Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei

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.