Kostas Iatrou

Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery.

Insect cell-based expression systems are prominent amongst current expression platforms for their ability to express virtually all types of heterologous recombinant proteins. Stably transformed insect cell lines represent an attractive alternative to the baculovirus expression system, particularly for the production of secreted and membrane-anchored proteins. For this reason, transformed insect cell systems are receiving increased attention from the research community and the biotechnology industry. In this article, we review recent developments in the field of insect cell-based expression from two main perspectives, the production of secreted and membrane-anchored proteins and the establishment of novel methodological tools for the identification of bioactive compounds that can be used as research reagents and leads for new pharmaceuticals and insecticides.

A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high‐throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone‐like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure‐activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell‐based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression

ABSTRACT Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

Cooperative interactions between odorant-binding proteins of Anopheles gambiae

To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.

Bombyx mori DNA/RNA non-specific nuclease: Expression of isoforms in insect culture cells, subcellular localization and functional assays

A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses.

Anopheles gambiae odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents

Insect odorant binding proteins (OBPs) are the first components of the olfactory system to encounter and bind attractant and repellent odors emanating from various sources for presentation to olfactory receptors, which trigger relevant signal transduction cascades culminating in specific physiological and behavioral responses. For disease vectors, particularly hematophagous mosquitoes, repellents represent important defenses against parasitic diseases because they effect a reduction in the rate of contact between the vectors and humans. OBPs are targets for structure-based rational approaches for the discovery of new repellent or other olfaction inhibitory compounds with desirable features. Thus, a study was conducted to characterize the high resolution crystal structure of an OBP of Anopheles gambiae, the African malaria mosquito vector, in complex with N,N-diethyl-m-toluamide (DEET), one of the most effective repellents that has been in worldwide use for six decades. We found that DEET binds at the edge of a long hydrophobic tunnel by exploiting numerous non-polar interactions and one hydrogen bond, which is perceived to be critical for DEET’s recognition. Based on the experimentally determined affinity of AgamOBP1 for DEET (Kd of 31.3xa0μΜ) and our structural data, we modeled the interactions for this protein with 29 promising leads reported in the literature to have significant repellent activities, and carried out fluorescence binding studies with four highly ranked ligands. Our experimental results confirmed the modeling predictions indicating that structure-based modeling could facilitate the design of novel repellents with enhanced binding affinity and selectivity.

Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells

A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophilas OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties.

The orphan nuclear receptor BmHR3A of Bombyx mori: hormonal control, ovarian expression and functional properties.

Ovarian development in the domesticated silkmoth, Bombyx mori, is induced by the molting hormone 20-hydroxy-ecdysone (20E) shortly after larval-pupal ecdysis. Studies of the ecdysone response in Drosophila and other insects have shown that 20E exerts its effects initially by the induction of a small number of early genes, including the orphan nuclear receptors HR3, that transduce and amplify the hormone signal. Here we show that the silkmoth orphan receptor BmHR3A acts in the 20E-induced regulatory cascade in the ovary during pupal and pharate adult development in a manner different than that observed in the classical ecdysone regulatory hierarchy in Drosophila salivary glands at the end of the third instar. While other isoforms of BmHR3 are induced as early gene products in the ecdysone response, BmHR3A is induced 2 days after 20E administration in the silkmoth ovary and, thus, behaves as late product. The period of accumulation of BmHR3A in ovarian follicular cells occurs during vitellogenesis and coincides with the period of transcriptional expression of the ESP (egg-specific protein) gene, whose product constitutes a major component of the egg yolk, while it is reciprocal to the period of expression of BmGATAbeta, a gene encoding a regulator of late chorion gene expression. Bandshift experiments demonstrate that BmHR3A binds specifically to RORE (Retinoic acid-related Orphan receptor Response Element)-like sequences in the promoters of both genes, thus suggesting a direct role for BmHR3A in regulating the expression of BmGATAbeta and ESP genes during vitellogenesis. Finally, we show that BmHR3A functions as a constitutive transcriptional activator in a B. mori derived cell line. We propose that BmHR3A may function as a regulator of vitellogenesis in the silkmoth ovary.

Comparison of the activity of non‐steroidal ecdysone agonists between dipteran and lepidopteran insects, using cell‐based EcR reporter assays

BACKGROUNDnDiacylhydrazine (DAH) analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. These DAHs have been shown to manifest their toxicity via interaction with the ecdysone receptor (EcR) in susceptible insects, as does the natural insect moulting hormone 20-hydroxyecdysone (20E). A notable feature is their high activity and specificity, particularly against lepidopteran insects, raising the question as to whether non-lepidopteran-specific analogues can be isolated. However, for the discovery of ecdysone agonists that target other important insect groups such as Diptera, efficient screening systems that are based on the activation of the EcR are needed.nnnRESULTSnIn this study, a dipteran-specific reporter-based screening system with transfected S2 cells of Drosophila melanogaster Meig. was developed in order to discover and evaluate compounds that have ecdysone agonistic or antagonistic activity. A library of non-steroidal ecdysone agonists containing different mother structures with DAH and other related analogues such as acylaminoketone (AAK) and tetrahydroquinoline (THQ) was tested. None of the compounds tested was as active as 20E. This is in contrast to the very high activity of several DAH and AAK congeners in lepidopteran cells (Bombyx mori L.-derived Bm5 cells). The latter agrees with a successful docking of a DAH, tebufenozide, in the binding pocket of the lepidopteran EcR (B. mori), while this was not the case with the dipteran EcR (D. melanogaster). Of note was the identification of two THQ compounds with activity in S2 but not in Bm5 cells. Although marked differences in activity exist with respect to the activation of EcR between dipterans and lepidopterans, there exists a positive correlation (R = 0.724) between the pLC(50) values in S2 and Bm5 cells. In addition, it was found through protein modelling that a second lobe was present in the ligand-binding pocket of lepidopteran BmEcR but was lacking in the dipteran DmEcR protein, suggesting that this difference in structure of the binding pocket is a major factor for preferential activation of the lepidopteran over the dipteran receptors by DAH ligands.nnnCONCLUSIONSnThe present study confirmed the marked specificity of DAH and AAK analogues towards EcRs from lepidopteran insects. THQ compounds did not show this specificity, indicating that dipteran-specific ecdysone-agonist-based insecticides based on the THQ mother structure can be developed. The differences in activity of ecdysone agonists in dipteran and lepidopteran ecdysone-reporter-based screening systems are discussed.

Sex-biased expression of odorant receptors in antennae and palps of the African malaria vector Anopheles gambiae

At the heart of the odor recognition process in all animals are G-protein-coupled receptors, which are seven-transmembrane domain proteins that initiate G-protein-mediated signaling cascades when activated by their ligands. Odorant receptors (ORs) are a large, diverse family of proteins with some 80 members in the mosquito Anopheles gambiae. With the assumption that more sensilla on female antennae are tuned to human odors than on male antennae, comparison of specific OR mRNA levels in male and female antennae can provide an indication as to which receptors may be stimulated by host odors. We have used RT PCR and quantitative real-time PCR (qRT PCR) to investigate sex-biased expression levels of 80 A. gambiae ORs in male and female antennae and maxillary palps. On the basis of prevalence of expression in female antennae and on a strong female relative to male expression bias we identified a short list of ORs that are likely involved in host odor recognition by female mosquitoes.