Structure-function studies of phosphatidylcholine transfer protein

Abstract

Phosphatidylcholine 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.