Superfamily Of Transport Proteins Research Articles

ApoM suppresses kidney renal clear cell carcinoma growth and metastasis via the Hippo-YAP signaling pathway

Renal cell carcinoma is one of the most common malignancies worldwide, and kidney renal clear cell carcinoma (KIRC) is the most common histopathological type of renal cell carcinoma. However, the mechanism of KIRC progression remains poorly understood. Apolipoprotein M (ApoM) is a plasma apolipoprotein and a member of the lipid transport protein superfamily. Lipid metabolism is essential for tumor progression, and its related proteins can be used as therapeutic targets for tumors. ApoM influences the development of several cancers, but its relationship with KIRC remains unclear. In this study, we aimed to investigate the biological function of ApoM in KIRC and to reveal its potential molecular mechanisms. We found that ApoM expression was significantly reduced in KIRC and was strongly correlated with patient prognosis. ApoM overexpression significantly inhibited KIRC cell proliferation in vitro, suppressed the epithelial mesenchymal transition (EMT) of KIRC cells, and decreased their metastatic capacity. Additionally, the growth of KIRC cells was inhibited by ApoM overexpression in vivo. In addition, we found that overexpression of ApoM in KIRC attenuated Hippo-YAP protein expression and YAP stability and thus inhibited KIRC growth and progression. Therefore, ApoM may be a potential target for the treatment of KIRC.

Overexpression of ATP-binding cassette transporters ABCG10, ABCH3 and ABCH4 in Aphis craccivora (Koch) facilitates its tolerance to imidacloprid

Aphis craccivora (Koch), a globally pest that causes significant threat to the legumes, has developed different degrees of resistance to a variety of insecticides. The ATP-binding cassette (ABC) transporters comprise a multifunctional transporter protein superfamily which play important roles in the transport and detoxification of xenobiotic compounds in insects. However, whether ABC transporters take part in the tolerance of imidacloprid in A. craccivora is still unknown. In order to investigate the functions of ABC transporters in the imidacloprid tolerance, fifty- eight ABC transporters were identified in the transcriptome and genome of A. craccivora and the toxicity of imidacloprid against A. craccivora was significantly increased after application the inhibitors of verapamil and Ko143. The relative expression levels of ABCG5, ABCG6, ABCG10, ABCH3, ABCH4, ABCH8 and ABCH10 were significantly up-regulated in response to imidacloprid treatment with LC15, LC50 and LC85 concentrations, and the expression patterns of these seven ABC transporters were further analyzed at different developmental stages and in different tissues of A. craccivora by quantitative real-time PCR (RT-qPCR). Furthermore, knockdown of ABCG10, ABCH3 and ABCH4 significantly increased the mortality of A. craccivora to imidacloprid. Our results reveal the key functions of ABC transporters in the tolerance of imidacloprid and provide valuable information regarding the development of improved management strategies in A. craccivora.

Longitudinal study of Staphylococcus aureus genotypes isolated from bovine clinical mastitis

Bovine clinical mastitis is an important problem for the dairy industry, and Staphylococcus aureus is a common mastitis-causing pathogen in many countries. Detailed knowledge on genetic variation of Staph. aureus strains within the bovine population, including changes over time, can be useful for mastitis control programs, because severity of disease and effects on milk production are at least partly strain-associated. Therefore, the major aim of this study was to compare sequence types of Staph. aureus isolated from cases of bovine clinical mastitis from 2002 to 2003 with sequence types of a more recent set of isolates collected from 2013 to 2018, using core genome multi-locus sequence typing (cgMLST). We also wanted to compare antibiotic resistance genes of isolates from the 2 sets, to identify changes that may have occurred over time in the Staph. aureus population. A total of 157 isolates of Staph. aureus, almost equally distributed between the 2 time periods, were subjected to high-throughput sequencing and cgMLST. The results showed that the most prevalent sequence types found among the 2002 to 2003 isolates belonged to the clonal complexes CC97, CC133, and CC151, and that those complexes still dominated among the isolates from 2013 to 2018. However, a population shift from CC133 to CC97 and CC151 over time was observed. Likewise, no important differences in prevalence of antibiotic resistance genes were found between the 2 sets of isolates. As expected, genes belonging to the major facilitator superfamily of transporter proteins, and multidrug and toxic compound extrusion transporters, were very common. Moreover, several genes and mutations conferring resistance to fosfomycin were present, but not in CC97 isolates. The β-lactamase gene blaZ was found in only 3 out of 81 isolates from 2002 to 2003 and 1 out of 76 isolates in 2013 to 2018. In conclusion, the results indicate that mastitis-associated Staph. aureus strains circulating among dairy cows in Sweden exhibit a remarkable genotypic persistence over a time frame of close to 15 yr.

