No / The genome of Caenorhabditis elegans encodes multiple homologues of the two major families of mammalian equilibrative and concentrative nucleoside transporters. As part of a programme aimed at understanding the biological rationale underlying the multiplicity of eukaryote nucleoside transporters, we have now demonstrated that the nematode genes ZK809.4 (ent-1) and K09A9.3 (ent-2) encode equilibrative transporters, which we designate CeENT1 and CeENT2 respectively. These transporters resemble their human counterparts hENT1 and hENT2 in exhibiting similar broad permeant specificities for nucleosides, while differing in their permeant selectivities for nucleobases. They are insensitive to the classic inhibitors of mammalian nucleoside transport, nitrobenzylthioinosine, dilazep and draflazine, but are inhibited by the vasoactive drug dipyridamole. Use of green fluorescent protein reporter constructs indicated that the transporters are present in a limited number of locations in the adult, including intestine and pharynx. Their potential roles in these tissues were explored by using RNA interference to disrupt gene expression. Although disruption of ent-1 or ent-2 expression alone had no effect, simultaneous disruption of both genes yielded pronounced developmental defects involving the intestine and vulva.

NorM multidrug transport proteins belong to the multiple antibiotics and toxins extrusion (MATE) family of secondary active transporters. Members of this family are present across all species including bacteria, plants and humans. In bacteria, their over-expression can lead to antibiotic resistance, whereas in the human body, the transporters can alter the plasma levels of drugs. NorM proteins are therefore relevant for the pharmacokinetic properties of drugs. Previously, NorM from Vibrio cholerae (NorM-VC) was shown to export drug (ethidium) in an antiport reaction that is coupled to the simultaneous uptake of protons and sodium ions down their electrochemical gradients across the plasma membrane. But NorM from Pseudomonas stutzeri (NorM-PS) was shown to transport DAPI by utilising proton cycling exclusively. NorM-VC and NorM-PS share 42% identical amino-acid residues and yet their functions differ in terms of their ion coupling properties. These differences in functionality of two highly homologous proteins provide an excellent opportunity to carry out a comparative study. The work presented in this thesis investigates the energetics of drug transport processes by NorM-VC and NorM-PS and the structural basis for ion-coupled drug transport by NorM-VC. Ethidium efflux assays in intact Lactococcus lactis cells were used to study the effect of the magnitude and composition of the proton- and sodium-motive force on transport activity. Furthermore, ethidium binding assays were used to study partial reactions in drug efflux processes. These biochemical data were supplemented by computational studies and analyses of current protein structures. Based on the observations detailed here, a novel transport model for NorM-VC is proposed, which explains published findings for NorM-VC and other MATE transporters. The model represents a potentially universal mechanism for MATE transporters that can be used to predict further structure-function relationships in this important family of member transporters.

In this study, several aspects of nitrate assimilation and transport have been studied using the filamentous fungus Aspergillus nidulans, which has been shown to be safe laboratory organism as judged by it's pathogenicity towards insect larvae. In silico analysis of the A. nidulans genome sequence, identified two putative genes designated cnxL and cnxK that might be involved in molybdenum cofactor (a component of nitrate reductase) biosynthesis as well as two putative nitrate reductases encoding genes niaB and niaC. All four genes are hitherto unknown. Although many features of these proteins provided clues of functionality, biochemical and genetical approaches employed in this present study failed to elicit expression of any of these four genes. A NrtA protein structure model was developed based on residue homology with the E. coli GlpT a protein, the structure of which has been solved. The results of thiol cross-linking of three double cysteine mutants in four NrtA essential residues, R87, R368, N168 and N459, indicated that the molecular distance between R87 and R368 is ~ 0.4 Å, R368 and N168 ~ 6.2 Å, R87 and N459 is ~ 2.2 Å. Another important observation was the change in the confirmation of Tm 2 and Tm 8 in the presence of nitrate. This shift resulted in an increase of ~ 2 Å gap between the residues R87 and R368. Distances between amino acid residue pairs estimated using such molecular rulers contradicted the NrtA existing model. Cysteine-scanning mutagenesis studies were extended to the generation of a library of single cysteine mutants of NrtA residues spanning Tm 2 and Tm 8. The majority of single cysteine mutants possessed wild type NrtA protein expression levels but unfortunately most were found to be loss-of-function. Consequently, thiol chemistry of this crop of mutants was not perused. Attempts were also made to overexpress and crystallise the bacterial nitrate transporters but none of the transporter tested proved to be a successful candidate for crystallisation. In this regard, bacterial nitrate transporters, NarU (E. coli), Nar (Bacillus cereus), NarK1 and NarK2 (Pseudomonas aeruginosa) and NarK2 (Thermus thermophilus) fused with GFP were expressed in E. coli and used in crystallisation trials. Although this approach has proved successful for a number of membrane proteins, unfortunately was not helpful with regard to the purification of any of the above bacterial nitrate transporters to yield protein expression levels required for successful protein crystallography. Finally, the effects of potential nitrate transport inhibitors were studied on net nitrate transport by NrtA and NrtB proteins of A. nidulans. The results indicated that chlorate had more of an inhibitory effect on NrtA net nitrate transport than that by NrtB. Chlorite and sulphite equally affected net nitrate transport by either NrtA or NrtB proteins while caesium strongly inhibited the net nitrate transport by NrtB transporter.

