked for cell toxicity by performing an MTT assay using MG63 cell line and the molecular mechanism of targeting efficiency was assessed by an in silico evaluation method. The results of the study confirmed the polymeric interlinking in the nanocomposite and the successful preparation of a composite material using biodegradable excipients. SEM and AFM studies indicated that the nanocomposite was within the 80–200 nm size range. MTT assay indicated that the developed nanocomposite was not cytotoxic and well tolerated by the MG63 cell line. In vivo, studies in validation with molecular docking evaluation suggested that the nanocomposite was effectively targeted to the bone and enhanced the bone density by blocking Farnesyl Diphosphate Synthetase (FDPS) in the mevalonate pathway which is essential for inhibiting the osteoclastic activity and promoting osteogenesis.

In silico pharmacodynamics, toxicity profile and biological activities of the Saharan medicinal plant Limoniastrum feei

In-silico study was performed to find the pharmacodynamics, toxicity profiles and biological activities of three phytochemicals isolated from Limoniastrum feei (Plumbagenaceae). Online pharmacokinetic tools were used to estimate the potential of Quercetin, kaempferol-3-O-β-D-glucopyranoside (astragalin) and quercitin-7-O-β-D-glucopyranoside as specific drugs. Then the prediction of potential targets of these compounds were investigated using PharmMapper. Auto-Dock 4.0 software was used to investigate the different interactions of these compounds with the targets predicted earlier. The permeability of quercetin was found within the range stated by Lipinski ׳s rule of five. Hematopoietic prostaglandin (PG) D synthase (HPGDS), farnesyl diphosphate synthetase (FPPS) and the deoxycytidine kinase (DCK) were potential targets for quercetin, astragalin and quercetin 7, respectively. Quercetin showed antiallergic and anti-inflammatory activity, while astragalin and quercetin 7 were predicted to have anticancer activities. The activity of Astragalin appeared to be mediated by FPPS inhibition. The inhibition of DCK was predicted as the anticancer mechanisms of quercetin 7. The compounds showed interesting interactions and satisfactory binding energies when docked into their targets. These compounds are proposed to have activities against a variety of human aliments such as allergy, tumors, muscular dystrophy, and diabetic cataracts.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

P1-01-04: Immunological Effects of Bisphosphonates on gdT Cells in Breast Cancer.

Abstract Background: Human γδT cells play an important role in the first-line of defence. Accumulating data demonstrate that γδ T cells recognize nonpeptide antigens such as IPP and HMBPP and activated γδT cells have potent cytotoxic activity against tumor cells. Nitrogen-containing bisphosphonates like zoledronic acid inhibit farnesyl diphosphate synthetase, resulting in the accumulation of upstream metabolites such as IPP. Previous studies on the adoptive transfer of ex-vivo amplified γδ T cells demonstrated the safety and feasibility of γδT cell-based immunotherapy for metastatic cancer. According to ABCSG-12 clinical trial, zoldedoric acid improved DFS of patients with early-stage breast cancer under adjuvant endocrine therapy. Whereas a precise mechanism remained unclear, it is likely that γδT cells are involved in this anti-tumor effect in breast cancer. Materials and Methods: Peripheral blood mononuclear cells derived from 63 (54 untreated non-metastatic and 9 metastatic) patients with breast cancer were examined for the baseline population of γδT cells and their proliferative responses to zoledronic acid in vitro. Tumor-infiltrating lymphocytes were isolated from 20 tumor biopsies and 2 pleural effusions. Peripheral blood γδT cells were stimulated with 5 μM zoledronic acid and cultured in the presence of 100 IU/ml IL-2 for 10 days. Amplified γδ T cells were analyzed for cytokine production and cytotoxic activity. Results: The proportion of γδ (TCRVδ2 -bearing) T cells among peripheral blood CD3+ cells was 4.2 ± 5.0%. Although it was equivalent to the previously reported value of 4.3 ± 2.2% in healthy volunteers, 14% (9/63) of patients exhibited an increase in the number of peripheral blood γδT cells at baseline. The level of TCRVδ2 T cells with potent anti-tumor activity was significantly higher than that of TCRVδ1 T cells (4.2± 5.0% vs. 1.5 ± 1.8%, p<0.001), whereas they were present in tumor-infiltrating cells at essentially the same levels (4.4± 9.6% vs. 3.7 ± 9.7%, p=0.434). Percentage of TCR Vδ2+ CD3+ cells among amplified γδ T cells in vitro and cytokine (IFN-γ and TNF-α) production by them were significantly proportional to the initial level of TCRVδ2+/CD3+ ratio in peripheral blood. Upon treatment with zoledronic acid, breast cancer cell lines became susceptible to γδ T cells. Conclusions: γδ (TCR Vδ2-bearing) T cells increased in some patients with breast cancer and the TCRVδ2+/CD3+ ratio at baseline was correlated with the efficiency of proliferation and cytotoxic activity when treated with zoledronic acid. Zoledronic acid efficiently sensitized breast tumor cells to be lyzed by γδ T cells. The present results suggest that γδ T cells stimulated with zoledronic acid can be utilized for immunotherapy against breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-04.

