Dataset Information

MicroID: A Novel Biotin Ligase Enables Rapid Proximity Ligation Proteomics

ABSTRACT: Identifying protein-protein interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity ligation method that uses a promiscuous biotin ligase to detect protein-protein interactions in cells in a highly reproducible manner. Recent advancements in proximity ligation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2 to create a smaller, highly active, biotin ligase that we named MicroID. Truncation of the c-terminal of BioID2 and mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller, highly active construct with lower baseline labeling. Several additional point mutations improved the function of our modified MicroID construct compared to BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID is the smallest biotin ligase (180 AA for microID vs. 257 AA for miniTurbo and 338 AA for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurboID. MicroID also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we developed a MicroID mutant that has lower labeling efficiency, but significantly reduced biotin scavenging compared to BioID2. Finally, we demonstrate utility of MicroID in mass spectrometry experiments by localizing MicroID constructs to subcellular organelles and measuring protein-protein interactions.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: James Londino

LAB HEAD: James David Londino

PROVIDER: PXD031146 | Pride | 2022-08-12

REPOSITORIES: Pride

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Publications

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.

Johnson Benjamin S BS Chafin Lexie L Farkas Daniela D Adair Jessica J Elhance Ajit A Farkas Laszlo L Bednash Joseph S JS Londino James D JD

Molecular & cellular proteomics : MCP 20220608 7

Identifying protein-protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an "abortive" biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, sta ...[more]

PMID: 35688383

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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.

Dataset Information

MicroID: A Novel Biotin Ligase Enables Rapid Proximity Ligation Proteomics

Publications

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.

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