Role of nuclear receptors in the biology of Schistosoma mansoni: SmCAR and SmTR2/4

In an effort to identify schistosome nuclear receptors, especially, the heterodimeric binding partners for SmRXR1 and SmRXR2, two schistosome nuclear receptors, SmCAR and SmTR2/4 were identified and studied in this research. SmCAR is a 702 amino acid protein exhibiting the highest homology with mouse constitutive androstane receptor (mCAR). SmCAR contains a novel P box (ESCKA), which is unique to invertebrates. The same P box has been found in DmDHR96 and CeDAF-12. This is relevant as the P Box is critical in recognizing and discriminating the core half site sequence in the hormone response element (HRE) for nuclear receptors. SmCAR mRNA is expressed at every parasitic developmental stage studied, and SmCAR protein is widely distributed in the adult schistosome worm, especially in the subtegumental cells. In vitro DNA binding assays demonstrated that SmCAR binds to a p14-S template, a HRE identified in the schistosome p14 gene upstream region, as a monomer. These data and previous studies of the DNA binding of DmDHR96 and CeDAF-12 together suggest that AGTGCA probably serves as a high affinity DNA binding half site for the ESCKA family members.;Yeast-two hybrid and in vitro pull down and co-immunoprecipitation experiments demonstrated that SmCAR interacts with SmRXR1 but not with SmRXR2. Study of schistosome-mouse CAR chimeras demonstrated that SmCAR DBD shares certain functional DNA binding specificity with its vertebrate homologues but also has some DNA binding properties specific to SmCAR DBD. In addition, SmCAR DBD exhibited the heterodimeric DNA binding properties. Therefore, we propose that SmCAR binds to its cognate sequence via at least two mechanisms, binding to DNA as a monomer or as a heterodimer with SmRXR1.;SmTR2/4 is a 1943 amino acid protein, the largest nuclear receptor reported to date. Homology search and phylogenetic analysis demonstrated that SmTR2/4 belongs to the TR2/TR4 group in nuclear receptor subfamily II. As reported for its homologues, SmTR2/4 binds to the DR-3, DR-4 hormone response elements in an in vitro DNA binding assay, suggesting a functional conservation among the TR2/TR4 family members in terms of DNA binding specificity. Study in this thesis will help define the role of nuclear receptors in the biology of S. mansoni.