Ginger phytochemicals mitigate the obesogenic effects of a high-fat diet in mice: a proteomic and biomarker network analysis.

Natural dietary anti-obesogenic phytochemicals may help combat the rising global incidence of obesity. We aimed to identify key hepatic pathways targeted by anti-obsogenic ginger phytochemicals fed to mice. Weaning mice were fed a high-fat diet containing 6-gingerol (HFG), zerumbone (HFZ), a characterized rhizome extract of the ginger-related plant Alpinia officinarum Hance (high fat goryankang, HFGK) or no phytochemicals (high-fat control, HFC) for 6 wks and were compared with mice on a low-fat control diet (LFC). Increased adiposity in the HFC group, compared with the LFC group, was significantly (p<0.05) reduced in the HFG and HFGK groups without food intake being affected. Correlation network analysis, including a novel residuals analysis, was utilized to investigate relationships between liver proteomic data, lipid and cholesterol biomarkers and physiological indicators of adiposity. 6-Gingerol significantly increased plasma cholesterol but hepatic farnesyl diphosphate synthetase, which is involved in cholesterol biosynthesis was decreased, possibly by negative feedback. Acetyl-coenzyme A acyltransferase 1 and enoyl CoA hydratase, which participate in the β-oxidation of fatty acids were significantly (p<0.05) increased by consumption of phytochemical-supplemented diets. Dietary ginger phytochemicals target cholesterol metabolism and fatty acid oxidation in mice, with anti-obesogenic but also hypercholesterolemic consequences.

Overcoming multidrug-resistance in cancer: Statins offer a logical candidate

About seven million people die of cancer every year. This is largely due to development of drug resistance, particularly multidrug resistance, in the tumor cells. Multidrug resistance (MDR) arises due to over-expression of MDR proteins in the cancer cells, which cause efflux of anticancer drugs from the cells using ATP. MDR proteins are members of the family of ABC transporters that occur universally, and are structurally and functionally conserved during evolution. In Drosophila, the germ cell attractant peptide is secreted by an ABC transport protein, mdr49. Recently, the peptide has been shown to undergo conjugation with the lipid geranylgeranyl before secretion. If conjugation with the lipid is inhibited, mdr49 protein is unable to transport the peptide. Similarly, in the case of yeast mating factor pheromone, farnesylation is required to occur before the export of the pheromone by ste6 protein, an ABC transporter. In view of the homology of mdr49 and ste6 proteins with mammalian MDR proteins, we postulate that the drug transporters also require their ligands to be conjugated to a lipid. This view finds support from the studies with synthetic inhibitors of geranylgeranyl-/farnesyl-diphosphate synthetase or transferase: The inhibitors are reported to overcome multidrug resistance in cancer cell lines or xenografts in animals. Thus, the MDR transporters also appear to require their substrates to be conjugated with a lipid. Statins are the widely used inhibitors of HMG-CoA reductase. By depleting precursors of the mevalonate pathway, statins can prevent the formation of lipids like geranylgeranyl and farnesyl. Accordingly, they should also be able to overcome multidrug resistance in cancer. A few reports in the literature indicate that they appear to do so. Statins are in wide clinical use, and their pharmacology is well known. Besides, statins per se have mild beneficial effect on the outcome of the disease. We propose that statins should be seriously investigated for their ability to overcome multidrug resistance in cancer. This should be done after careful standardization of the protocol of simultaneous treatment with anticancer drugs and a statin.

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

Characterization and functional analysis of cis-acting elements of the human farnesyl diphosphate synthetase (FDPS) gene 5′ flanking region

Farnesyl diphosphate synthetase (FDPS) is a key enzyme in the isoprenoid pathway responsible for cholesterol biosynthesis, post-translational protein modifications and synthesis of steroid hormones, whose expression is regulated by phorbol esters and polyunsaturated fatty acids. Genomic comparison of the 5′ upstream sequence of the FDPS genes identifies conserved binding sites for NF-Y, SP1, SRE3, and YY1 regulatory elements in rat, mouse, dog and chimpanzee. Two additional specific consensus sequences, upstream of the core promoter that had not been analysed previously, are shared only by human and chimpanzee genomes. The work presented here aimed at characterizing these genomic sequence elements in the human FDPS promoter region and their contribution to gene expression. We have characterized functionally the minimal basal promoter of the human FDPS gene by means of deletion mutants and we have identified two cis-acting elements which modulate the FDPS gene expression and are recognized by Pax5 and OCT-1 transcription factors.

Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3‐L1 adipocyte differentiation

In the present study, we performed a differential proteomic analysis using 2-DE combined with MS to clarify the molecular mechanism for the suppressive effect of chitosan oligosaccharides (CO) during differentiation of adipocyte 3T3-L1. Cell differentiation was significantly inhibited by CO at the concentration of 4 mg/mL. Protein mapping of adipocyte homogenates by 2-DE revealed that numerous protein spots were differentially altered in response to CO treatment. Out of 50 identified proteins showing significant alterations, six were up-regulated and 44 were down-regulated by CO treatment in comparison to control mature adipocytes. Among them, most of the proteins are associated with lipid metabolism, cytoskeleton, and redox regulation, in which the levels of farnesyl diphosphate synthetase (FDS), dedicator of cytokinesis 9 (DOCK9), and chloride intracellular channel 1 (CLIC1) were significantly reduced (>two-fold) with CO treatment. These results have not previously been examined in the context of adipogenesis, and thus can be used as novel biomarkers. Taken together with immunoblot analysis, it was concluded that the inhibitory effect of CO on adipocyte differentiation was mediated by C/EBPalpha and PPARgamma pathway through significant downregulations of important adipogenic molecules such as fatty acid binding protein and glucose transporter 4.

Calcium ionophore treatment impairs the sterol-mediated suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, and farnesyl diphosphate synthetase.

We report that the sterol-mediated suppression of the mRNA levels of three cholesterogenic enzymes, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and farnesyl diphosphate (FPP) synthetase is partially overcome by the calcium ionophore A23187. Addition of A23187 to the human monocytic leukemia cell line THP-1 in the presence of fetal calf serum led to rapid increases in mRNA concentration of up to 40-fold for HMG-CoA synthase and 15-fold for HMG-CoA reductase with little or no change in FPP synthetase mRNA levels. Treatment of HepG2 cells with A23187 resulted in approximately 2-4-fold increases in the mRNA levels for these three enzymes. The increases in HMG-CoA synthase and HMG-CoA reductase mRNAs were maximal after treatment of THP-1 cells with 10 micrograms/ml A23187 for 3 h. The stimulation was blocked by actinomycin D but not by cycloheximide treatment. Ionophore treatment had no effect on the half-lives of the mRNAs for HMG-CoA reductase and HMG-CoA synthase. Surprisingly, the addition of A23187 to THP-1 cells incubated in the presence of 25-hydroxycholesterol and mevalonic acid also led to significant increases in the mRNA levels for HMG-CoA reductase and HMG-CoA synthase. Finally, the stimulation of these mRNA levels by A23187 was reduced in cells in which protein kinase C had been inactivated by preincubation of the cells with a phorbol ester. Taken together, these data suggest that A23187 treatment results in increased transcription of HMG-CoA reductase, HMG-CoA synthase, and, in some cell types, FPP synthetase by a mechanism that does not involve de novo protein synthesis. We speculate that A23187 treatment results in the modification of a trans-acting factor(s) which is common for the transcription of all these genes.

Introduction of terpene-producing ability in a wine strain of Saccharomyces cerevisiae

The terpene-producing ability due to mutation in the ERG 20 gene coding for the farnesyl diphosphate synthetase in Saccharomyces cerevisiae was introduced into a wine yeast by crossing its meiotic progeny with an erg20 laboratory strain. After three backcross generations, strains displaying improved technological properties and more typical terpenic profile were obtained. These strains, which contribute a strong muscatel-type aroma to processed grape juice, are henceforth available for industrial applications.

Sterol pathway in yeast. Identification and properties of mutant strains defective in mevalonate diphosphate decarboxylase and farnesyl diphosphate synthetase

Yeast mutant strains auxotrophic for ergosterol and blocked in mevalonate diphosphate decarboxylase (erg19) and farnesyl diphosphate (FPP) synthetase (erg20) were isolated. The main feature of the mutants blocked in FPP synthetase is their ability to excrete prenyl alcohols, such as geraniol and farnesol. The isolation of the functional ERG20 gene allowed us to show that farnesyl diphosphate synthetase could be a rate limiting enzyme in ergosterol biosynthesis in yeast.

