Dataset Information

Engineered allostery in a light-regulated version of TurboID for precise spatiotemporal control of proximity labeling in living cells

ABSTRACT: Lee SY, Chea JS, Zhao BX, Xu C, Udeshi ND, Roh H, Kim C, Cho K, Carr SA, Ting A. 2022 The incorporation of light-responsive domains into engineered proteins has produced optogenetic tools with the ability to regulate protein localization, interactions, and function with light. We introduced this mode of regulation into proximity labeling (PL), which has been a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through a combination of structure-guided screening and yeast display directed evolution, we inserted the light sensitive LOV domain into a surface exposed loop of the PL enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. We showed that "LOV-Turbo" works in multiple organelles and cell types and can dramatically reduce background labeling in biotin-rich environments such as living neurons. We utilized LOV-Turbo's reversibility to perform pulse-chase labeling followed by organelle fractionation to discover endogenous proteins that traffick between endoplasmic reticulum, nuclear, and mitochondrial compartments under cellular stress. Lastly, we showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from proximal luciferase in living cells, opening the door to chemical and/or interaction dependent PL. Overall, LOV-Turbo increases the spatial and temporal precision of PL and expands the scope of experimental questions that can be addressed using PL.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000090683 | MassIVE | Wed Nov 09 14:24:00 GMT 2022

REPOSITORIES: MassIVE

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Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

Lee Song-Yi SY Cheah Joleen S JS Zhao Boxuan B Xu Charles C Roh Heegwang H Kim Christina K CK Cho Kelvin F KF Udeshi Namrata D ND Carr Steven A SA Ting Alice Y AY

bioRxiv : the preprint server for biology 20230309

The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions, and function with light. We integrated optogenetic control into proximity labeling (PL), a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the PL enzyme TurboID to rapidly and reversibly control its lab ...[more]

PMID: 36945504

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Project description:Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.