Broadening the proximity labeling landscape with next generation enzymes, chemistries and probes

About This Grant

Project Summary Proximity labeling is a powerful technique that creates localized reactive intermediates to "tag" nearby proteins in their native cellular environment, facilitating proteomic analysis. It has revolutionized our understanding of subcellular proteomes and the discovery of novel protein-protein interactions. My lab uses chemical and genetic tools to uncover molecular interactions between organs in the body, and how they change under homeostatic and pathophysiological processes. A key part of our research involves applying and developing techniques like proximity labeling to map these interactions in living organisms, particularly in mouse models. Despite its success and adaptation to cell culture systems, proximity labeling for proteomics has limited applications in vivo, where tissues have overwhelming background interference from endogenous biotinylated proteins. Additionally, the potential for using proximity labeling to study biomolecules beyond proteins, such as lipids, remains largely unexplored. However, extending proximity labeling to lipids could significantly enhance lipidomics, just as it has advanced proteomics. To address these challenges, we propose developing new chemical probes and enzymes to optimize proximity labeling for in vivo applications, and expand its use to lipids. The former will involve engineering a class of adenylate-forming enzymes, which we will test both in E. coli and with purified proteins in vitro. We will also create novel chemical substrates that do not rely on biotin-based enrichment and ensure they are bioavailable, non-toxic, and suitable for proximity labeling in mammalian cell cultures and mouse models. In addition, we will investigate the mechanisms behind these engineered enzymes and identify the specific protein sites where labeling occurs from the reactive intermediates they generate. Ultimately, this innovative approach will enhance the power of proximity labeling, allowing us to explore physiological processes that can only be studied in living organisms. At the same time, we are working to integrate proximity labeling into existing lipidomics techniques by developing new probes and reactive chemistries that can specifically label lipids in live cells. We will also optimize enzyme and photocatalyst-mediated processes to generate these reactive intermediates within cells. Finally, we will incorporate these methods into current lipidomic workflows, enabling lipid detection via mass spectrometry. This effort will significantly broaden the range of biomolecules that can be studied through proximity labeling, deepening our knowledge gained through lipidomics and the impact of lipid species on cellular functions.