Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

AIMS: The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia. METHODS AND RESULTS: The human-yeast hybrid SQS, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in Saccharomyces cerevisiae strain deleted of its own SQS gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end products, ubiquinone and dolichol, was down-regulated. CONCLUSIONS: The S. cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast strain expressing hybrid SQS with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties.

Life with too much polyprenol: polyprenol reductase deficiency.

Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G>A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis.

Cloning and characterization of Rab Escort Protein (REP) from Arabidopsis thaliana.

Intracellular vesicular trafficking is regulated by Rab proteins, small GTPases that require posttranslational geranylgeranylation for biological activity. This covalent modification is catalyzed by Rab geranylgeranyl transferase (RabGGTase) and proceeds only in the presence of accessory Rab Escort Protein (REP). In this communication, we report the cloning and characterization of REP gene of Arabidopsis thaliana. Highest expression of REP mRNA was detected in leaves and flowers in contrast to stems and roots. AtREP is recognized by anti-rat REP1 serum. Interaction of AtREP with the protein substrate is presented, as well as a structural model obtained through homology modeling, based on the known structure of rat REP1.

Targeting extracellular pyrophosphates underpins the high selectivity of nisin.

The spread of infectious diseases and the increase in antibiotic resistance represent a life-threatening global development that calls for new approaches to control microorganisms. Of all potential targets, the essential and unique pathway of bacterial cell wall synthesis, targeted by the first known antibiotic penicillin, remains a perfect candidate for the development of new antibiotics. Here we show that the lantibiotic nisin exercises its antibacterial action by targeting peptidoglycan intermediates' extracellular pyrophosphate, unique to bacterial cell wall precursors. We show that nisin sequesters cell wall precursors found in the outer leaflet of bacterial plasma membranes, Lipid II and undecaprenyl pyrophosphate, into stable complexes. We propose a model of antibacterial action for nisin in which the terminal amino group of Ile1 targets the pyrophosphate groups of the bacterial cell wall precursors, where it docks via a hydrogen bond. The pyrophosphate moiety, a highly conserved chemical group different from the L-Lys-D-Ala-D-Ala docking motif for vancomycin, has no biochemical analogs with comparable properties and is unlikely to be susceptible to bacterial adaptations akin to those responsible for resistance to penicillins and vancomycin.

Regulatory aspects of coenzyme Q metabolism.

A number of factors are involved in the regulation of the amount and distribution of coenzyme Q in cells and tissues. These factors modify preferentially the biosynthetic mechanism in order to keep up an optimal tissue concentration of the lipid. The amount of substrate provided by the mevalonate pathway is able to both up- and down-regulate the velocity of synthesis. At the translation level, regulation occurs by receptor-mediated ligand binding and appears most clearly upon treatment with hormones and peroxisomal inducers. There are a number of pathophysiological conditions when these mechanisms of regulation are modified and explain the decreased coenzyme Q tissue concentrations. It is of considerable interest to establish appropriate physiological, hormonal and drug-mediated conditions in order to counteract disturbed cellular functions caused by coenzyme Q deficiency

Search for polyprenols in leaves of evergreen and deciduous Ericaceae plants.

Various species and cultivars of Ericaceae family were checked for the presence of long-chain polyprenols in their leaves. In the genus Rhododendron no polyprenols were found in the ever-green species, while they were present in the deciduous type. The polyprenols were of chain-length of 14-20 isoprene residues and they occurred in the form of acetic acid esters. The polyprenol accumulation is discussed with respect to senescence of leaves.

A novel family of longer chain length dolichols present in oleate-induced yeast Saccharomyces cerevisiae.

The typical size of the yeast dolichol family ranges from 14 to 19 isoprene units D((14-19)) with dolichol(16) being the dominating species. Induction of peroxisome proliferation by growing the cells in medium containing oleate as carbon source induces the synthesis of an additional family of longer dolichols D((19-24)) with D(21) being the most prominent. This phenomenon is abolished in the peroxisome biogenesis deficient strain in which the PEX1 gene (encoding Pex1p peroxin) has been disrupted. The total amount of dolichols in pex1Delta cells is lower than in the wild-type cells, as is the amount of phosphatidylcholine. Moreover, the levels of 3-hydroxy-3-methylglutaryl CoA reductase and farnesyl diphosphate synthase, two key enzymes in dolichol biosynthesis, are decreased in the absence of a functional PEX1 gene. The presence of longer dolichols in oleate-induced Saccharomyces cerevisiae cells, the absence of this additional family in peroxisome deficient cells, and a decrease of the total amount of dolichols in these cells indicate the involvement of peroxisomes in the biosynthesis of dolichols in this organism.

