The focus of this thesis is the lipidation by N-myristoyltransferase (NMT) in the plant pathogenic fungus Zymoseptoria tritici. The work presented here was performed at Imperial College London in the group led by Prof. Ed Tate in collaboration with Syngenta AG.
Z. tritici NMT was characterized and IC50 values were determined for a number of small-molecule inhibitors. A number of NMT inhibitors from the Imperial College in-house collection were tested in an enzymatic assay against the enzyme. A few of the compounds that showed lowest IC50 values were chosen for use in an in vitro assay in Z. tritici cultures. A few inhibitors were found to possess EC50 values in the range of 20-50 μM. A separate experiment confirmed, through metabolic labelling of NMT substrates, that one of the inhibitors, IMP-1088, was able to reduce myristoylation activity in cultured Z. tritici.
Metabolic labelling with alkyne-tag modified myristic acid was optimized via an in-gel fluorescence method. A complete workflow including the pull-down of the labelled myristoylated proteins and their proteomics analysis was performed. The first experimental evidence for myristoylation of twenty-four Z. tritici proteins in proteomics experiments was obtained using label-free and tandem mass tag (TMT) approaches. The TMT proteomics was used to demonstrate that myristoylation was decreased upon IMP-1088 treatment.
The whole proteome analysis of Z. tritici cultures upon IMP-1088 treatment revealed significant changes in the expression of several proteins. We used a BLAST tool to find counterparts of Z. tritici proteins in Saccharomyces cerevisiae and GO terms were assigned based on S. cerevisiae terms. Several GO terms, such as decrease in cell metabolism and over-representation of GST protein URE2 analogues, were found among the most significantly upregulated terms. These terms provide the foundation for hypothesis for understanding the low translation of inhibition from NMT enzyme to Z. tritici cultures.