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.

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.

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.

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.

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.

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.

The integrated Membrane Yeast Two-Hybrid technology (iMYTH) was employed to screen the ABCD family of ABC Transporters in S. cerevisiae. The two ABCD members, Pxa1p and Pxa2p were screened against yeast libraries which detected many interactors involved in expected biological processes in accordance with Pxa1p and Pxa2p’s annotated function (i.e. fatty acid metabolism), and unexpected new interactors that may elucidate a new role for the transporters (e.g. oxidative protection). Members of the glutaredoxins and thioredoxins, and associated proteins were found to interact with Pxa1p and Pxa2p. This may indicate that Pxa1p and Pxa2p also play a role in managing the redox environment, protecting against reactive oxygen species. The iMYTH technology was also used to show that Pxa2p is able to form homodimers, and that Pxa1p localization is directly or indirectly dependent on Pxa2p.

The integrated Membrane Yeast Two-Hybrid technology (iMYTH) was employed to screen the ABCD family of ABC Transporters in S. cerevisiae. The two ABCD members, Pxa1p and Pxa2p were screened against yeast libraries which detected many interactors involved in expected biological processes in accordance with Pxa1p and Pxa2p’s annotated function (i.e. fatty acid metabolism), and unexpected new interactors that may elucidate a new role for the transporters (e.g. oxidative protection). Members of the glutaredoxins and thioredoxins, and associated proteins were found to interact with Pxa1p and Pxa2p. This may indicate that Pxa1p and Pxa2p also play a role in managing the redox environment, protecting against reactive oxygen species. The iMYTH technology was also used to show that Pxa2p is able to form homodimers, and that Pxa1p localization is directly or indirectly dependent on Pxa2p.

Heart disease is leading cause of death in the United States. High-density lipoprotein (HDL) levels are inversely correlated with the prevalence of coronary heart disease. The anti-atherogenic properties of HDL are associated with its role in the pathway of the reverse cholesterol transport, which removes cholesterol from peripheral cells for transport back to hepatocytes. The formation of HDL is facilitated by ATP cassette transporter ABCA1 and apolipoprotein A-I, the major protein of HDL particles. However, the underlying molecular mechanism behind the biogenesis of HDL is not well understood. To provide further understanding of this mechanism, we developed two ABCA1 expression systems in both Sf9 insect cells and FreestyleTM 293-F human cells for functional and structural studies. We designed all constructs of ABCA1 to contain a C-terminal rho1d4 tag that bound to an affinity matrix of rho1d4 antibodies for successful purification. To reconstitute ABCA1 in a detergent-free environment that models the native membrane, we developed three reconstitution systems for ABCA1: saposin A nanodiscs, peptidisc, and amphipol A8-35. Biochemical and structural studies were carried out to understand the mechanism behind ABCA1’s function. We demonstrated a potential direct interaction of ABCA1 and apolipoprotein A-I with a pull-down experiment. Two cryo-electron microscopy data collections were obtained of ABCA1 in a detergent environment in the presence of ATP with the goal of determining the structure of ABCA1’s active state. We produced a 12 Å reconstruction of ABCA1 from this first data collection. This low-resolution structure confirmed the general structure that currently exists for ABCA1. Processing the data helped us streamline and troubleshoot the electron microscopy workflow pipeline for future data collections. Unfortunately, the second data collection had astigmatism issues that prevented particle alignment during data analysis. However, these data collections provided considerable insight into the ideal sample freezing and grid preparation conditions that affect data collection and data processing. More transmembrane protein structures are being solved each year but there remain many obstacles and challenges in ABCA1 purification and grid preparation that affect the ability to perform functional studies and high-resolution structure determination. Our developmental work has helped move forward our biochemical understanding of ABCA1 to achieve these aims. The more that is learned about this important membrane protein the more likely it is that future consistent production of ABCA1 will be accomplished to answer the questions of how ABCA1 mediates the formation of HDL particles.