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Proximity Biotinylation for Investigating Protein Interactions in Living Cells

This journal club presentation explores the use of proximity biotinylation, specifically the BioID and APEX methods, to label and investigate protein interactions in living cells. The advantages and limitations of these techniques are discussed, along with their applications in capturing GPCR protein interaction networks. The presentation concludes that proximity biotinylation is a valuable tool for spatiotemporal analysis of protein interactions in living cells.

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Proximity Biotinylation for Investigating Protein Interactions in Living Cells

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  1. JOURNAL CLUB PRESENTATION Rex D.A.B Graduate student YU-IOB Center for Systems Biology and Molecular Medicine August 09, 2017

  2. Concept

  3. Proximity biotinylation Biotin-protein ligase (BirA) used to label interacting proteins having a biotin acceptor peptide (BAP) Investigate specific pairwise interactions BioID, a mutant version of BirA Labeling takes 6 to 24 hours Unsuitable for addressing temporal dynamics of protein interactions Proximity labeling using peroxidase enzymes Labeling takes only 30 minutes Peter Lonn., Trends in Biochemical Sciences. 2017

  4. Proximity biotinylation • Ascorbateperoxidase that oxidizes phenol derivatives to radicals • Reactive biotin phenoxyl radicals covalently react with Trp, Tyr, His, Cys (investigate weak/transient interactions) Dae In Kim., Trends in Cell Biology. 2016

  5. APEX (Ascorbate peroxidase) • Originally identified in soy bean • Localize subcellular compartments of live cells • Maintains protein complex and membrane integrity • Living cellular protein-­protein interactions (PPIs) • This concept therefore has been used for proximal labeling

  6. Spatiotemporally ResolveProtein Interaction Networks in Living Cells

  7. APEX Captures GPCR Protein Interaction Networks

  8. APEX Captures GPCR Protein Interaction Networks

  9. APEX Captures GPCR Protein Interaction Networks

  10. Differentiated GPCR Interactors from General Compartmental Proteins

  11. Differentiated GPCR Interactors from General Compartmental Proteins

  12. Specific Deconvolution Identifies Interacting Partners for the Delta Opioid Receptor

  13. Specific Deconvolution Identifies Interacting Partners for the Delta Opioid Receptor

  14. Conclusion • Method to spatially identify protein interaction networks in living cells • Quantitative proteomics applied to ‘‘deconvolve’’ APEX proximity labeling data • Ligand-stimulated remodeling of GPCR networks captured with sub-minute resolution • Previously unknown GPCR-linked proteins identified and functionally validated

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