Assay for Rab geranylgeranyltransferase using size exclusion chromatography.

A simple chromatographic assay for Rab geranylgeranyltransferase (Rab GGTase) has been developed. The method involves separation of the reaction mixture on a Sephadex G-25 superfine minicolumn. Addition of 2-propanol to the assay results in substantial (approximately 90%) decline of formation of noncovalent lipid-protein complexes, increasing reproducibility and reliability of the method. The activity of Rab prenyltransferase was measured in crude and partially purified enzyme preparations from wheat seedlings; measurements for several other plants and rat brain cytosol fractions are also presented. This method can be routinely applied to evaluate the activity of different protein prenyltransferases.

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway.

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase.

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product. The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity. Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely. This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling. We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.

Single polyprenol and dolichol isolation by semipreparative high-performance liquid chromatography technique.

A new method of separation of single polyprenols (or dolichols) from a mixture of isoprenoid alcohols is described. Application of a high-performance liquid chromatography (HPLC) apparatus equipped with a semipreparative ODS column resulted in preparation of long-chain (dihydro)polyprenols of high purity (>95%). This approach substantially decreases the time scale of the conventional chromatographical preparative procedure. The method can be widely used in chemical and biochemical projects, where single polyprenols or dolichols are required.

Modulation of properties of phospholipid membranes by the long-chain polyprenol (C(160)).

The electrical measurements of phospholipid bilayers and the studies of phospholipid vesicles by using the transmission electron microscopy (TEM) showed that dotriacontaprenol (C(160)) isolated from leaves of Spermatophyta influences some properties of membranes. The current-voltage characteristics, the membrane conductance-temperature relationships, the membrane breakdown voltage and the membrane capacitance have been measured for different mixtures of C(160)/DOPC. The membrane conductance, the activation energy of ion migration across the membrane and the membrane thickness were determined. Dotriacontaprenol decreases the membrane breakdown voltage, the activation energy and the membrane capacitance, and increases the membrane conductance and the membrane hydrophobic thickness. The analysis of TEM micrographs shows several characteristic structures, which have been described. The results indicate that dotriacontaprenol increases the membrane elasticity and modulates the surface curvature of the membranes by the formation of fluid microdomains. We suggest that the long polyprenols facilitate the formation of transmembrane, ions-conductive pores.

Half-life of ubiquinone and plastoquinone in spinach cells.

The half-life of plastoquinone (PQ), ubiquinone-9 (UQ-9) and ubiquinone-10 (UQ-10) in spinach tissue was determined. This was achieved by monitoring the decay of radioactivity incorporated into these lipids from a labeled precursor. The half-life of PQ was 15 h while for UQ-9 and UQ-10 it was longer, i.e. 30 h. The values of half-lives of PQ and UQ suggest a high rate of turnover of these lipids in spinach cells.

The regulation of activity of main mevalonic acid pathway enzymes: farnesyl diphosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and squalene synthase in yeast Saccharomyces cerevisiae.

The co-regulation of the main mevalonic acid pathway enzymes was investigated in the yeast Saccharomyces cerevisiae. It was found that a 6-fold increase in FPPS activity compared with that of the wild-type strain FL100 did not cause significant changes in HMG-CoA reductase activity, while the amounts of synthesized dolichols and ergosterol increased by 80 and 32%, respectively. The disruption of the SQS gene in the strain grown in the presence of ergosterol repressed the activities of both FPP synthase and HMG-CoA reductase to a comparable degree, whereas in the same strain starved for ergosterol the activity of FPPS was 10-fold higher and HMG-CoA reductase activity was practically unchanged. We show that FPPS is the enzyme that regulates the flow rate of synthesized mevalonic acid pathway products independent of HMG-CoA reductase and SQS.

