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dc.contributor.authorAgate, Diana S.
dc.date.accessioned2018-07-12T17:33:42Z
dc.date.available2018-07-12T17:33:42Z
dc.date.issued2006
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6587.;Advisors: David E. Cohen.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3196705
dc.identifier.urihttps://hdl.handle.net/20.500.12202/809
dc.description.abstractPhosphatidylcholine transfer protein (PC-TP) is a member of the START domain protein superfamily that exclusively binds phosphatidylcholines. The aim of this thesis was to explore the structure and function of this highly specific lipid-binding protein.;A yeast two-hybrid screen identified 3 transcription factors that interact with PC-TP at days 9.5--10.5 of development: Pax3, Tbx1, and Tsc2. In situ hybridization displayed a diffuse expression pattern of PC-TP throughout the E10.5 mouse embryo. The interaction between PC-TP and Pax3 was confirmed by immunoprecipitation from cotransfected HEK 293T cell lysates. Confocal laser scanning mircroscopy demonstrated that the colocalization of PC-TP and Pax3 to the nucleus in HEK 293T cells was dependent on the presence of the Pax3 homeodomain.;To test the significance of a disulfide bond found within the crystal structure of PC-TP in complex with dilinoleoyl-phosphatidylcholine, we utilized site-directed mutagenesis to create two mutant forms of PC-TP, C63S and C63A, which were incapable of forming a disulfide linkage. Intermembrane phosphatidylcholine transfer activities of the mutated forms of PC-TP were measured in vitro. Compared with wild type PC-TP, the C63A and C63S transfer abilities were only modestly reduced, suggesting that this bond may not be functionally significant for optimal binding and transfer of phosphatidylcholine by PC-TP.;New Zealand Obese (NZO) mice are characterized by juvenile-onset obesity and maturity-onset diabetes. Nucleotide sequence analysis of Pctp revealed a polymorphism in the coding region of the NZO allele that resulted in an R120H substitution. Using site-directed mutagenesis to recreate the mutation in recombinant PC-TP, we assessed the in vitro phosphatidylcholine transfer activity of R120H PC-TP. The R120H substitution eradicated phosphatidylcholine transfer activity, suggesting that a spontaneous mutation in Pctp may predispose one to obesity and diabetes.;These studies have given insight into the binding mechanisms and specificity PC-TP has for phosphatidylcholine, as well as propose novel roles in embryogenesis, diabetes, and obesity in mice. A broader implication of these studies suggest an evolutionary divergence of START from other functional domains created lipid-sensing molecules whose multiple functions are dictated by the binding partner.
dc.publisherProQuest Dissertations & Theses
dc.subjectBiochemistry.
dc.titleStructure-function studies of phosphatidylcholine transfer protein
dc.typeDissertation


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