Isolation and properties of yeast mutants affected in farnesyl diphosphate synthetase

Two yeast mutant strains auxotrophic for ergosterol and blocked in farnesyl diphosphate synthetase (EC 2.5.1.1) were isolated. Genetic analysis has shown that these mutant strains carry additional mutations in the ergosterol pathway besides erg20-1 and erg20-2 which affect FPP synthetase. The novel feature of these mutants is their ability to excrete prenyl alcohols (farnesol and geraniol). As geraniol is toxic for yeast cells, the above leaky mutations in FPP synthetase have to be associated with others in the sterol pathway, in order to slow down geraniol synthesis.

Farnesyl diphosphate synthetase: Molecular cloning, sequence, and expression of an essential gene from Saccharomyces cerevisiae

Farnesyl diphosphate (FPP) synthetase is a key enzyme in isoprenoid biosynthesis which supplies C15 precursors for several classes of essential metabolites including sterols, dolichols, and ubiquinones. The structural gene for FPP synthetase was isolated on a 4.5-kilobase EcoRI genomic restriction fragment from the yeast Saccharomyces cerevisiae. The clone encodes a 40,483-dalton polypeptide of 342 amino acids with a high degree of similarity to the protein encoded by a putative rat liver clone of FPP synthetase (Clarke, C. F., Tanaka, R. D., Svenson, K., Wamsley, M., Fogelman, A. M., and Edwards, P. A. (1987) Mol. Cell Biol. 7, 3138–3146) and to an active site protein fragment from avian liver FPP synthetase (Brems, D. N., Bruenger, E., and Rilling, H. C. (1981) Biochemistry 20, 3711–3718). When cloned into the yeast shuttle vector YRp17, the 4.5-kilobase EcoRI fragment directed a 2–3-fold over-expression of FPP synthetase activity in transformed yeast cells. The levels of expression were independent of culture growth phase and orientation of the insert, indicative of a functional promoter in the clone. Disruption of the FPP synthetase gene from a diploid yeast strain, followed by dissection and analysis of tetrads, demonstrates that the gene is an essential, single copy number gene in yeast. The gene for FPP synthetase resides on chromosome XI as judged from Southern blots of separated yeast chromosomes.

Isolation and characterization of isoprene mutants of Escherichia coli

Isoprenoid compounds are found in all organisms. In Escherichia coli the isoprene pathway has three distinct branches: the modification of tRNA; the respiratory quinones ubiquinone and menaquinone; and the dolichols, which are long-chain alcohols involved in cell wall biosynthesis. Very little is known about procaryotic isoprene biosynthesis compared with what is known about eucaryote isoprene biosynthesis. This study approached some of the questions about isoprenoid biosynthesis and regulation in procaryotes by isolating and characterizing mutants in E. coli. Mutants were selected by determining their resistance to low levels of aminoglycoside antibiotics, which require an electron transport chain for uptake into bacterial cells. The mutants were characterized with regard to their phenotypes, map positions, enzymatic activities, and total ubiquinone content. In particular, the enzymes studied were isopentenyldiphosphate delta-isomerase (EC 5.3.3.2), farnesyldiphosphate synthetase (EC 2.5.1.1), and higher prenyl transferases.

Determination of isopentenyl diphosphate and farnesyl diphosphate in tissue samples with a comment on secondary regulation of polyisoprenoid biosynthesis

A double-isotope dilution procedure is described for the determination of two isoprenoid precursors, isopentenyl and farnesyl diphosphate. Recovery of each is determined by the addition of the appropriate radioactive diphosphate to the tissue sample. After partial purification, each is coupled by a prenyltransferase with a cosubstrate of known specific activity. The products, doubly labeled farnesyl and geranylgeranyl diphosphates, are cleaved to the parent alcohols by alkaline phosphatase. The resulting polyprenols are isolated by reversed-phase thin-layer chromatography and their radioisotopic content is determined. The levels of these precursors have been measured in livers of rats and mice that have been maintained on several different diets. The concentration of each was about 0.5 μmol/g wet tissue and varied as much as 10-fold under the different test conditions. The levels of isopentenyl diphosphate isomerase, farnesyl diphosphate synthetase, and squalene synthetase were also measured in these animals. The changes in levels of these enzymes, in conjunction with the variation in substrate concentrations, are such that they could substantially influence the rate of cholesterol synthesis in liver.

ChemInform Abstract: Methane‐ and Difluoromethanediphosphonate Analogues of Geranyl Diphosphate: Hydrolysis‐Inert Alternate Substrates.

AbstractThe reactions of compounds (I) with isopentenyl diphosphate catalyzed by avian liver farnesyl diphosphate synthetase, leading to farnesyl diphosphate as the exclusive product in each case, are studied kinetically using 3H and 14C radiolabel.