{"id":{"repo_id":"dundee","oai_identifier":"oai:discovery.dundee.ac.uk:studenttheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4"},"canonical_url":"https://search.dev.ndltd.org/etd/dundee/oai:discovery.dundee.ac.uk:studenttheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4","repository":{"repo_id":"dundee","name":"University of Dundee","base_url":"https://discovery.dundee.ac.uk/ws/oai"},"display":{"title":"Assessment of <i>N</i>-myristoyltransferase and the <i>N</i>-myristoylomeas a potential chemotherapeutic target in<i>Trypanosoma cruzi</i>","abstract":"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.","abstract_html":"As there is a need for fully validated drug targets in &lt;i&gt;Trypanosoma cruzi&lt;/i&gt;, the genetic andbiochemical essentiality of &lt;i&gt;N&lt;/i&gt;-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&lt;i&gt;NMT&lt;/i&gt; null parasite. It was only possible to achieve this in the presence of an ectopiccopy of &lt;i&gt;NMT&lt;/i&gt; under constitutive expression, providing the strongest evidence that thisgene is essential for the proliferation of the epimastigote. While both NMT and &lt;i&gt;N&lt;/i&gt;-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, &lt;i&gt;N&lt;/i&gt;-myristoylation in thisparasite was found to be primarily associated with nascent protein synthesis, astreatment with cycloheximide reduced the number of &lt;i&gt;N&lt;/i&gt;-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 &lt;i&gt;N&lt;/i&gt;-myristoylated proteins detected in parasitestreated with this compound. Mechanism of action studies revealed a cytokinesis defectcaused by the inhibition of &lt;i&gt;N&lt;/i&gt;-myristoylation and NMT. Overexpression of NMT wasable to rescue these cells from this phenotype confirming that it is NMT mediated. The&lt;i&gt;N&lt;/i&gt;-myristoylated proteins comprising the &lt;i&gt;N&lt;/i&gt;-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 &lt;i&gt;N&lt;/i&gt;-myristoylome of &lt;i&gt;T. cruzi&lt;/i&gt; studied to date and provide severalhypotheses, by which the inhibition of NMT leads to the observed cytokinesis defect.","abstract_has_math":false,"creators":["Roberts, Adam"],"institution":"University of Dundee","degree_name":"Doctor of Philosophy","degree_level":"Doctoral Thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Fairlamb, Alan"],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014","date_published":"2014","updated_at":"2026-07-24T02:07:59Z","subjects":["N-myristoyltransferase","N-myristoylation","Trypanosoma cruzi","Chemotherapy","Proteomics","click chemistry","Kinetics"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["oai:discovery.dundee.ac.uk:studenttheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4"],"render_values":[{"text":"oai:discovery.dundee.ac.uk:studenttheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4","href":null,"code":true}]}]},"links":{"outbound_url":"https://discovery.dundee.ac.uk/en/studentTheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Fairlamb, Alan"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Biotechnology and Biological Sciences Research Council","Wellcome Trust"]},{"key":"dc:creator","label":"Author","values":["Roberts, Adam"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014"]},{"key":"dc:date.issued","label":"Date","values":["2014"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["Biological Chemistry and Drug Discovery"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Dundee"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://discovery.dundee.ac.uk/en/studentTheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral Thesis"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["N-myristoyltransferase","N-myristoylation","Trypanosoma cruzi","Chemotherapy","Proteomics","click chemistry","Kinetics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2015-12-31"]},{"key":"dc:rights.embargoreason","label":"Dc Rights Embargoreason","values":["/dk/atira/pure/core/document/studentthesisembargoreason/commercialexploitation"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["oai:discovery.dundee.ac.uk:studenttheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4","https://discovery.dundee.ac.uk/en/studentTheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://discovery.dundee.ac.uk/files/6210570/Roberts_2014_electronic_thesis.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:title","label":"Title","values":["Assessment of <i>N</i>-myristoyltransferase and the <i>N</i>-myristoylomeas a potential chemotherapeutic target in<i>Trypanosoma cruzi</i>"]}]}],"canonical_facts":{"dc:contributor.advisor":["Fairlamb, Alan"],"dc:contributor.sponsor":["Biotechnology and Biological Sciences Research Council","Wellcome Trust"],"dc:creator":["Roberts, Adam"],"dc:date":["2014"],"dc:date.issued":["2014"],"dc:description.abstract":["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."],"dc:identifier":["oai:discovery.dundee.ac.uk:studenttheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4","https://discovery.dundee.ac.uk/en/studentTheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4"],"dc:identifier.uri":["https://discovery.dundee.ac.uk/files/6210570/Roberts_2014_electronic_thesis.pdf"],"dc:language":["eng"],"dc:publisher.department":["Biological Chemistry and Drug Discovery"],"dc:publisher.institution":["University of Dundee"],"dc:relation.isreferencedby":["https://discovery.dundee.ac.uk/en/studentTheses/0751872a-abe8-4de0-86f6-5f26fe9ab6e4"],"dc:rights.embargodate":["2015-12-31"],"dc:rights.embargoreason":["/dk/atira/pure/core/document/studentthesisembargoreason/commercialexploitation"],"dc:subject":["N-myristoyltransferase","N-myristoylation","Trypanosoma cruzi","Chemotherapy","Proteomics","click chemistry","Kinetics"],"dc:title":["Assessment of <i>N</i>-myristoyltransferase and the <i>N</i>-myristoylomeas a potential chemotherapeutic target in<i>Trypanosoma cruzi</i>"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral Thesis"],"dc:type.qualificationname":["Doctor of Philosophy"]},"updated_at":"2026-07-24T02:07:59Z"}