Dissecting protein and lipid networks with membrane-centric proximity labeling

About This Grant

Cellular membranes serve as dynamic platforms for cell communication and signaling. On each plasma and organelle membrane, lipid–protein and protein–protein interactions are spatiotemporally organized to regulate key biological processes, such as receptor activation and oncogenic signaling. Despite their critical roles, these membrane-specific interactions remain challenging to study due to their transient and context-dependent nature. This proposal aims to develop a novel membrane-centric proximity labeling approach to systematically map lipid–protein and protein–protein networks in live cells. Proximity labeling is a powerful technology for mapping the protein interactome and spatial proteome in living systems. However, conventional tools, such as TurboID and APEX2, lack the spatiotemporal control required to target and capture the dynamic and heterogeneous membrane microenvironment. To address this limitation, I will engineer conditionally activated proximity labeling enzymes that sense their membrane microenvironment and directly couple it to their activity. The first phase (K99) will focus on developing Antigen-Controlled TurboID (ACTurbo), a proximity labeling enzyme activated upon binding to protein of interest, and applying it to dissect compartment-specific μ-opioid receptor signaling pathways in neurons. The second phase (R00) will develop Lipid-Activated Biotin Ligases (LABL) to enable phosphoinositide-centric mapping of oncogenic lipid signaling. This work will establish an innovative and versatile framework for studying lipid–protein and protein–protein interactions in their physiological context, addressing fundamental gaps in membrane biology. By systematically mapping lipid–protein networks on live membranes, this project will provide insights into how membrane dynamics regulate cellular signaling in both normal and degenerative conditions, with potential implications for therapeutic interventions. To achieve this goal, I will integrate my expertise in lipid biology, membrane biology, chemical biology, and protein engineering, bolstered by guidance from my primary mentor, Dr. Alice Ting. To facilitate my proposed research and training, I have assembled a team of leading experts, including Dr. David Baker (computational protein design), Dr. Ilme Schlichting (structural biology), Dr. Ivan Soltesz (neuroscience), and Dr. Ruth Huttenhain (GPCR proteomics). Upon completing this proposal, I will be fully equipped to establish my independent research program focused on developing molecular tools to decipher and manipulate the complex and dynamic networks of membranes, proteins, and lipids.