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bioRxiv · 10.1101/2021.12.15.472799

Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes

Fedoryshchak, R. O.·Gorelik, A.·Shen, M.·Shchepinova, M. M.·Perez-Dorado, I.·Tate, E. W.
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Abstract

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in cell biology. Despite development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human N-myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray co-crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and in situ intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions driven by the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein FSP1 and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome, which may prove broadly applicable to other PTMs.

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Fedoryshchak, R. O., Gorelik, A., Shen, M., Shchepinova, M. M., Perez-Dorado, I., Tate, E. W.. 2021-12-15. Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes. https://doi.org/10.1101/2021.12.15.472799

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