Precise and In Vivo-Compatible Spatial Proteomics via Bioluminescence-Triggered Photocatalytic Proximity Labeling

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Protein localization and interactions are crucial for cellular function.
• Traditional proximity labeling (PL) methods face limitations like toxicity, high background, and restricted in vivo applications.
• Visible-light-triggered PL is hindered by light-induced background and limited in vivo use.

Purpose of the Study:

• To develop an in vivo-compatible proximity labeling technology for precise mapping of membraneless organelles and transient protein-protein interactions.
• To achieve subminute temporal resolution for studying dynamic biological events.
• To overcome limitations of existing PL methods.

Main Methods:

• Development of BRET-ID, combining a genetically encoded photocatalyst and NanoLuc luciferase.
• Utilizing bioluminescence resonance energy transfer (BRET) to locally generate blue light and activate the photocatalyst.
• Employing singlet oxygen for protein oxidation and subsequent analysis via a streamlined chemoproteomic workflow.

Main Results:

• BRET-ID enabled precise mapping of ER membrane proteins with high spatial specificity.
• The technology provided 1-minute snapshots of dynamic GPCR interactions during ligand-induced endocytosis.
• BRET-ID identified novel stress granule components, including RICTOR, in stressed cells and tumor xenografts.

Conclusions:

• BRET-ID is a powerful, genetically encoded tool for in vivo proximity labeling.
• It allows for precise mapping of protein localization and molecular interactions with high spatial and temporal resolution.
• BRET-ID facilitates the study of dynamic cellular processes and the discovery of novel protein interactions in living organisms.