Key result
A novel method using Tamavidin 2-REV efficiently enriched biotinylated peptides, identifying over 4,000 STING-proximal proteins and revealing dynamic interaction profiles during STING activation.
Why the study?
Conventional BioID rarely detects biotinylated peptides due to overly strong biotin-streptavidin binding, limiting the global identification of proteins that spatiotemporally interact with STING.
The use of Tamavidin 2-REV enables efficient and specific enrichment of biotinylated peptides in BioID screening, providing detailed insights into STING signaling pathways.
Does not yet inform STING-targeted CV therapies; leaves open hypothesis generation for dynamic interactome studies in inflammation.
Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.
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Motani et al. (2020) studied this question. Tamavidin 2-REV enrichment method for BioID vs. Conventional BioID method using streptavidin beads was evaluated on Identification and quantification of biotinylated peptides of STING-proximal proteins. A novel method using Tamavidin 2-REV efficiently enriched biotinylated peptides, identifying over 4,000 STING-proximal proteins and revealing dynamic interaction profiles during STING activation.
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