Coordination of Substrate Binding and Protonation in the N.gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism.

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N.gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N.gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.

A guide to plasma membrane solute carrier proteins.

This review aims to serve as an introduction to the solute carrier proteins (SLC) superfamily of transporter proteins and their roles in human cells. The SLC superfamily currently includes 458 transport proteins in 65 families that carry a wide variety of substances across cellular membranes. While members of this superfamily are found throughout cellular organelles, this review focuses on transporters expressed at the plasma membrane. At the cell surface, SLC proteins may be viewed as gatekeepers of the cellular milieu, dynamically responding to different metabolic states. With altered metabolism being one of the hallmarks of cancer, we also briefly review the roles that surface SLC proteins play in the development and progression of cancer through their influence on regulating metabolism and environmental conditions.

Drugging drug resistance with bystander-assisted immunotherapy.

Abstract Acquired drug resistance is a longstanding challenge that reduces the efficacy of chemotherapeutic drugs. In contrast, the efficacies of modern immunotherapies do not directly correlate to the prevalence of chemo-resistant phenotypes. Here we improve upon this paradigm by targeting the action of immunotherapeutics to two general mechanisms of acquired drug resistance: irregular metabolism and drug efflux mediated by the ABC superfamily of transport proteins. In chemo-resistant prostate cancer cells, we demonstrate that these two mechanisms act in concert to selectively metabolize our newly developed class of enzyme-directed prodrug to the Toll-Like Receptor 7 immunotherapeutic Imiquimod. Following metabolism, liberated Imiquimod undergoes drug efflux to the extracellular space where it activates bystander immune cells in local proximity. In-vitro, we characterize this process of Bystander-Assisted ImmunoTherapy (BAIT) using an AT3B-1 chemo-resistant prostate cancer model with RAW-Blue and JAWSII reporter immune cell lines. Co-culture of AT3B-1 cancer cells with reporter immune cells results in immunogenicity mediated selectively by cancer cells. This results in enhanced NF-κB transcription, expression of cell surface markers, and secreted cytokines indicative of a cell-mediated immune response. Our prodrug is non-immunogenic with healthy cells alone and the enzyme-directing groups are stable for over 3 days in serum. Taken together, our results demonstrate that BAIT co-opts common mechanisms of drug resistance to elicit immunogenicity mediated by cancer cells themselves. We anticipate that BAIT will find use as a new mechanism of action that exploits drug resistant phenotypes to generate an immune response.

Bioinformatic characterization of the Anoctamin Superfamily of Ca2+-activated ion channels and lipid scramblases

Our laboratory has developed bioinformatic strategies for identifying distant phylogenetic relationships and characterizing families and superfamilies of transport proteins. Results using these tools suggest that the Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca2+-permeable Stress-gated Cation Channel (CSC) families; as well as four families of functionally uncharacterized proteins, which we refer to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families. We have constructed protein clusters and trees showing the relative relationships among the seven families. Topological analyses suggest that the members of these families have essentially the same topologies. Comparative examination of these homologous families provides insight into possible mechanisms of action, indicates the currently recognized organismal distributions of these proteins, and suggests drug design potential for the disease-related channel proteins.

A Fluorescent Transport Assay Enables Studying AmpG Permeases Involved in Peptidoglycan Recycling and Antibiotic Resistance.

Inducible AmpC β-lactamases deactivate a broad-spectrum of β-lactam antibiotics and afford antibiotic resistance in many Gram-negative bacteria. The disturbance of peptidoglycan recycling caused by β-lactam antibiotics leads to accumulation of GlcNAc-1,6-anhydroMurNAc-peptides, which are transported by AmpG to the cytoplasm where they are processed into AmpC inducers. AmpG transporters are poorly understood; however, their loss restores susceptibility toward β-lactam antibiotics, highlighting AmpG as a potential target for resistance-attenuating therapeutics. We prepare a GlcNAc-1,6-anhydroMurNAc-fluorophore conjugate and, using live E. coli spheroplasts, quantitatively analyze its transport by AmpG and inhibition of this process by a competing substrate. Further, we use this transport assay to evaluate the function of two AmpG homologues from Pseudomonas aeruginosa and show that P. aeruginosa AmpG (Pa-AmpG) but not AmpP (Pa-AmpP) transports this probe substrate. We corroborate these results by AmpC induction assays with Pa-AmpG and Pa-AmpP. This fluorescent AmpG probe and spheroplast-based transport assay will enable improved understanding of PG recycling and of permeases from the major facilitator superfamily of transport proteins and may aid in identification of AmpG antagonists that combat AmpC-mediated resistance toward β-lactam antibiotics.

