Molecular tools for cholesterol metabolite signaling

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.