Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/37923
Title: STRUCTURAL STUDIES OF TRANSCRIPTIONAL REPRESSOR DOMAIN (TRD) OF MBD1 AND MIDA PROTEINS
Authors: UMAR FAROOK SHAHUL HAMEED
Keywords: TRD, MBD1, MCAF1, MPG, HDAC3, MIDA
Issue Date: 25-Jan-2013
Source: UMAR FAROOK SHAHUL HAMEED (2013-01-25). STRUCTURAL STUDIES OF TRANSCRIPTIONAL REPRESSOR DOMAIN (TRD) OF MBD1 AND MIDA PROTEINS. ScholarBank@NUS Repository.
Abstract: Epigenetics is an important field of genetic modification that controls cell function, development and sustenance. Methylation of CpG nucleotides is an important modification that could alter the expression of particular genes, mostly tumor suppressor genes. In mammalian genomes, the promoter region of almost 70% of genes is rich in CpG nucleotides known as ¿CpG islands.¿ MBD1 plays an important role in cell development and function by suppressing genes that are methylated at CpG regions by binding to methylated DNA and recruiting other repressor proteins to form heterochromatin. MBD1 is composed of methylated DNA binding domain (MBD), cysteine rich CXXC zing finger domain and transcriptional repressor domain (TRD). The TRD of MBD1 is very essential for gene silencing, since it is responsible for recruiting other repressor proteins, like MCAF/AM family and MPG proteins to form heterochromatin. In our current study we have characterized the structure of TRD using NMR and also analyzed its function by in vitro studies. TRD is found to be an intrinsically unstructured protein with less defined secondary structures and the titration of unlabelled truncated form of MCAF1 and MPG with labeled TRD showed weak binding,as seen from ITC data. From the binding studies using NMR, we identified the important residues of TRD that are involved in interaction with its binding partners. These results were further validated by pull down assay experiments. Our results show that MBD1 recruits repressor proteins in a transient manner, rather than tight binding, which would otherwise result in constitutive gene repression. MidA (Mitochondrial dysfunction ProteinA) is present in mitochondria of Dictyostelium and is a close homolog to human protein C2orf56. The protein is reported to be important for mitochondrial Complex I formation and also has a methyl transferase domain, which possibly methylates one of the subunits of mitochondrial Complex I. Mitochondrial Complex I is composed of approximately 45 subunits and it plays an important role in oxidative phosphorylation and many reports confirm that mitochondrial diseases are mostly due to malfunctioning of Complex I. MidA is reported to be important for normal cell functioning in cells, since knock down could result in severe defects in cell morphology and functioning of Dictyostelium, where formation of Complex I is affected drastically. Protein MidA has been proposed to methylate NDUFS2, a subunit of Complex I. We have determined the crystal structure of the apo MidA protein and in complex with SAM and SAH, the co-factor and the modified form, respectively. MidA has characteristics of a macromolecular methyl transferase, with its N-terminal domain binding to co-factors and its C-terminal domain could possibly bind to substrate.
URI: http://scholarbank.nus.edu.sg/handle/10635/37923
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