Rapid multiplexed phenotypic screening identifies drug resistance functions for three novel efflux pumps in Acinetobacter baumannii.

Drug efflux pumps are one of the key machineries in bacterial drug resistance. Although quite a few of these transport systems have been functionally characterized in various organisms, due to large-scale genome sequencing efforts and improved prediction pipelines there are increasing numbers of putative drug efflux genes annotated. For phenotype identification of the proteins encoded by these genes, we developed a novel high-throughput phenotype screening strategy and demonstrated its utility in identifying phenotypes for putative efflux systems encoded by the human pathogen Acinetobacter baumannii. Seventeen putative drug efflux systems from A. baumannii were heterologously expressed in Escherichia coli. For rapid and economical phenotype screening we employed a combination of multiplexed Biolog Phenotype Microarrays and quantitative PCR. Using this method we screened these putative drug efflux pumps against 240 antimicrobial conditions, equating to 4080 simultaneous phenotypic tests. Of the 17 putative drug efflux systems, phenotypes were confirmed for two pumps and novel drug resistance phenotypes were identified for three new A. baumannii drug transporters, which exemplified the power of this method as a high-throughput screening technique. One of the phenotypically characterized putative drug efflux systems was classified within the ATP-binding cassette superfamily of transport proteins and represents the first drug resistance protein characterized from this superfamily in A. baumannii. The remaining two proteins were members of the major facilitator superfamily of efflux pumps. This method has broad potential for high-throughput phenotype characterization of putative drug efflux systems in a range of organisms.

Inhibition of ABC transport proteins by oil sands process affected water

The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI+ and ESI− mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O+, SO+, and NO+ chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2−), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates this effect and inhibition of the activity of MRPs by OSPW from Base Mine Lake does not occur at environmentally relevantconcentrations.

The LysE Superfamily of Transport Proteins Involved in Cell Physiology and Pathogenesis.

The LysE superfamily consists of transmembrane transport proteins that catalyze export of amino acids, lipids and heavy metal ions. Statistical means were used to show that it includes newly identified families including transporters specific for (1) tellurium, (2) iron/lead, (3) manganese, (4) calcium, (5) nickel/cobalt, (6) amino acids, and (7) peptidoglycolipids as well as (8) one family of transmembrane electron carriers. Internal repeats and conserved motifs were identified, and multiple alignments, phylogenetic trees and average hydropathy, amphipathicity and similarity plots provided evidence that all members of the superfamily derived from a single common 3-TMS precursor peptide via intragenic duplication. Their common origin implies that they share common structural, mechanistic and functional attributes. The transporters of this superfamily play important roles in ionic homeostasis, cell envelope assembly, and protection from excessive cytoplasmic heavy metal/metabolite concentrations. They thus influence the physiology and pathogenesis of numerous microbes, being potential targets of drug action.

Implications of Mycobacterium Major Facilitator Superfamily for Novel Measures against Tuberculosis.

Major facilitator superfamily (MFS) is an important secondary membrane transport protein superfamily conserved from prokaryotes to eukaryotes. The MFS proteins are widespread among bacteria and are responsible for the transfer of substrates. Pathogenic Mycobacterium MFS transporters, their distribution, function, phylogeny, and predicted crystal structures were studied to better understand the function of MFS and to discover specific inhibitors of MFS for better tuberculosis control.

Why Is the GMN Motif Conserved in the CorA/Mrs2/Alr1 Superfamily of Magnesium Transport Proteins?

Members of the CorA/Mrs2/Alr1 superfamily of transport proteins mediate magnesium uptake in all kingdoms of life. Family members have a low degree of sequence conservation but are characterized by a conserved extracellular loop. While the degree of sequence conservation in the loop deviates to some extent between family members, the GMN family signature motif is always present. Structural and functional data imply that the loop plays a central role in magnesium selectivity, and recent biochemical data suggest it is crucial for stabilizing the pentamer in the magnesium-free (putative open) conformation. In this study, we present a detailed structure-function analysis of the extracellular loop of CorA from Thermotoga maritima, which provides molecular insight into how the loop mediates these two functions. The data show that loop residues outside of the GMN motif can be substituted if they support the pentameric state, but the residues of the GMN motif are intolerant to substitution. We conclude that G(312) is absolutely required for magnesium uptake, M(313) is absolutely required for pentamer integrity in the putative open conformation, and N(314) plays a role in both of these functions. These observations suggest a molecular reason why the GMN motif is conserved throughout the CorA/Mrs2/Alr1 superfamily.

