Browsing by Subject "ATP-Binding Cassette Transporters"
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Item ABC transporters and human disease: From cholestasis to heart disease(2003-01-16) Hobbs, Helen H.Item Characterization of the Reaction Cycle of MJ0796: A Model Archaeal Adenosine Triphosphate-Binding Cassette Transporter Nucleotide Binding Domain(2006-12-20) Moody, Jonathan Edward; Thomas, Philip J.Adenosine-triphosphate binding cassette (ABC) transporters couple nucleotide hydrolysis to vectorial transport of solutes across lipid bilayers. These proteins, found in all kingdoms of life, have been implicated in a variety of human genetic disorders and engender drug resistance in cancer cells and infectious prokaryotes. Despite the widely varying solutes transported by these protein machines, a conserved functional mechanism is suggested by the high degree of amino acid conservation found in the nucleotide binding domains of all ABC transporters. Using two model archaeal ABC transporter nucleotide binding domains, MJ0796 and MJ1267, from Methanocaldococcus jannaschii the highly conserved Walker A, Walker B, and LSGGQ motifs were probed using site-directed mutagenesis. Catalytic carboxylate mutants, MJ0796-E171Q and MJ1267-E179Q, exhibited nucleotide-dependent dimerization upon analytical gel filtration and equilibrium centrifugation experiments. This self-association was negatively affected by changes in the electrostatic environment, as shown using alanine substitutions at these loci as well as altering the ionic conditions of the experiments. The MJ0796-E171Q protein was crystallized, and its structure solved to 1.9 angstrom resolution. The structure reveals an ATP sandwich dimer with two nucleotides bound at the dimeric interface, with each binding site composed of Walker A and B residues from one monomer and LSGGQ residues from the opposing monomer. A proposed reaction cycle based upon the MJ0796-E171Q dimer structure was probed using Walker A, Walker B, and LSGGQ point mutants. Mixtures of the Walker A mutant MJ0796-K44A with LSGGQ mutant MJ0796-S147F, both hydrolytically deficient in isolation, did not exhibit activity. In stark contrast, mixtures of MJ0796-S147F and MJ0796-E171Q did exhibit 25% wild type activity, suggesting a mechanism whereby two nucleotide binding events and a single hydrolysis event complete the minimal reaction cycle. This also suggests that during wild type hydrolysis, two nucleotides are hydrolyzed per cycle. These heterodimeric mutant mixtures were further analyzed using tryptophan fluorescence emission and anisotropy. Mixing experiments were performed using a full transporter system, the lipoprotein release machinery from Escherichia coli. A modified ABC transporter reaction cycle is presented.Item The Roles of ATP-Binding Cassette Transporters G5 and G8 in Liver X Receptor-Mediated Sterol Trafficking(2007-12-03) York, Jennifer Lynn; Hobbs, Helen H.The liver X receptor (LXR) is a nuclear receptor that plays a critical role in orchestrating the trafficking of sterols between tissues. Treatment of wild type mice with a potent and specific nonsteroidal LXR agonist, T0901317, is associated with increased biliary cholesterol secretion, decreased fractional cholesterol absorption, and increased fecal neutral sterol excretion. The following studies show that expression of two target genes of LXRalpha , the ATP-binding cassette (ABC) transporters Abcg5 and Abcg8, is required for the increase in sterol excretion, the decrease in fractional cholesterol absorption, and the increase in fecal neutral sterol excretion associated with LXR agonist treatment. Mice lacking ABCG5 and ABCG8 (G5G8-/- mice) and wild type littermate controls were treated for 7 days with T0901317. In control animals, the LXR agonist produced a 3-fold increase in biliary cholesterol concentration, a 25% reduction in fractional cholesterol absorption, and a 4-fold elevation in fecal neutral sterol excretion. In contrast, treatment of G5G8-/- mice with the LXR agonist did not significantly affect any of these parameters. These results demonstrate that ABCG5 and ABCG8 are required for LXR agonist-associated changes in dietary and biliary sterol trafficking and that increased expression of these proteins promotes cholesterol excretion in vivo.Item Somatic Stem Cell Populations and Studies on the Functional Role and Regulation of ABCG2(2005-12-20) Tunison, Mary Katherine; Garry, Daniel J.ATP binding cassette transporters use ATP to transport substances such as sterols, peptides, drugs and other toxins across membranes. Abcg2 is a member of the G family of transporters that was identified in breast cancer cells due to its ability to efflux chemotherapeutic drugs. Abcg2 can also efflux Hoechst 33342, resulting in a side population phenotype when stained cells are sorted by FACS. Recent studies have suggested that Abcg2 may be a marker for stem and progenitor cells. This paper presents experiments that were undertaken to further evaluate the functional role and regulation of Abcg2. Preliminary data obtained from a microarray performed on main and side population cells indicated that side population cells were enriched for genes that are important in cytoprotection and cell cycle control. To confirm this observation, cell cycle analysis was performed on C2C12 myoblasts transfected with an Abcg2 overexpressing plasmid. Those cells that overexpressed Abcg2 and consequently effluxed Hoechst 33342 dye were arrested in G0/G1 phase of the cell cycle, consistent with the microarray results. To determine whether Abcg2 played a cytoprotective role I attempted to measure the viability of main population versus side population cells when exposed to the oxidative compound menadione. These experiments were inconclusive, most likely due to limitations of the cell line used. To assess the regulation of Abcg2 I examined evolutionary conservation between the upstream regions of mouse and human Abcg2. Conserved regions were identified up to 11kb upstream of the mouse gene. In these regions, putative binding sites were discovered for transcription factors that are involved in stem and progenitor cell regulation. Present studies support that Abcg2 is a marker for stem and progenitor cells. Future studies will uncover additional functional roles of the transporter.