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

Poly-saturated dolichols from filamentous fungi modulate activity of dolichol-dependent glycosyltransferase and physical properties of membranes

Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here, we confirm, using an LC-ESI-QTOF-MS analysis that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Also the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS

Polyprenols Are Synthesized by a Plastidial cis-Prenyltransferase and Influence 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 Δrer2 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

Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite

cis‐prenyltransferase 3 and α/β‐hydrolase are new determinants of dolichol accumulation in Arabidopsis

Dolichols (Dols), ubiquitous components of living organisms, are indispensable for cell survival. In plants, as well as other eukaryotes, Dols are crucial for posttranslational protein glycosylation, aberration of which leads to fatal metabolic disorders in humans and male sterility in plants. Until now, the mechanisms underlying Dol accumulation remain elusive. In this study, we have analysed the natural variation of the accumulation of Dols and six other isoprenoids among more than 120 Arabidopsis thaliana accessions. Subsequently, by combining QTL and GWAS approaches, we have identified several candidate genes involved in the accumulation of Dols, polyprenols, plastoquinone and phytosterols. The role of two genes implicated in the accumulation of major Dols in Arabidopsis—the AT2G17570 gene encoding a long searched for cis‐prenyltransferase (CPT3) and the AT1G52460 gene encoding an α/β‐hydrolase—is experimentally confirmed. These data will help to generate Dol‐enriched plants which might serve as a remedy for Dol‐deficiency in humans

Synthesis of Dolichols in Candida albicans is Co-Regulated With Elongation of Fatty Acids

Abstract: Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl re-ductase Dfg10 together with some other unknown enzymes. The aim of this study was to identi-fy such enzymes in Candida. The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans, which were similar, respective-ly, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae. Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dol-ichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis-prenyltransferases from the dolichol branch but to a lower expression of genes encoding en-zymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation

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-Mur-NAc-pentapeptide and heptaprenyl phosphate (C35-P, shortchain homolog of undecaprenyl phosphate, the endogenous substrate ofMraY)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 UDPMurNAc-pentapeptide-DNS and C35-P to the enzyme is required before the release of the two products, Lipid I andUMP.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 inMraY(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

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

Efficient, non-toxic gene delivery by negatively charged polyprenyl-based lipoplexes: Application in RNA delivery and the effects on cell physiology

The development in the field of DNA and RNA delivery into cells and progress in understanding pathogenesis of many diseases resulted in nucleic acids becoming actually drugs and their delivery one of the top molecular biology techniques applicable in clinics. Still, one of the major challenges facing the development of gene therapy is lack of efficient and safe gene vectors. We have examined a new class of polyprenyl-based cationic lipids for gene transfer. Studies have shown that semisynthetic polyprenyltrimethylammonium iodides (PTAI) in formulations with co-lipids (DOPE, DC-cholesterol, DOPC) have the ability to effectively transfect plasmid DNA in a wide range of cell types in vitro both in the presence and absence of serum. Although generally it is considered that bigger lipoplexes bearing positive zeta potential are more efficient, our data clearly demonstrate that small (90 – 150 nm), negatively charged (about -30 mV) polyprenyl-based lipoplexes are efficient and have parameters making them promising candidates for in vivo gene delivery. As it was demonstrated that lipofection procedure may have several side effects on cell physiology, we tested the effects of PTAI formulation on cell motility, proliferation, viability and gap junctional intercellular coupling (GJIC). We have tested four derivatives: amino-Pren-7, amino-Pren-8, amino-Pren-11 and amino-Pren-15. Cell motility of a model DU-145 (human prostate cancer) cells was estimated by time-laps monitoring of movement of individual cells and GJIC intensity measured using donor cells labelled with calcein plated onto monolayers of acceptor cells transfected with PTAI-based lipoplexes. The dynamics of calcein transfer from donor to acceptor cells was analyzed. Antimicrobial activity was evaluated by colony reduction assay and the hemolytic activity against human red blood cells (RBCs) was tested using PBS suspension prepared from fresh blood. The results show that lipoplexes based on PTAI have no effects on cell physiology that is cell viability, proliferation and morphology. Moreover, they also occurred to have no effect on GJIC and cell motility (24 hours after transfection all the cells cover the distance of about 210-240 μm showing a displacement of 70-80 μm). Some PTAI-based vectors exhibit potent bactericidal activity against Streptococcus aureus and Escherichia coli, while showing no toxic effect on eukaryotic cells, which can be beneficial during prolonged storage of formulations. Furthermore, (as we suggest in vivo application of PTAI vectors) we have also proved their safety towards human RBSs, which membranes are not disrupted in the presence of all the examined concentrations of PTAI-based lipoplexes. Moreover, the formulations tested in plasmid DNA transfer into cells are also effective in gene silencing techniques utilizing RNA delivery. We have successfully introduced shRNA inducing GFP gene silencing into DU145, XC (rat sarcoma) and B16F10 (mouse melanoma) cells expressing pEGFP-C1 plasmid achieving GFP gene silencing. Additionally, PTAI-based formulations can be safely stored for extended periods (up to 18 months) at 4°C. In conclusion, lipoplexes based on PTAI provide ability to introduce DNA or RNA into cells with satisfying efficiency, easily and safety, as they exhibit no toxic activity and no side effects on cell proliferation, motility and GJIC. What is more, PTAI-based formulations show advantages important for convenient use (both – DNA and RNA delivery, antimicrobial activity, prolonged storage) and in vivo applications (no RBCs rupture in the presence of PTAI-based lipoplexes, effectiveness in the presence of serum)