Potential Application of Network Descriptions for Understanding Conformational Changes and Protonation States of ABC Transporters

The ABC (ATP Binding Cassette) transporter protein superfamily comprises a large number of ubiquitous and functionally versatile proteins conserved from archaea to humans. ABC transporters have a key role in many human diseases and also in the development of multidrug resistance in cancer and in parasites. Although a dramatic progress has been achieved in ABC protein studies in the last decades, we are still far from a detailed understanding of their molecular functions. Several aspects of pharmacological ABC transporter targeting also remain unclear. Here we summarize the conformational and protonation changes of ABC transporters and the potential use of this information in pharmacological design. Network related methods, which recently became useful tools to describe protein structure and dynamics, have not been applied to study allosteric coupling in ABC proteins as yet. A detailed description of the strengths and limitations of these methods is given, and their potential use in describing ABC transporter dynamics is outlined. Finally, we highlight possible future aspects of pharmacological utilization of network methods and outline the future trends of this exciting field.

Abstract 812: Identification of novel anticancer drugs as substrates of organic anion transporting polypeptide 1B3 using a cell viability assay

Abstract Organic anion transporting polypeptide 1B3 (OATP1B3) belongs to a superfamily of transport proteins that mediate the uptake of a wide range of endogenous and exogenous compounds into cells. Under normal conditions OATP1B3 is selectively expressed at the basolateral membrane of human hepatocytes. Recently, OATP1B3 expression has been detected in a variety of different cancers and because of its multispecificity OATP1B3 is thought to be a potential therapeutic target to enhance drug delivery into cancer cells. Thus far, only a few anticancer drugs have been identified as substrates of OATP1B3 including docetaxel, paclitaxel, methotrexate and imatinib. Given that OATP1B3 can potentially be used as a target for anticancer drug delivery we need to identify additional anticancer drug substrates of OATP1B3. Therefore, we aimed at establishing a cell based high throughput assay to screen the NCI approved oncology drug set for novel cytotoxic substrates of OATP1B3. Using a luminescent cell viability assay, we measured the viability of wild-type and OATP1B3-expressing Chinese Hamster Ovary (CHO) cells in the absence or presence of the anticancer drugs. After we identified positive hits we further characterized them with respect to concentration dependencies. We identified etoposide, bleomycin, oxaliplatin and plicamycin as compounds that kill OATP1B3-expressing CHO cells at much lower concentrations than wild-type CHO cells suggesting that these compounds are substrates of OATP1B3. Using secondary assays we confirmed that etoposide is a substrate of OATP1B3 and we are currently testing the remaining compounds for direct uptake by OATP1B3. In conclusion, this cell-based viability assay is a good tool to identify novel cytotoxic OATP1B3 substrates that can potentially be used to treat OATP1B3-expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 812. doi:1538-7445.AM2012-812

Twelve transmembrane helices form the functional core of mammalian MATE1

MATE1 and MATE2/2‐K, the mammalian organic cation/H+ (OC/H+) exchangers, are key players in renal and hepatic secretion of cationic drugs. They belong to a superfamily of transport proteins (Multidrug And Toxin Extruders) with 1000+ members. The x‐ray structure of the prototypic MATE family member, NorM from Vibrio cholera, shows a protein fold comprised of 12 transmembrane helices (TMHs), as predicted by hydropathy analysis of the majority of MATE transporters. However, the mammalian MATEs are predicted to have a C‐terminal 13th TMH. Here we confirmed the presence of the 13th TMH in mammalian MATE1. Whereas the C‐termini of epitope‐tagged, full length human, rabbit and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane, truncation of these proteins at or near the junction between the 13th TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane, (ii) had a cytoplasmic C‐terminus, and (iii) supported substrate transport. The latter observation indicates that the functional ‘core structure’ of mammalian MATE consists of the first 12 TMHs, whereas the 13th TMH is not necessary for essential function. Therefore, we used the x‐ray structure of NorM to develop a homology model of the 3D structure of hMATE1 that proved to be stable in Molecular Dynamic simulations when placed (in silico) in a hydrated phospholipid bilayer.

