Chemical Synthesis and Semisynthesis of Lipidated Proteins.

Lipidation is a ubiquitous modification of peptides and proteins that can occur either co- or post-translationally. An array of different lipid classes can adorn proteins and has been shown to influence a number of crucial biological activities, including the regulation of signaling, cell-cell adhesion events, and the anchoring of proteins to lipid rafts and phospholipid membranes. Whereas nature employs a range of enzymes to install lipid modifications onto proteins, the use of these for the chemoenzymatic generation of lipidated proteins is often inefficient or impractical. An alternative is to harness the power of modern synthetic and semisynthetic technologies to access lipid-modified proteins in a pure and homogeneously modified form. This Review aims to highlight significant advances in the development of lipidation and ligation chemistry and their implementation in the synthesis and semisynthesis of homogeneous lipidated proteins that have enabled the influence of these modifications on protein structure and function to be uncovered.

Flagellar targeting of an arginine kinase requires a conserved lipidated protein intraflagellar transport (LIFT) pathway in Trypanosoma brucei.

Both intraflagellar transport (IFT) and lipidated protein intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility, and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies have revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei, the causative agent of African sleeping sickness. TbArl13 catalyzes two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 has been identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting to and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacted with TbUnc119 in a GTP-dependent manner, suggesting functional specialization of Arl3-GTPases in T. brucei These results establish the function of TbUnc119 as a myristoylated cargo carrier and support the presence of a conserved LIFT pathway in T. brucei.

Evolution and expression of the zebrafish unc119 paralogues indicates a conserved role in cilia.

UNC119 proteins are required for ciliary trafficking in a process called lipidated protein intraflagellar targeting (LIFT) or through vesicle transport. However, although unc119 has been studied in a variety of contexts, either organismal constraints or genetic redundancy has largely restricted their study in ciliary contexts. One possible solution for this is to use the zebrafish, however, the unc119 genes have not been well described in this species. In our study, we show in a condensed species tree that the presence of unc119 genes correlates with the presence of cilia across eukaryotes and that phylogenetic evidence suggests there are three subgroups of UNC119 proteins. Zebrafish contain all three of these subgroups: two vertebrate-specific UNC119A proteins, one vertebrate-specific UNC119B protein, and one UNC119. Expression analyses show that each of the zebrafish unc119 genes are maternally-expressed and have overlapping but distinct expression in ciliated tissues, such as the eye, pronephric duct, and spinal cord. Overall, these findings set the foundation for future studies into the use of the zebrafish to study unc119 gene knock-outs, particularly from a ciliary perspective.