Development of enzymatic labelling methods has been driven by the importance of studying molecular structures and interactions to comprehend cellular processes. Methyltransferases (MTases), which regulate genetic expression by transferring a methyl group from the cofactor S-adenosyl-L-methionine (SAM) to DNA, histones and various proteins, have been shown to accept SAM analogues with an alternative alkyl group on the sulfonium centre. These alkyl groups can be transferred to the substrate, and with a further reaction can be selectively functionalized. Thus, MTases together with SAM analogues have emerged as novel labelling tools. The project aims to use MTases to obtain an orthogonal system that can selectively use a SAM cofactor analogue to transfer functional chains to proteins with a specific motif. To achieve selectivity of the system, the SAM analogue cofactor was modified on the ribose ring; to obtain a new transferase activity of the system, the transferable methyl on the sulfonium centre was changed to a different substituent. SAM analogues were produced enzymatically with hMAT2A by using 3'-deoxy-ATP and methionine or ethionine. Mutants of SET8 and novel substrates were designed to have modifications at residues in the active site, within the vicinity of the ribose ring of SAM, and were assessed for selective activity with the new analogue cofactor. The results showed that the new cofactor 3'-deoxy-S-adenosyl-L-methionine (3'dSAM) was efficient in the mono-methylation of the substrate peptide RFRKVL, and that the mutant SET8 C270V exhibited over 13 fold MTase activity in presence of 3'dSAM and the RFRKVL substrate, in comparison with the activity with the WT sequence RHRKVL and the SAM cofactor. In addition, glutathione S-transferase (GST) was used as a model protein to express the motif RFRKVL, to transform it into a potential substrate for SET8. Assessment of the MTase activity of SET8, 3'dSAM and the novel GST substrate indicated mono-methylation of the substrate. Moreover, the motif showed no interference with GST native activity. Based on the observations, a new enzymatic system shows higher selectivity with a new analogue cofactor over SAM to effectively methylate proteins expressing the consensus RFRKVL. Work on substrates, enzymes and cofactors should continue to obtain a functional-chain transferase activity of the enzymatic system.

Transcription factor LSF is an oncogene in Hepatocellular Carcinoma (HCC). HCC is the sixth most common cancer worldwide and the second highest cause of cancer-related death globally. LSF is overexpressed in human HCC cell lines, compared to normal hepatocytes, and expression levels show significant correlation with the stage and grades of the disease. Current treatments for HCC are insufficient, especially given the frequency of late stage diagnoses. Therefore, it is necessary to understand the molecular mechanism of HCC disease to aid in targeted and effective treatments. Most investigations of the regulation of LSF activity have focused on its post-translational modifications in response to cellular proliferation and signal transduction. Chromatin modifications and epigenetic mechanisms of LSF-mediated gene regulation had not been investigated. Given that alterations of epigenetic writers or readers have been demonstrated in a large fraction of HCC patient samples, I examined the connection between LSF and epigenetic regulators. In particular, LSF is shown to interact with DNA methyltransferase 1 (DNMT1) and Ubiquitin like with PHD and Ring Finger Domains (UHRF1), with consequences for global DNA methylation and transcription patterns. Additionally, I identified unexpected, pairwise associations between LSF, histone methyltransferase SET8, and tubulin, both in vitro and in vivo. The interactions were identified by proteomics analyses, co-localization, co-immunoprecipitation, and direct protein-protein interaction studies in vitro. Strikingly, both LSF and SET8 associate with microtubules, leading to the discovery that SET8 methylates α-tubulin at several novel, specific lysines. This suggests parallels between regulation of chromatin by the histone code and regulation of microtubule function by the tubulin code. Surprisingly, LSF enhances tubulin methylation by SET8 in vitro and FQI1, a specific LSF small molecule inhibitor, reduces tubulin methylation. Furthermore, LSF promotes, and FQI1 inhibits, tubulin polymerization in vitro. Taken together, these findings suggest that SET8 is a microtubule-associated methyltransferase that LSF recruits to microtubules to enhance tubulin modification. The results indicate that both LSF and SET8 have cellular implications beyond their roles in gene transcription and histone methylation. Finally, this discovery of the dual functions for LSF and SET8 set up the possibility for connections between epigenetic and cytoskeleton modifications in cancer. / 2021-12-24T00:00:00Z