As there is a need for fully validated drug targets in <i>Trypanosoma cruzi</i>, the genetic andbiochemical essentiality of <i>N</i>-myristoyltransferase (NMT) was assessed. The geneticrequirement was assessed using a classical gene replacement strategy, attempting tosequentially replace the endogenous alleles with drug resistance genes to generate an<i>NMT</i> null parasite. It was only possible to achieve this in the presence of an ectopiccopy of <i>NMT</i> under constitutive expression, providing the strongest evidence that thisgene is essential for the proliferation of the epimastigote. While both NMT and <i>N</i>-myristoylationwere detected in all lifecycle stages, there were subtle differences in theexpression of several myristoylated proteins. However, at least ~10 myristoylatedproteins were common throughout the lifecycle. In addition, <i>N</i>-myristoylation in thisparasite was found to be primarily associated with nascent protein synthesis, astreatment with cycloheximide reduced the number of <i>N</i>-myristoylated proteins detected. The sensitivity of epimastigotes to the inhibitor DDD85646 correlated with theexpression of NMT, suggesting it to be the target in the parasite. This was confirmedby the dose-dependent depletion of <i>N</i>-myristoylated proteins detected in parasitestreated with this compound. Mechanism of action studies revealed a cytokinesis defectcaused by the inhibition of <i>N</i>-myristoylation and NMT. Overexpression of NMT wasable to rescue these cells from this phenotype confirming that it is NMT mediated. The<i>N</i>-myristoylated proteins comprising the <i>N</i>-myristoylome of the epimastigote wereidentified using the myristic acid analog, azidomyristate and a chemical proteomicsapproach. Combining label-free and SILAC methodologies, 38 proteins were enrichedfrom azidomyristate labelled cells, 35 of which were predicted to have a glycine afterthe initial methionine. The findings from these experiments have led to the mostcomprehensive <i>N</i>-myristoylome of <i>T. cruzi</i> studied to date and provide severalhypotheses, by which the inhibition of NMT leads to the observed cytokinesis defect.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642885 |
| Date | January 2014 |
| Creators | Roberts, Adam |
| Contributors | Fairlamb, Alan |
| Publisher | University of Dundee |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://discovery.dundee.ac.uk/en/studentTheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4 |
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