Modulation of mdr‐1 Expression and Activity by Verapamil and Resveratrol Potentiates the Cytotoxicity of Docetaxel and Doxorubicin in Solid Tumor Cell Lines

P‐glycoprotein (P‐gp) is a member of ATP‐dependent membrane transport protein superfamily encoded by mdr‐1 gene family. In tumor cells, expression of P‐gp results in reduced intracellular drug concentrations, which decreases the cytotoxicity of antitumor drugs. Herein, we evaluated the added value of combining two P‐gp blockers (verapamil and resveratrol) with docetaxel and doxorubicin and the possible underlying mechanism(s) in solid tumor cell lines (MCF‐7, HeLa and HepG2). Verapamil possessed weaker anti‐cancer properties than resveratrol (IC50 of 92.1 to 107.6 μM and 35.0 to 84μM, respectively). Doxorubicin and docetaxel IC50's of 0.48 to 0.72 μM and 25.9 to 77.8 nM, respectively. Verapamil combination with doxorubicin and docetaxel significantly decreased the IC50's of both agents to 0.034 to 0.12 μM and 2.5 to 6.0 nM, respectively. Resveratrol increased the potencies of both chemotherapeutic agents to IC50 of 0.12 to 0.34 μM and 7.2 to 53.02 nM, respectively. The interaction of resveratrol with both agents was additive in HeLa and Hep‐G2 cell lines. Surprisingly, only resveratrol but not verapamil interfered with the expression of mdr‐1 and several apoptotic genes BAX, BcL2 and p53. P‐gp efflux activity was significantly inhibited by resveratrol and verapamil with subsequent accumulation of P‐gp substrate within the intracellular compartment. In conclusion, resveratrol was comparable to verapamil in potentiating the anti‐cancer effects of doxorubicin and docetaxel in solid tumor.

Expression and Function of Human MRP1 (ABCC1) Is Dependent on Amino Acids in Cytoplasmic Loop 5 and Its Interface with Nucleotide Binding Domain 2

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.

Multidrug Resistance Mediated by Half ABC Transporter ABCG2*

Cancers are often intrinsically resistant or become resistant to treatment with conventional chemotherapy. This broad-spectrum resistance results in large part,but not solely,from overexpression of a variety of members of the ATP-binding cassette (ABC) superfamily of transport proteins. ABCG2 (ABCP/MXR/BCRP),as a half ABC transporter,is a novel member of the ABC membrane transporters,which can actively transport a wide spectrum of drugs with varying chemical structures or cellular targets. ABCG2 is overexpressed in the intestine,liver,placenta,and the blood-brain barrier,where it plays a role in protecting the organs from potentially harmful ingested toxins. Moreover,ABCG2 is also highly expressed in hematopoietic stem cells,it seems that ABCG2 has a key function in stem cell regulation or protection from a variety of xenobiotics. Multiple of SNP (single nucleotide polymorphisms) in ABCG2 may affect absorption and distribution,altering the effectiveness and toxicity of drugs in patients. Like other members of the ABC transporter superfamily,such as MDR1 and MRP1,ABCG2 is expressed in a variety of malignancies. Overexpression of ABCG2 in tumor cells confers cancer multidrug resistance to a variety of newly developed anticancer drugs including mitoxantrone,topotecan,epirubicin,anthracyclines and camptothecins. Furthermore,ABCG2 are advocated for use as potentially selectable markers in stem cell based gene therapy. Given the profound impact of ABCG2 on multidrug resistance in tumor cells and on in vivo pharmacology and toxicology,a complete understanding of the mechanism and biological function of ABCG2 will be important for understanding its normal physiology as well as potentially for the development of novel chemotherapeutic treatment strategies. The discovery,the biochemistry,the normal physiology and the current insight of human ABCG2 were introduced.

Identification of multi-drug resistance associated proteins MRP1 (ABCC1) and MRP3 (ABCC3) from rainbow trout ( Oncorhynchus mykiss)

The MRPs (multi-drug resistance associated proteins; ABCC subfamily) are members of the ATP binding cassette (ABC) superfamily of transport proteins and act as cellular efflux transporters of a wide variety of substrates, in particular glutathione, glucuronide and sulphate conjugates of diverse compounds. Together with P-gp (P-glycoprotein; MDR1; ABCB1) and BCRP (breast cancer resistance protein, ABCG2) the MRPs are highly important as cellular defense against toxicants and confer multixenobiotic resistance (MXR). In aquatic ecotoxicology MXR research has mostly focussed on P-glycoprotein while the other relevant ABC transporters are widely under-appreciated. We here present complete MRP1 (ABCC1) and partial MRP3 (ABCC3) cDNAs from rainbow trout ( Oncorhynchus mykiss). We identified 4398 bp of the MRP1 and 3786 bp of the MRP3 open reading frames (ORF) by screening the NCBI EST database and by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using RNA from RTgill-W1 cells. Identities with human homologs are 56% for MRP1 and 64% for MRP3.