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dc.contributor.authorMok, Leoen
dc.date2010-02-12 11:14:11.381
dc.date.accessioned2010-02-12T18:24:18Z
dc.date.available2010-02-12T18:24:18Z
dc.date.issued2010-02-12T18:24:18Z
dc.identifier.urihttp://hdl.handle.net/1974/5434
dc.descriptionThesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-02-12 11:14:11.381en
dc.description.abstractThe ATP-binding cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport, which is the elimination of excess sterols from peripheral cells and their transport to the liver for elimination. Early studies failed to detect significant ATPase activity, prompting the suggestion that ABCA1 was an ATP-regulated receptor, rather than an active transporter. We have provided evidence that ABCA1 can bind ATP and trap its hydrolysis product, ADP, in the presence of either ortho-vanadate or beryllium fluoride and Mg2+ or Mn2+. We have also shown that both nucleotide-binding domains (NBDs) trap nucleotide comparably, suggesting that ABCA1 is a functional ATPase. In addition, we have shown that ABCA1 can directly transport 25-hydroxycholesterol (25-OHC) in an ATP-dependent manner using a membrane vesicle uptake assay, and can do so when the physiological substrate acceptor apoA-I is replaced with BSA as a non-specific binding protein. Although more than 50 naturally occurring missense mutations and polymorphisms in ABCA1 have been identified in individuals with HDL-C levels within the lowest 5th percentile of the general population, the extent to which many of these mutations affect ABCA1 function is not known and cannot be predicted. Naturally occurring extracellular loop (ECL) mutations W590S and C1477R have both been shown to effectively eliminate the ability to mediate lipid efflux, despite the fact that the W590S mutant protein retains the ability to bind apoA-I. We show that neither mutant can transport nor efflux 25-OHC, whether in the presence of apoA-I or BSA, despite apparently full retention of the ability to bind and trap nucleotide. This suggests that these two ECL mutations inhibit transport by a mechanism that is independent of their effect on apoA-I binding. By introduction of naturally occurring mutations in the NBDs, we show that although some mutations associated with Tangier Disease, such as N935S, essentially eliminate nucleotide trapping and substrate translocation, other polymorphisms such as L1026P and T2073A associated with low HDL-C, appear to be fully functional. Lastly, we observed differences in the behaviour of both wild-type and mutant forms of ABCA1-GFP depending on whether they were expressed in insect or mammalian cell lines.en
dc.format.extent4174205 bytes
dc.format.mimetypeapplication/pdf
dc.language.isoengen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectATP-binding cassette transporteren
dc.subjectTangier Diseaseen
dc.subjectAtherosclerosisen
dc.subjectHeart diseaseen
dc.subject25-hydroxycholesterolen
dc.subjectCholesterolen
dc.subjectPhospholipiden
dc.subjectApolipoproteinen
dc.subjectHDLen
dc.subjectLipid effluxen
dc.titleIn Vitro Characterization of the Function of ABCA1: Effects of Naturally Occurring Mutationsen
dc.typethesisen
dc.description.degreePhDen
dc.contributor.supervisorDeeley, Roger G.en
dc.contributor.departmentPathology and Molecular Medicineen
dc.degree.grantorQueen's University at Kingstonen


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