Biochemical and Structural Studies of the Role of FKBP(L)s in the fly and Mammalian Circadian Cycle
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Abstract
FK506 binding-like proteins (FKBPLs) are proteins that structurally resemble the immunophilins. Their functions in cell signaling include protein folding, molecular trafficking, RNA splicing and cell cycle regulation among others. Bride of Doubletime (BDBT) previously designated as CG17282 in the flybase was identified as a circadian clock component that stimulates DBT’s circadian function. Here, we have used biochemical and structural approaches to characterize BDBT. We determined the x-ray crystal structure of BDBT and the analysis showed that it is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that interacts with DOUBLETIME and tetratricopeptide repeats at its C-terminal region that may mediate protein-protein interaction. We used similar techniques to identify BDBT vertebrate functional homologues FKBP51, FKBP52 and FKBPL. In vitro kinase assays showed that all three proteins stimulate CK1δ phosphorylation activity towards casein. Among these three proteins, FKBP51 was seen to bind mammalian casein kinase 1 delta (CK1δ) with much stronger affinity than the other FKBPs. From genetic studies, ShRNA to fkbpl in mammalian U20S cell model showed reduced amplitude and longer circadian period. Further site-directed mutagenesis revealed to us that certain residues, which are important for nuclear localization of CK1δ affect its interaction with FKBP51. Altogether, our work illustrates novel role for FKBP(L)s as molecular clock components in flies and mammals.
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Introduction -- Materials and methodology -- Crystal structure of BDBT reveals a non-canonical FK506 binding protien as a circadian clock regulator -- FKBP51, FKBP52 and FKBPL interact with CK1δ to regulate its function in the mammalian clock -- Final discussion
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Ph.D.