Katarzyna Gawarecka

Candida albicans cis-prenyltransferase Rer2 is required for protein glycosylation, cell wall integrity and hypha formation.

cis-Prenyltransferase is the first enzyme of the mevalonate pathway committed to the biosynthesis of dolichol in eukaryotes. The RER2 gene encoding cis-prenyltransferase (Rer2p) in the human fungal pathogen Candida albicans was characterized. In addition, the ORF19.5236 encoding the second cis-prenyltransferase, which putatively is responsible for the synthesis of longer polyisoprenoids chains, was identified. When cultivated under repressive conditions, the conditional mutant strain expressing the RER2 gene from the regulatable MET3 promoter contained only 4% of cis-prenyltransferase activity and markedly diminished amounts of dolichols, as compared to the wild-type strain. Moreover, transcriptomal analyses revealed changes in the expression of 300 genes, mainly involved in transport, response to stress, filamentous growth and organelle organization. Growth of the conditional strain was blocked completely at 37 °C. The strain was hypersensitive to a wide range of inhibitors, which suggested glycosylation defects and compromised cell wall integrity. Moreover, the rer2 conditional mutant grown in the repressive conditions, unlike the same strain in the absence of repressor, failed to form hyphae. The results indicate that dolichols are essential not only for protein glycosylation and cell wall integrity but also for growth and development of C. albicans.

New insight into the catalytic mechanism of bacterial mray from enzyme kinetics and docking studies

Phospho-MurNAc-pentapeptide translocase (MraY) catalyzes the synthesis of Lipid I, a bacterial peptidoglycan precursor. As such, MraY is essential for bacterial survival and therefore is an ideal target for developing novel antibiotics. However, the understanding of its catalytic mechanism, despite the recently determined crystal structure, remains limited. In the present study, the kinetic properties of Bacillus subtilis MraY (BsMraY) were investigated by fluorescence enhancement using dansylated UDP-MurNAc-pentapeptide and heptaprenyl phosphate (C35-P, short-chain homolog of undecaprenyl phosphate, the endogenous substrate of MraY) as second substrate. Varying the concentrations of both of these substrates and fitting the kinetics data to two-substrate models showed that the concomitant binding of both UDP-MurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I and UMP. We built a model of BsMraY and performed docking studies with the substrate C35-P to further deepen our understanding of how MraY accommodates this lipid substrate. Based on these modeling studies, a novel catalytic role was put forward for a fully conserved histidine residue in MraY (His-289 in BsMraY), which has been experimentally confirmed to be essential for MraY activity. Using the current model of BsMraY, we propose that a small conformational change is necessary to relocate the His-289 residue, such that the translocase reaction can proceed via a nucleophilic attack of the phosphate moiety of C35-P on bound UDP-MurNAc-pentapeptide.

POLYPRENOL REDUCTASE2 Deficiency Is Lethal in Arabidopsis Due to Male Sterility

Arabidopsis PPRD1 and -2, orthologs of human SRD5A3 (steroid 5α reductase type 3), encode polyprenol reductases responsible for conversion of polyprenol to dolichol. Dolichol is a required cofactor for protein glycosylation, the most common posttranslational modification modulating the stability and biological activity of proteins in all eukaryotic cells. We have identified and characterized two genes, PPRD1 and -2, which are orthologous to human SRD5A3 (steroid 5α reductase type 3) and encode polyprenol reductases responsible for conversion of polyprenol to dolichol in Arabidopsis thaliana. PPRD1 and -2 play dedicated roles in plant metabolism. PPRD2 is essential for plant viability; its deficiency results in aberrant development of the male gametophyte and sporophyte. Impaired protein glycosylation seems to be the major factor underlying these defects although disturbances in other cellular dolichol-dependent processes could also contribute. Shortage of dolichol in PPRD2-deficient cells is partially rescued by PPRD1 overexpression or by supplementation with dolichol. The latter has been discussed as a method to compensate for deficiency in protein glycosylation. Supplementation of the human diet with dolichol-enriched plant tissues could allow new therapeutic interventions in glycosylation disorders. This identification of PPRD1 and -2 elucidates the factors mediating the key step of the dolichol cycle in plant cells which makes manipulation of dolichol content in plant tissues feasible.

Polyprenols Are Synthesized by a Plastidial cis -Prenyltransferase and Influence Photosynthetic Performance