The placenta provides a protective barrier for the developing foetus during gestation. Physiological barriers including the placenta, liver, kidney, intestine and blood-brain barrier are known to express ATP-Binding cassette transporters (ABC transporters) and metabolising enzymes. These specialised proteins have the ability to transport or metabolise xenobiotics. There is evidence to suggest that ABC transporters and metabolising enzymes are located at the interface between the maternal and foetal blood supplies (a cell layer referred to as the syncytiotrophoblast) and therefore may help protect the foetus from harmful xenobiotics. During new compound development prenatal developmental toxicity testing forms an important part of safety assessment. In order to predict potential toxicity of a new chemical entity to humans, rodent and non-rodent species are currently used. This thesis investigates the rat and human placental barrier properties in order to help facilitate our knowledge of species differences and contribute to our understanding of the limitations of these surrogate models. The approaches taken include: genomic analyses using microarray data to compare the overall expression of ABC transporters and metabolising enzymes throughout gestation in both species, immunohistochemical techniques to localise transporters and metabolising enzymes in the rat placenta, and in vitro functionality assays of selected transporters performed in rat and human placental cell line models. The main findings have shown a similar mRNA expression level of ABCG2/BCRP (breast cancer resistance protein) throughout gestation in the rat and human, however different mRNA expression levels of other transporters (slco4a1/oatp4a1 in particular) and metabolising enzymes were also highlighted. Immunohistochemistry localised selected transporters to the syncytiotrophoblast region of the rat placenta (the interface of maternal and foetal circulations). Functional in vitro assays were successfully utilised in rat and human placental cell lines which showed functional ABCB1/P-gp in both species. Overall, these findings provide a genomic characterisation of the rat and human protective placental barrier properties and show transporter functionality in in vitro cell-based assays which will prove useful in prenatal and developmental toxicity tests. Alternatives to using animals have been explored by using functional in vitro assays which could potentially be implored during the new compound discovery phase. This could help to make animal testing more selective for given compounds and ensures the new chemical entity is being tested in the model closest resembling the human.

The ATP binding cassette (ABC) transporters represent the largest family of transmembrane proteins and play important roles in human inherited disease such as the multi-organ disease cystic fibrosis and cholesterol transport disorder Tangier’s disease. These proteins are also implicated in conferring multidrug resistance, rendering many cancer therapies ineffective, as well as contributing to the pathogenicity of some organisms. The yeast ABC proteins, Pdr12p, a weak acid efflux pump, and Ste6p, the a-factor exporter, were screened for interacting partners using the integrated membrane yeast two-hybrid (iMYTH) system to gain further insight into their biological function. Two interactors were identified for Ste6p, however, the Pdr12p screen identified 13 novel interactions, most notable of which are three other ABC transporters, Pdr5p, Pdr10p and Pdr11p. Subsequent functional analysis of double deletion mutants supports a genetic interaction between Pdr12p and Pdr10p as the pdr12Δ pdr10Δ strain showed resistance to increasing concentrations of weak organic acids.

