Structural and functional analyses of the FAM46 proteins.

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FAM46C (Family with sequence similarity 46, C) is one of the most frequently mutated genes in multiple myeloma (found in over 20% of the patients). Mutations of other FAM46 family members (A, B and D) are also associated with various human diseases. Despite the strong connections with diseases, the functions of FAM46 in either physiological or pathological settings are unknown. The goal of the project is to fill the knowledge gap on the functions of the FAM46 proteins and the underlying mechanisms, paving the way for developing therapeutic strategies for the associated diseases. The project is based on our preliminary work showing the direct physical interactions of FAM46 with polo-like kinase 4 (Plk4) and BRCA2 and CDKN1A(p21) interacting protein (BCCIP). Plk4 is the master regulator of centrosome duplication and ciliogenesis. BCCIP has been shown to be involved in regulating DNA repair and centrosome duplication. These together suggest that the FAM46 proteins regulate centrosome duplication, ciliogenesis and DNA repair. Centrosomes organize the bi-polar spindle formation and proper chromosome segregation in the cell cycle. In non-dividing cell, centrosomes mediate the formation of primary cilia, specialized signaling organelles critical for organogenesis and tissue homeostasis. A role in regulating these processes are consistent with the strong connection between FAM46 mutations and cancer. These hypotheses will be tested in three aims by combining X-ray crystallographic, biophysical, biochemical and cell-based functional approaches. Aim 1 will be focused on biophysical and structural analyses of the FAM46/Plk4 interaction. These studies will reveal the binding mode between FAM46 and Plk4, and identify key residues that can be tested by mutations in functional assays. In addition, potential mutual regulation of the enzymatic activities between FAM46 and Plk4 will be analyzed. Aim 2 will be focused on biophysical and structural analyses of the FAM46/BCCIP interaction. The crystal structure of the FAM46 and BCCIP complex will be determined to elucidate their interaction in atomic detail. Structural analyses will also be directed at the potential FAM46, Plk4 and BCCIPα tripartite complex. Mutational analyses will follow to test the binding mode and interface residues. The goal of Aim 3 is to analyze the roles of the interactions among FAM46, Plk4 and BCCIPα in regulating centrosome duplication, ciliogenesis and DNA repair. Fluorescence microscopy will be used to probe the localization of FAM46, Plk4 and BCCIPα, and their colocalization in and out of centrosomes in various stages of the cell cycle. The regulation of centrosome duplication, ciliogenesis and DNA repair by FAM46 through the interactions with Plk4 and BCCIP will also be analyzed by fluorescence microscopy.