A plastidial cis-prenyltransferase synthesizes a family of polyprenols that accumulate in thylakoid membranes and play a central role in the efficiency of photosynthetic performance. Plants accumulate a family of hydrophobic polymers known as polyprenols, yet how they are synthesized, where they reside in the cell, and what role they serve is largely unknown. Using Arabidopsis thaliana as a model, we present evidence for the involvement of a plastidial cis-prenyltransferase (AtCPT7) in polyprenol synthesis. Gene inactivation and RNAi-mediated knockdown of AtCPT7 eliminated leaf polyprenols, while its overexpression increased their content. Complementation tests in the polyprenol-deficient yeast ∆rer2 mutant and enzyme assays with recombinant AtCPT7 confirmed that the enzyme synthesizes polyprenols of ∼55 carbons in length using geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate as substrates. Immunodetection and in vivo localization of AtCPT7 fluorescent protein fusions showed that AtCPT7 resides in the stroma of mesophyll chloroplasts. The enzymatic products of AtCPT7 accumulate in thylakoid membranes, and in their absence, thylakoids adopt an increasingly “fluid membrane” state. Chlorophyll fluorescence measurements from the leaves of polyprenol-deficient plants revealed impaired photosystem II operating efficiency, and their thylakoids exhibited a decreased rate of electron transport. These results establish that (1) plastidial AtCPT7 extends the length of GGPP to ∼55 carbons, which then accumulate in thylakoid membranes; and (2) these polyprenols influence photosynthetic performance through their modulation of thylakoid membrane dynamics.

Effects of liposomes with polyisoprenoids, potential drug carriers, on the cardiovascular and excretory system in rats

BACKGROUND The unpredictable side effects of a majority currently used drugs are the substantial issue, in which patients and physicians are forced to deal with. Augmenting the therapeutic efficacy of drugs may prove more fruitful than searching for the new ones. Since recent studies show that new cationic derivatives of polyisoprenoid alcohols (APrens) might exhibit augmenting properties, we intend to use them as a component of liposomal drug carriers. In this study we investigate if these compounds do not per se cause untoward effects on the living organism. METHODS Male Sprague-Dawley rats received for four weeks daily injections (0.5 ml sc) of liposomes built of dioleoyl phosphatidylethanolamine (DOPE), liposomes built of DOPE and APren-7 (ratio 10:1) or water solvent. Weekly, rats were observed in metabolic cages (24h); blood and urine were sampled for analysis; body weight (BW) and systolic blood pressure (SBP) were determined. After chronic experiment, kidneys and heart were harvested for histological and morphometric analysis. RESULTS The 4-week BW increments were in the range of 97 ± 4 to 102 ± 4%, intergroup differences were not significant. Microalbuminuria was the lowest in the group receiving liposomes with APren-7 (0.22 ± 0.03 mg/day). Water and food intake, plasma and urine parameters were similar in all groups. CONCLUSIONS Newly designed liposomes containing APren-7 did not affect functions of the excretory and cardiovascular systems, and renal morphology; therefore we find them suitable as a component of liposomal drug carriers.

Analysis of Plant Polyisoprenoids

Polyisoprenoid alcohols are representatives of high-molecular terpenoids. Their hydrocarbon chains are built of 5 to more than 100 isoprene units giving rise to polymer molecules that differ in chain-length and/or geometrical configuration. Plants have been shown to accumulate diverse polyisoprenoid mixtures with tissue-specific composition. In this chapter, methods of analysis of polyisoprenoid alcohols in plant material are described, including isolation and purification of polyisoprenoids from plant tissue, fast semiquantitative analysis of the polyisoprenoid profile by thin-layer chromatography (straight phase adsorption and reversed phase partition techniques), and quantification of polyisoprenoids with the aid of high performance liquid chromatography. This approach results in full characterization of complex polyisoprenoid mixtures accumulated in various plant tissues and other matrixes.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus

BackgroundCationic derivatives of polyprenols (trimethylpolyprenylammonium iodides – PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine.MethodsSeveral vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.ResultsThe mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid.ConclusionThe animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

Purpose Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. Methods AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. Results All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation—considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. Conclusion Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

Efficient and non-toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology.

One of the major challenges limiting the development of gene therapy is an absence of efficient and safe gene carriers. Among the nonviral gene delivery methods, lipofection is considered as one of the most promising. In the present study, a set of cationic polyprenyl derivatives [trimethylpolyprenylammonium iodides (PTAI)] with different lengths of polyprenyl chains (from 7, 8 and 11 to 15 isoprene units) was suggested as a component of efficient DNA vehicles.

High-resolution NMR studies of antibiotics in cellular membranes

The alarming rise of antimicrobial resistance requires antibiotics with unexploited mechanisms. Ideal templates could be antibiotics that target the peptidoglycan precursor lipid II, known as the bacterial Achilles heel, at an irreplaceable pyrophosphate group. Such antibiotics would kill multidrug-resistant pathogens at nanomolecular concentrations without causing antimicrobial resistance. However, due to the challenge of studying small membrane-embedded drug–receptor complexes in native conditions, the structural correlates of the pharmaceutically relevant binding modes are unknown. Here, using advanced highly sensitive solid-state NMR setups, we present a high-resolution approach to study lipid II-binding antibiotics directly in cell membranes. On the example of nisin, the preeminent lantibiotic, we show that the native antibiotic-binding mode strongly differs from previously published structures, and we demonstrate that functional hotspots correspond to plastic drug domains that are critical for the cellular adaptability of nisin. Thereby, our approach provides a foundation for an improved understanding of powerful antibiotics.Antibiotics that target the peptidoglycan precursor lipid II are promising templates for next-generation antibiotics. Here authors use solid-state NMR and monitor lipid II-binding antibiotics, such as nisin, directly in cell membranes.