Turning the spotlight on protein-lipid interactions in cells

Abstract

Protein function is largely dependent on coordinated and dynamic interactions of the protein with biomolecules including other proteins, nucleic acids and lipids. Although powerful methods for global profiling of protein-protein and protein-nucleic acid interactions are available, proteome-wide mapping of protein-lipid interactions is still challenging and rarely performed. The emergence of bifunctional lipid probes with photoactivatable and clickable groups offers new chemical tools for globally profiling protein-lipid interactions under cellular contexts. In this review, we summarize recent advances in the development of bifunctional lipid probes for studying protein-lipid interactions. We also highlight how in vivo photocrosslinking reactions contribute to the characterization of lipid-binding proteins and lipidation-mediated protein-protein interactions.

Figure 1

Photocrosslinking strategy for studying protein-lipid interactions. (a) Radiolabeled photoactivatable lipid probes are incorporated into biological membranes in vitro or in vivo. Photoirradiation induces crosslinking between the probe and interacting proteins. After immunoprecipitation, the protein of interest is analyzed by autoradiography. (b) Bifunctionalized photoactivatable and clickable lipid probes are incorporated into biological membranes in vitro or in vivo. Photoirradiation induces crosslinking between the probe and interacting proteins. The protein of interest is subjected to immunoprecipitation and a click reaction with azide-fluorophore, and then analyzed by in-gel fluorescence. Alternatively, the whole proteome is subjected to a click reaction with azide-fluorophore or azide-biotin for in-gel fluorescence detection or affinity enrichment, respectively. The enriched protein-lipid complexes are digested and identified by mass spectrometry. The lipid head with a photoactivatable group is shown in orange. Note that while the scheme only depicts the capture of integral membrane proteins, this strategy is also effective for crosslinking peripheral proteins that bind lipids.

Figure 2

Representative photoactivatable lipid probes for studying protein-lipid interactions. (a) Bifunctional lipid probes for studying protein-phosphatidylcholine (1, 2) and protein-PI(3,4,5)P₃ (3) interactions in vitro. (b) Tritium-labeled photoactivatable probes for capturing proteins that interact with cholesterols (4, 5, 6), sphingosine (7) and phosphatidylcholine (8) in vivo. (c) Bifunctional lipid probes for global detection and profiling of cholesterol-binding (9) and glycerolipid-binding (10) proteins in vivo.

Figure 3

In-cell photocrosslinking strategy for capturing palmitoylation-mediated protein-protein interaction by using bifunctional fatty acid x-alk-16. Bifunctional fatty acid x-alk-16 is first incubated with live cells. The incorporation of x-alk-16 into Spalmitoylated proteins of interest (POI) is detected by in-gel fluorescence after immunoprecipitation and CuAAC with azide-fluorophore (az-dye). In-cell photocrosslinking enables formation of covalent complexes between x-alk-16-modified POI and interacting partners. After immunoprecipitation the crosslinked complexes are characterized by western blotting or mass spectrometry.