The ATP binding cassette (ABC) transporters represent the largest family of transmembrane proteins and play important roles in human inherited disease such as the multi-organ disease cystic fibrosis and cholesterol transport disorder Tangier’s disease. These proteins are also implicated in conferring multidrug resistance, rendering many cancer therapies ineffective, as well as contributing to the pathogenicity of some organisms. The yeast ABC proteins, Pdr12p, a weak acid efflux pump, and Ste6p, the a-factor exporter, were screened for interacting partners using the integrated membrane yeast two-hybrid (iMYTH) system to gain further insight into their biological function. Two interactors were identified for Ste6p, however, the Pdr12p screen identified 13 novel interactions, most notable of which are three other ABC transporters, Pdr5p, Pdr10p and Pdr11p. Subsequent functional analysis of double deletion mutants supports a genetic interaction between Pdr12p and Pdr10p as the pdr12Δ pdr10Δ strain showed resistance to increasing concentrations of weak organic acids.

Pancreatic cancer (PC) is known to be highly resistant to chemotherapy. Transporters, which regulate the influx and efflux of substrates across the plasma membrane, may play a role in PC drug resistance. ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which play major roles in cancer drug resistance. Given that 90% of PC express a mutant K-ras oncogene and that PC are highly hypoxic, I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote drug resistance in PC. Using normal and PC cell lines either overexpressing mutant K-ras or subjected to hypoxic treatment, mRNA expression was profiled for 48 ABC transporters. My findings indicate that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in PC cells in part by inducing the expression of ABC transporters. Similar to ABC transporters, I investigated whether amino acid transporters would mediate drug resistance in PC. The Xc⁻ amino acid transporter (Xc⁻) mediates cellular uptake of cystine for the biosynthesis of glutathione, a major detoxifying agent. Because the Xc⁻ has been regulates the growth of various cancer cell types, and Xc⁻ is expressed in the pancreas, I postulated that the Xc⁻ may be involved in growth and drug resistance in PC. The Xc⁻ transporter is differentially expressed in normal pancreatic tissues and is overexpressed in PC in vivo. Using PC cell lines, I found that cystine uptake via the Xc⁻ was required for growth and survival in response to oxidative stress, and that expression of the Xc⁻ correlated with gemcitabine resistance. Accordingly, inhibition of Xc⁻ expression via siRNA reduced PC cell proliferation and restored sensitivity to gemcitabine. I also identified the anti-inflammatory drug sulfasalazine as a mixed inhibitor of the Xc⁻, which acts to inhibit cell proliferation via reducing Xc⁻ activity and not by reducing NFKB activity. My findings thus indicate that the Xc⁻ plays a role in PC growth in partby contributing to glutathione synthesis to promote PC cell proliferation, survival, and drug resistance.

No description available.

Pancreatic cancer (PC) is known to be highly resistant to chemotherapy. Transporters, which regulate the influx and efflux of substrates across the plasma membrane, may play a role in PC drug resistance. ABC transporters are a large family of transmembrane proteins with diverse physiological functions, several of which play major roles in cancer drug resistance. Given that 90% of PC express a mutant K-ras oncogene and that PC are highly hypoxic, I postulated that constitutive K-ras activation and/or hypoxia may correlate with ABC transporter expression, which in turn may promote drug resistance in PC. Using normal and PC cell lines either overexpressing mutant K-ras or subjected to hypoxic treatment, mRNA expression was profiled for 48 ABC transporters. My findings indicate that expression of mutant K-ras and hypoxic treatment, as well as long-term exposure to chemotherapy, may contribute to the development of drug resistance in PC cells in part by inducing the expression of ABC transporters. Similar to ABC transporters, I investigated whether amino acid transporters would mediate drug resistance in PC. The Xc⁻ amino acid transporter (Xc⁻) mediates cellular uptake of cystine for the biosynthesis of glutathione, a major detoxifying agent. Because the Xc⁻ has been regulates the growth of various cancer cell types, and Xc⁻ is expressed in the pancreas, I postulated that the Xc⁻ may be involved in growth and drug resistance in PC. The Xc⁻ transporter is differentially expressed in normal pancreatic tissues and is overexpressed in PC in vivo. Using PC cell lines, I found that cystine uptake via the Xc⁻ was required for growth and survival in response to oxidative stress, and that expression of the Xc⁻ correlated with gemcitabine resistance. Accordingly, inhibition of Xc⁻ expression via siRNA reduced PC cell proliferation and restored sensitivity to gemcitabine. I also identified the anti-inflammatory drug sulfasalazine as a mixed inhibitor of the Xc⁻, which acts to inhibit cell proliferation via reducing Xc⁻ activity and not by reducing NFKB activity. My findings thus indicate that the Xc⁻ plays a role in PC growth in partby contributing to glutathione synthesis to promote PC cell proliferation, survival, and drug resistance.

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.