Identification of Protein Interactors of Cholesteryl Esters

Abstract

Cholesterol and cholesteryl esters form the major sterol component of an animal cell. While there are extensive studies on understanding protein-cholesterol interactions, the protein interactors of cholesteryl esters mostly remain unknown. Therefore, the possible signalling mechanisms for these biomolecules are largely elusive. In this study, we present a chemoproteomic strategy to identify the interactors of cholesteryl esters on a proteome-wide scale. For this purpose, we employ a photoreactive bioorthogonal cholesteryl ester probe to map these interactors. Our results demonstrate two ways to effectively deliver the cholesteryl ester probe into different cell lines, which enables analysis of these lipid-protein interactions. We also show a difference in these interactions in different cell lines, thus hinting at possibilities of identifying different protein interactors in different cells. Moreover, our microscopy data demonstrates that these interactions occur at a pan-cellular level and are not restricted to the plasma membrane. Lastly, the proteomics analyses could identify initial hits of proteins in the N2a cell line with both the probes. Taken together, we establish methods to study cholesteryl ester-protein interactome, which will enable identification of possible signalling roles of cholesteryl esters in the future.