Subcellular localization of plastoquinone and ubiquinone synthesis in spinach cells.

In vivo labeling of spinach etiolated leaves with [(3)H]mevalonate followed by rapid cell fractionation procedure showed that ER-Golgi membranes are involved in transport of plastoquinone (PQ) and ubiquinone (UQ) to plastids and mitochondria, respectively. Translocation of these lipids was inhibited by agents which affect protein and lipid intracellular transport causing structural and functional disintegration of the ER-Golgi system (monensin, brefeldin) and interfere with mitochondrial energy conservation (carbonyl cyanide m-chlorophenylhydrazone), but was not affected by colchicine which influences the organization of the cytoskeletal network. Colchicine treatment resulted in marked stimulation of PQ and UQ synthesis. Results of experiments with pre-exposure of etiolated seedlings to light suggest that translocation processes are dependent on the plastid developmental state and their capacity as acceptors of PQ. Thus, the experiments indicate that biosynthesis and transport of PQ and UQ involve multiple cellular compartments and that kinetics of the transport process is dependent on the actual physiological conditions.

Polyprenols in hairy roots of Coluria geoides.

Long-chain polyisoprenoid alcohols built from several up to more than 100 isoprenoid units are common constituents of all living organisms. They were found mostly in plants, bacteria, yeasts and mammalian cells. In vitro hairy root culture of Coluria geoides was obtained from plants transformed with Agrobacterium rhizogenes. Growth was optimal at 0.75% (w/v) glucose and at 22 degrees C. Dry samples of roots were extracted and lipid content was analysed by HPLC. According to our estimation, polyprenols are accumulated in roots of C. geoides cultivated in vitro as a mixture of several prenologues with the dominating prenol composed of 16 isoprenoid units. The content of polyprenols in tissue was approx. 300 microg/g of dry weight.

Ras protein of the slime mold Physarum polycephalum is farnesylated in vitro.

Physarum Ppras1 protein was efficiently prenylated by prenyltransferases of spinach. Surprisingly in spite of the C-terminal sequence (CLLL) specific for geranylgeranylation the protein was preferentially farnesylated. Consequences of this observation are discussed.

Phosphorylation of farnesol in rat liver microsomes: properties of farnesol kinase and farnesyl phosphate kinase.

As farnesol may serve as a nonsterol endogenous regulator of the mevalonate pathway, the possibility that a kinase specific for its phosphorylation is present in the rat liver was investigated. In the 10,000 g supernatant of rat liver, farnesyl monophosphate was synthesized in the presence of ATP. The Km value for farnesol was 2.3 microM. Various detergents inhibited the activity of the enzyme. The farnesol kinase was present in rough and in smooth I microsomes, but not in smooth II microsomes, mitochondria, peroxisomes, Golgi, or plasma membranes. The enzyme was associated with the inner, luminal surface of the vesicles. Further analyses have demonstrated that an enzymatic mechanism exists which catalyzes the phosphorylation of farnesyl-P to farnesyl-PP. Activity of the farnesyl phosphate kinase resulted in the phosphorylation of the monophosphate by CTP but not by ATP, GTP, or UTP. This enzyme is activated by low concentrations of detergents. Treatment with proteases and chemical probes indicate that this second phosphorylation reaction probably takes place on the outer, cytoplasmic surface of microsomal vesicles. These results demonstrate that rat liver microsomes contain two enzymes for the consecutive phosphorylation of farnesol to farnesyl-PP.

The diversity of polyprenol pattern in leaves of fruit trees belonging to Rosaceae and Cornaceae.

The polyprenol pattern in leaves of fruit trees belonging to the Rosaceae (genera: Prunus, Malus) and Cornaceae (genus: Cornus) families is presented. The content of polyprenyl acetates varied within plant species between 10-50 mg per gram of dry weight. In genus Prunus, Cornus and in representatives of species Malus domestica, a mixture of polyprenols composed of 18, 19, 20, 21 isoprene units was found. In six species of genus Prunus (sour-cherry): P. serrulata-spontanea, P. yedoensis, P. fruticosa. P. kurilensis, P. subhirtella and P. incisa the presence of a second polyprenol family, i.e. the group of prenologues consisting of prenol -35, -36, -37, etc. up to -42 was detected.

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.