Analysis of Mdr50: A Drosophila P-Glycoprotein/Multidrug Resistance Gene Homolog

doi:10.1006/geno.1993.1286

Genomics

Volume 17, Issue 1, July 1993, Pages 83-88

https://doi.org/10.1006/geno.1993.1286 Get rights and content

Abstract

Using degenerate oligonucleotides from conserved portions of the ATP-binding domain of the active transporter genes, a new member of this gene superfamily has been cloned from Drosophila DNA. The gene contains two sets of transmembrane domains and two ATP-binding domains and shows a high degree of similarity to the mammalian P-glycoprotein/multidrug resistance (MDR) genes. The gene is adjacent to Hsc5, locus mapped to chromosome 2, band 50, and is named Mdr50. Mdr50 represents the third MDR homolog identified in Drosophila . Conservation in the position of intervening sequences between Mdr50 and the human MDR genes provides further evidence for their common origin.

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Cited by (38)

Metabolite transport across the mammalian and insect brain diffusion barriers
2017, Neurobiology of Disease
Citation Excerpt :
Active efflux is achieved by the members of the ATP binding cassette (ABC) transporter family. In humans, this family comprises 49 members that are grouped into seven sub-families, several of which are also represented in Drosophila (Desalvo et al., 2014; Gerrard et al., 1993; Tapadia and Lakhotia, 2005; Wu et al., 1991, reviewed in Mayer et al., 2009; Qosa et al., 2015). In mammals, ABC transporters mainly export lipid-soluble substrates from endothelial cells, since these are able to diffuse through the plasma membrane, and thereby protect the brain from neurotoxic molecules.
The nervous system in higher vertebrates is separated from the circulation by a layer of specialized endothelial cells. It protects the sensitive neurons from harmful blood-derived substances, high and fluctuating ion concentrations, xenobiotics or even pathogens. To this end, the brain endothelial cells and their interlinking tight junctions build an efficient diffusion barrier. A structurally analogous diffusion barrier exists in insects, where glial cell layers separate the hemolymph from the neural cells. Both types of diffusion barriers, of course, also prevent influx of metabolites from the circulation. Because neuronal function consumes vast amounts of energy and necessitates influx of diverse substrates and metabolites, tightly regulated transport systems must ensure a constant metabolite supply. Here, we review the current knowledge about transport systems that carry key metabolites, amino acids, lipids and carbohydrates into the vertebrate and Drosophila brain and how this transport is regulated. Blood-brain and hemolymph-brain transport functions are conserved and we can thus use a simple, genetically accessible model system to learn more about features and dynamics of metabolite transport into the brain.
Multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of cardenolides
2017, Insect Biochemistry and Molecular Biology
Citation Excerpt :
The LD50 values for orally ingested cardenolides observed in our experiments on Drosophila were comparable to those reported for vertebrate animals when expressed relative to body weight for all three cardenolides (Table 1). Mdr49, -50 and −65 have previously been identified as the Drosophila orthologues of human P-glycoprotein (P-gp) (Gerrard et al., 1993; Wu et al., 1991). Apolar cardenolides such as digoxin are P-gp substrates in vertebrates and insects (Gozalpour et al., 2013; Petschenka et al., 2013).
In the struggle against dietary toxins, insects are known to employ target site insensitivity, metabolic detoxification, and transporters that shunt away toxins. Specialized insects across six taxonomic orders feeding on cardenolide-containing plants have convergently evolved target site insensitivity via specific amino acid substitutions in the Na/K-ATPase. Nonetheless, in vitro pharmacological experiments have suggested a role for multidrug transporters (Mdrs) and organic anion transporting polypeptides (Oatps), which may provide a basal level of protection in both specialized and non-adapted insects. Because the genes coding for these proteins are evolutionarily conserved and in vivo genetic evidence in support of this hypothesis is lacking, here we used wildtype and mutant Drosophila melanogaster (Drosophila) in capillary feeder (CAFE) assays to quantify toxicity of three chemically diverse, medically relevant cardenolides.
We examined multiple components of fitness, including mortality, longevity, and LD50, and found that, while the three cardenolides each stimulated feeding (i.e., no deterrence to the toxin), all decreased lifespan, with the most apolar cardenolide having the lowest LD50 value. Flies showed a clear non-monotonic dose response and experienced high levels of toxicity at the cardenolide concentration found in plants. At this concentration, both Mdr and Oatp knockout mutant flies died more rapidly than wildtype flies, and the mutants also experienced more adverse neurological effects on high-cardenolide-level diets. Our study further establishes Drosophila as a model for the study of cardenolide pharmacology and solidifies support for the hypothesis that multidrug and organic anion transporters are key players in insect protection against dietary cardenolides.
An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog
2015, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
This generally indicates the presence of a conserved BBB efflux mechanism between insects and vertebrates; given that sequence conservation reflects similar functionality [25]. Previously, three homologs of the human ABCB1 were identified in D. melanogaster with predicted amino acid identities over 40% and denoted as mdr49, 50 and 65 [26,27]. Interestingly, functional analysis of D. melanogaster mdr49 mutants revealed increased sensitivity to the P-gp substrate colchicine [26], and mdr65 mutants showed impaired xenobiotic efflux activity at the insect's brain barrier [25].
ABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models.
Transcriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses.
A protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers.
Findings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.
RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster
2015, Pesticide Biochemistry and Physiology
Citation Excerpt :
The glutathione-S-transferase genes, GstE6 and GstE5, were found to be over expressed in the 91-R strain and are likely necessary to offset the increased oxidative stress produced through constitutive over transcription of the P450 detoxification genes [38]. The ABC B-type multiple drug resistance (Mdr) genes, Mdr49, Mdr50, Mdr65 as well as the ABC C-type multidrug resistance-associated protein Mrp1 were selected for RNAi due to preliminary reverse transcription–quantitative real time PCR (RT-qPCR) results that showed their over transcription in the 91-R strain [39] and their prior implications in insecticide processing and resistance [40–45]. Additional ABC B-type transporter genes, CG11897, CG7806, and CG5789, were selected through a bioinformatics approach using Flybase.
4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance.
ABC transporters and their role in protecting insects from pesticides and their metabolites
2014, Advances in Insect Physiology
Citation Excerpt :
In D. melanogaster, there are also some indications suggesting that ABC transporters are involved in insecticide resistance. Due to the well-documented role of ABC transporters in MDR in bacterial and mammalian systems, speculations about a role in insecticide resistance came up after the first homologs of mammalian P-gps/MRPs belonging to the ABCB and ABCC subfamilies had been cloned from D. melanogaster (Gerrard et al., 1993; Grailles et al., 2003; Wu et al., 1991). In one mutant fly line expressing a truncated non-functional Mdr49, Wu et al. (1991) indeed could measure an increased sensitivity to colchicine, a known substrate of mammalian P-gps.
Insects are frequently exposed to toxic compounds either naturally produced by host plants to prevent feeding damage or artificially manufactured by man to control herbivores and vectors of diseases. As a result from co-evolutionary adaptation, many of these insects have developed strategies to avoid exposure or eliminate toxic effects by biotransformation and/or reduction of the effective cytosolic concentrations. The elimination of toxic compounds frequently involves phase I and phase II reactions which functionalise the molecules and increase their solubility in water. Excretion in turn may rely on the activity of ATP-binding cassette (ABC) transporters, integral membrane proteins of the ABC superfamily that utilise the energy derived from ATP hydrolysis to translocate a variety of different physiological metabolites and xenobiotics. In recent years, ABC transporters have raised special interest, because multidrug resistance-associated ABC genes particularly of subfamilies B and C have been linked with insecticide resistance. In addition, some ABC transporters have been shown to directly mediate toxic effects of insecticides and biopesticides, and several inhibitors may prove useful in potentiating insecticide toxicity. Increasing the knowledge on the specific physiological functions of ABC transporters and elucidation of ABC-mediated resistance mechanisms may help to identify novel compounds for insect control that are highly selective and environmentally safe.
Interaction of pesticides with p-glycoprotein and other ABC proteins: A survey of the possible importance to insecticide, herbicide and fungicide resistance
2008, Pesticide Biochemistry and Physiology
Citation Excerpt :
Both mdr49 and mdr65 are overexpressed in D. melanogaster tumours in a parallel to mammalian p-gps [37]. The role of mdr50 [39] is unclear at this time. Functional p-gps have been found in several agriculturally and medically important insect pests (see Table 1), as well as in many aquatic invertebrates and vertebrates, where they have been implicated in protection against pollutants [40].
P-glycoproteins (p-gps) are ubiquitous membrane proteins from the ABC (ATP-binding cassette) family. They have been found in many animals, bacteria, plants and fungi and are extremely important in regulating a wide range of xenobiotics including pesticides. P-gps have been linked to xenobiotic resistance, most famously in resistance to cancer drug treatments. Their wide substrate range has led to what is known as “multidrug resistance”, where resistance developed to one type of xenobiotic gives resistance to a different classes of xenobiotic. P-gps are a major contributor to drug resistance in mammalian tumours and infections of protozoan parasites such as Plasmodium and Leishmania. There is a growing body of literature suggesting that p-gps, and other ABC proteins, are important in regulating pesticide toxicity and represent potential control failure through the development of pesticide resistance, in both agricultural and medical pests. At the same time, aspects of their biochemistry offer new hope in pest control, in particular in furthering our understanding of toxicity and offering insights into how we can improve control without recourse to new chemical discovery.

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Regular ArticleAnalysis of Mdr50: A Drosophila P-Glycoprotein/Multidrug Resistance Gene Homolog

Abstract

Regular Article
Analysis of Mdr50: A Drosophila P-Glycoprotein/Multidrug Resistance Gene Homolog