Cholesteryl esters (CEs) are commonly considered inert storage forms of cholesterol, yet their capacity to directly engage cellular proteins has been underexplored. This work establishes an integrated chemical proteomic platform to systematically identify CE–protein interactions in mammalian cells. The authors aimed to provide a systems-level map of CE-associated proteins by deploying complementary labeling strategies that capture distinct biochemical and cellular contexts of esterified sterols.
Three orthogonal photoaffinity labeling strategies were developed and comparatively applied:
Metabolic assembly of a bifunctional CE probe. This approach leverages endogenous acyl-CoA:cholesterol acyltransferase activity to assemble a bifunctional CE probe inside cells, allowing the probe to follow native biosynthetic and trafficking routes.
Direct delivery of a structurally defined diazirine-alkyne CE analog. Here a chemically defined CE analog containing a photoactivatable diazirine and an alkyne handle is introduced to cells to permit UV-dependent crosslinking and downstream enrichment.
Fragment-assisted subtraction using sterol and fatty acyl control probes. This subtraction strategy uses control probes representing the sterol and fatty acyl fragments to resolve interactions that depend on the intact esterified scaffold rather than individual fragments.
Each strategy is orthogonal in that it interrogates CE interactions from different biochemical entry points and probe topologies, increasing the breadth of detectable engagements.
Before proteomic analysis, each probe system underwent rigorous validation. The authors applied lipidomics to confirm probe incorporation and assessed UV-dependent crosslinking to verify that probe activation resulted in covalent capture of interacting proteins. These validation steps were used to ensure the chemical behavior and cellular routing of each probe were compatible with downstream quantitative proteomics.
Following validation, the study applied quantitative proteomic workflows to identify proteins crosslinked to the CE probes. Data from the three probe modalities were integrated to assemble a composite interactome. Integration of complementary datasets was a central component of the study design, intended to balance the modality-specific biases of each labeling strategy and to expand the coverage of CE-associated proteins.
The multimodal approach identified a total of 495 CE-associated proteins. These proteins span multiple functional classes, including enzymes, transporters, scaffolding proteins, and members of canonical sterol-binding families. The reported dataset therefore expands the conceptual view of CEs from passive storage lipids to bioactive molecules capable of engaging diverse protein partners.
A notable observation was the limited overlap of protein hits across the three strategies. The authors interpret this result to mean that lipid–protein engagement is strongly conditioned by factors such as the biosynthetic origin of the CE, the topology of the probe used, and intracellular routing of the lipid probe. In other words, different labeling strategies capture complementary, and at times nonredundant, subsets of the overall CE interactome.
Functional and database annotation of the identified proteins revealed enrichment for druggable targets and proteins linked to disease. The annotations connect CE-associated proteins to pathways relevant to metabolism, neurology, and cardiovascular biology. These links suggest potential mechanistic routes by which esterified sterols could influence disease-associated processes or serve as handles for therapeutic targeting.
By providing the first systems-level map of CE–protein interactions, the study offers a resource for researchers interested in sterol biology and lipid-mediated regulation of cellular function. The multimodal chemical proteomics framework demonstrated here is generalizable and can be adapted to resolve interactomes of other complex lipid species, particularly where biosynthetic origin and intracellular trafficking shape molecular engagements.
This article is reported as a preprint and has not been peer reviewed. The abstract and manuscript indicate that supplementary material accompanies the preprint and contains additional experimental details and data. Specific lists of the 495 proteins, quantitative values, and detailed methods are provided in the full manuscript and its supplementary files; those specific data are not reproduced in this summary. Readers should consult the preprint and supplementary materials for full datasets and experimental parameters.
Collectively, the study introduces a validated, multimodal chemical proteomics pipeline and a wide-ranging inventory of CE-associated proteins, highlighting the context-dependent nature of lipid–protein interactions and their potential relevance to disease and therapeutic discovery.