|Institution:||University of Oslo|
|Full text PDF:||http://urn.nb.no/URN:NBN:no-25053
This study investigates the function of a novel human protein, which, based on secondary structure prediction, belongs to class I of SAM-dependent methyltransferases (MTases). No specific functions or substrates have yet been published for this protein, but results from our laboratory indicate that it is a protein methyltransferase, thus denoted PMTX1. In this work, several human cell lines with a knockout (KO) mutation in all alleles of the PMTX1-encoding gene were generated for future use as a tool for investigating the function of the gene in vivo. This was achieved using novel engineered Zinc finger nuclease (ZFN) technology which proved to be a rather efficient and practical method of targeted inactivation of the PMTX1-encoding gene. To complement the gene disruption, one of the KO cell lines generated by ZFN technology was used to make a cell line for stable and inducible expression of the PMTX1 wild type protein. The function of the PMTX1 protein was also investigated through several in vitro MTase assays which indicated that this enzyme preferably methylates protein substrates on lysine residues and has some activity on recombinant human histones. In an attempt to identify key residues of the PMTX1 protein involved in the binding of SAM and substrate, several mutant proteins were generated. Thermal Shift Assays (TSA) indicated that all PMTX1 mutants except one were correctly folded and that binding of SAM to PMTX1 increased its stability. By using in vitro MTase assays, it was determined whether the mutations introduced into PMTX1 influence their activity. Two different mutations of one conserved residue in the SAM-binding domain were shown to abolish MTase activity, while mutations of several conserved residues in the putative substrate-binding domain have moderately decreased the activity of the protein.