Issue 9, 2023

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

Abstract

Protein–protein interactions (PPIs) are essential and pervasive regulatory elements in biology. Despite the 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 the 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 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 in 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 1 (FSP1) and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome without the requirement for genetic modification, which may prove broadly applicable to other PTMs.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
06 nov 2022
Accepted
29 jan 2023
First published
30 jan 2023
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2023,14, 2419-2430

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

R. O. Fedoryshchak, A. Gorelik, M. Shen, M. M. Shchepinova, I. Pérez-Dorado and E. W. Tate, Chem. Sci., 2023, 14, 2419 DOI: 10.1039/D2SC06116C

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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