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Title: Chemical tools for probing protein N-myristoylation in protozoan parasites
Author: Wright, Megan Holly
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Protein N -myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal residue of specific proteins. This co - and post-translational modification is catalysed by myristoyl CoA:protein N -myristoyltransferase (NMT), an essential enzyme in eukaryotes. N -Myristoylated proteins have diverse roles, but typically localise to cellular membranes where many are thought to be involved in signalling or trafficking processes. NMT is a potential therapeutic target in diseases caused by parasitic protozoa, such as malaria, leishmaniasis and African sleeping sickness. N -Myristoylation is difficult to study by conventional biochemical methods. Recently chemical proteomics has emerged as a powerful technique for probing protein lipidation. In this approach chemically tagged myristic acid substrate analogues are incorporated metabolically into lipidated pro teins. Tagged proteins can then be selectively 'captured' after cell lysis via a bioorthogonal ligation reaction to install a variety of labels for further analysis. This thesis describes the development of such an approach for profiling protein lipidation in diverse systems, including human cells, Plasmodium , Leishmania and T. brucei parasites. This work includes the the synthesis of multifunctional probes for the capture of tagged proteins and characterisation of a tagged myristate analogue as a Plasmodium NMT substrate. These tools are applied in several contexts, with analysis by a combination of gel-based methods and mass spectrometry. The first de novo identification of N -myristoylated proteins in P. falciparum , T. brucei and L. donovani is described. The chemical proteomic methodology developed is also used to profile lipidation in the presence of putative NMT inhibitors to assess whether compounds act on -target in live parasites. The correlation between efficacy against the cell and on-target activity is analysed to explore NMT as a potential drug target in malaria and leishmaniasis.
Supervisor: Haslam, Stuart ; Mann, David ; Tate, Ed Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available