Expression and characterization of FAT1 and atrophin 1 proteins regulating planar cell polarity and MBD1 protein involved in lymphoma

Abstract

Fat, the first tumor suppressor gene to be discovered in Drosophila melanogester, is one of the most important regulators of planar cell polarity which controls the directional alignment of hair bristles and photoreceptors in the eyes of Drosophila. The mammalian counterpart of Fat known as Fat1 has been found to play a vital role during cerebral development, glomerular slit formation and gastrulation. Atrophin1 (also known as grunge) is a nuclear receptor which is predominately found in the nucleus but sometimes shuttles to the cytoplasm. The C-terminus of Atrophin is shown to interact with the C-terminal domain of Fat in the regulation of planar cell polarity. The precise role of these two important molecules in planar cell polarity is yet to be fully understood. Apart from its role in the Fat-Atrophin complex, Atrophin1 like proteins have been implicated in Dentatorbral Pallidoluysian Atrophy, a neural disorder. The structures of the C-terminal domains of Fat1 (160 aa) and Atrophin1 (196 aa) from Mus musculus (to be solved, separately and for their complex, using X-ray crystallography) will provide a pedestal for understanding the roles of Fat1 and Atrophin1 in the mechanism of regulation in planar cell polarity. MBD1 or Methyl binding domain 1 protein belongs to the class of Methyl CpG binding proteins (MBD 1-4 and MeCP2).The sequence similarity of these proteins is restricted only in their MBD domain, thus highlighting different roles. MBD1 has additional TRD and Zinc finger domains, which bind to non-methylated DNA and silence them, while the MBD domain silences hypermethylated DNA. The dual DNA binding capacity of MBD1 is of great importance in understanding tumorigenesis, very little of which is currently known. The solution structure of the human MBD domain in complex with DNA has been solved. Currently, we are cloning full length MBD1 (605 aa) from a human lymphoma cell line into the p Fast Bac Htb vector for baculovirus expression, after failure to express this protein in bacterial expression systems using the pET14b and pET32A vectors.