As there is a need for fully validated drug targets in
Trypanosoma cruzi, the genetic andbiochemical essentiality of
N-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
NMT null parasite. It was only possible to achieve this in the presence of an ectopiccopy of
NMT under constitutive expression, providing the strongest evidence that thisgene is essential for the proliferation of the epimastigote. While both NMT and
N-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,
N-myristoylation in thisparasite was found to be primarily associated with nascent protein synthesis, astreatment with cycloheximide reduced the number of
N-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
N-myristoylated proteins detected in parasitestreated with this compound. Mechanism of action studies revealed a cytokinesis defectcaused by the inhibition of
N-myristoylation and NMT. Overexpression of NMT wasable to rescue these cells from this phenotype confirming that it is NMT mediated. The
N-myristoylated proteins comprising the
N-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
N-myristoylome of
T. cruzi studied to date and provide severalhypotheses, by which the inhibition of NMT leads to the observed cytokinesis defect.
| Date of Award | 2014 |
|---|
| Original language | English |
|---|
| Awarding Institution | |
|---|
| Sponsors | Biotechnology and Biological Sciences Research Council & Wellcome Trust |
|---|
| Supervisor | Alan Fairlamb (Supervisor) |
|---|