N-terminal protein myristoylation represents the attachment of a C14 saturated fatty acid moiety to the N-terminal glycine of a target. Myristoylation is involved in various biological processes, including protein-protein-interactions, subcellular localization and reversible membrane binding. However, the regulation of subcellular localization of lipidated proteins is still not fully understood. A subgroup of myristoylated proteins requires assistance of chaperone proteins that can accommodate the lipid moiety within their cavity for cellular transport. Two homologues UNC119A and UNC119B belong to the group of chaperone proteins that specifically recognize and interact with the myristoylated N-terminus of a selected subset of lipidated proteins. However, to date only a few myristoylated proteins were shown to interact with UN119 chaperones, including the ciliary protein nephrocystin-3 and the rod photoreceptor transducin α subunit (Gnat1). Recently, evidence for the transport and activation of Src kinase, a very important oncogene, in complex with UNC119 proteins was obtained. The specificity of myristoylated cargo recognition by UNC119 proteins is not well understood. Due to the fact, that the protein-protein interactions between the myristoylated cargo and the chaperone are transient and in some cases characterized by weak affinity, the identification of new UNC119 binding partners is especially challenging.<br/>Inspired by the photoactivatable probes, developed for the farnesyl lipid moiety binding to the chaperone protein PDEδ, we designed and synthesized the first photoactivatable analogue of the myristate moiety. Upon photoactivation the myristoyl analogue establishes the covalent bond between the lipid probe and the hydrophobic pocket of UNC119. Using the Gnat1 and Src N-terminal peptides modified with the photoactivatable analogue, we were able to map the interacting part of the hydrophobic pocket of UNC119 proteins. Gnat1 probe binds both UNC119A and UNC119B proteins, whereas Src probe selectively labels UNC119B. Moreover, the interaction of the Gnat1 probe with both UNC119 proteins in cell lysate was shown in covalent pulldown experiments.

Syntheses and evaluation of photoactivatable myristoyl analogues for identification of new binding partners of UNC119 proteins

256th National Meeting (2018)

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