PROJECT SUMMARY/ABSTRACT
Cholesterol is a central molecule in human health, serving as a precursor for active metabolites, a specific ligand
for proteins, and a determinant of membrane architecture. Dysregulated cholesterol metabolism has wide-
reaching consequences and is a hallmark of nearly every major human disease, including neurodegeneration,
cancer, and immune disorders. Despite the immense roles of cholesterol and its metabolites in human health,
their molecular signaling mechanisms remain largely unknown. The main barrier to understanding and targeting
cholesterol metabolites in therapy is our lack of tools to define their precise interactions. To address this gap,
we have established innovative cholesterol metabolite probes that contain functionalities for chemoproteomics-
based target identification and microscopy-based subcellular localization. We have used these probes to
quantitatively profile the proteome-wide interactomes of oxysterol metabolites and specify their roles in specific
processes of cancer and immunity. Importantly, our tools have uncovered highly selective protein interactors,
including the tumorigenic and neuroactive sigma-2 orphan receptor (TMEM97), providing new opportunities to
target cholesterol metabolite activities in disease. Over the next 5 years, we will build upon our established tools
and technologies to interrogate oncogenic, immunomodulatory, and neuroactive cholesterol metabolites as well
as cholesterol itself. Specifically, we will design a next-generation chemoproteomics platform for multiplexed
cholesterol metabolite target profiling and pioneer an integrated fluorescence microscopy approach for
subcellular cholesterol metabolite imaging. Our tools and technologies will revolutionize the analysis of
cholesterol metabolite interactions, advancing cholesterol metabolism as a druggable target in human disease.