Journal
ACS INFECTIOUS DISEASES
Volume 2, Issue 6, Pages 427-441Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsinfecdis.6b00034
Keywords
human African trypanosomiasis; N-myristoylation; chemical proteomics; click chemistry; protein lipidation; target validation
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Funding
- U.K. Engineering and Physical Sciences Research Council
- Wellcome Trust [087792]
- U.K. Biotechnology and Biological Sciences Research Council [BB/D02014X/1]
- Biotechnology and Biological Sciences Research Council [BB/D02014X/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/J021199/1] Funding Source: researchfish
- BBSRC [BB/D02014X/1] Funding Source: UKRI
- EPSRC [EP/J021199/1] Funding Source: UKRI
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The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell.
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