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Current Protocols Mar 2023Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and...
Proteins frequently function in high-order complexes. Defining protein-protein interactions is essential to acquiring a full understanding of their activity and regulation. Proximity biotinylation has emerged as a highly specific approach to capture transient and stable interactions in living cells or organisms. Proximity biotinylation exploits promiscuous biotinylating enzymes fused to a bait protein, resulting in the biotinylation of adjacent endogenous proteins. Biotinylated interactors are purified under very strict conditions and identified by mass spectrometry to obtain a high-confidence list of candidate binding partners. AirID is a recently described biotin ligase specifically engineered for proximity labeling. This protocol details proximity biotinylation by AirID, using protein complexes that form during a type I interferon response as an example. It covers the construction and validation of AirID fusion proteins and the enrichment and identification of biotinylated interactors. We describe a variation on the protocol using splitAirID. In this case, AirID is split into two inactive fragments and ligase activity is only restored upon dimerization of the bait proteins. This permits selective detection of proteins that interact with homo- or heterodimeric forms of the bait. The protocol considers design strategies, optimization, and the properties of different biotin ligases to identify optimal conditions for each experimental question. We also discuss common pitfalls and how to troubleshoot them. These approaches allow proximity biotinylation to be a powerful tool for defining protein interactomes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Construction and functional validation of AirID fusion proteins Alternate Protocol: Construction and functional validation of splitAirID fusion proteins Support Protocol: Western blot for biotinylated proteins Basic Protocol 2: Biotinylation, enrichment, and identification of protein interactors.
Topics: Biotin; Biotinylation; Proteins; Blotting, Western; Ligases
PubMed: 36939277
DOI: 10.1002/cpz1.702 -
Open Biology Aug 2022Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ...
Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.
Topics: Animals; Biotinylation; Mammals; Mass Spectrometry; Mice; Models, Genetic; Proteomics; Secretome
PubMed: 35946312
DOI: 10.1098/rsob.220149 -
Molecular & Cellular Proteomics : MCP May 2020The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of... (Review)
Review
The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.
Topics: Animals; Biotinylation; Enzymes; Humans; Proteomics; Staining and Labeling; Substrate Specificity
PubMed: 32127388
DOI: 10.1074/mcp.R120.001941 -
Journal of Proteomics May 2023Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase...
Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system's inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilized avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions. SIGNIFICANCE: Proximity biotinylation screens using the last generation biotin ligase TurboID represent a powerful approach for the characterisation of transient protein-protein interaction and signaling networks. However, a constant and high basal biotinylation rate and the associated cytotoxicity often preclude the use of this method in proteomic studies. We report a protocol based on modulation of free biotin levels that prevents the deleterious effects of TurboID while allowing inducible biotinylation, even in subcellular compartments such as the endoplasmic reticulum. This optimised protocol greatly expands the applications of TurboID in proteomic screens.
Topics: Humans; Biotinylation; Biotin; Proteomics; Proteins; Ligases
PubMed: 36966971
DOI: 10.1016/j.jprot.2023.104886 -
Scientific Reports Jun 2022Protein-protein interaction (PPI) analysis is a key process to understand protein functions. Recently, we constructed a human protein array (20 K human protein beads...
Protein-protein interaction (PPI) analysis is a key process to understand protein functions. Recently, we constructed a human protein array (20 K human protein beads array) consisting of 19,712 recombinant human proteins produced by a wheat cell-free protein production system. Here, we developed a cell-free protein array technology for proximity biotinylation-based PPI identification (CF-PPiD). The proximity biotinylation enzyme AirID-fused TP53 and -IκBα proteins each biotinylated specific interacting proteins on a 1536-well magnetic plate. In addition, AirID-fused cereblon was shown to have drug-inducible PPIs using CF-PPiD. Using the human protein beads array with AirID-IκBα, 132 proteins were biotinylated, and then selected clones showed these biological interactions in cells. Although ZBTB9 was not immunoprecipitated, it was highly biotinylated by AirID-IκBα, suggesting that this system detected weak interactions. These results indicated that CF-PPiD is useful for the biochemical identification of directly interacting proteins.
Topics: Biotinylation; Humans; NF-KappaB Inhibitor alpha; Protein Array Analysis; Protein Interaction Mapping; Recombinant Proteins
PubMed: 35732899
DOI: 10.1038/s41598-022-14872-w -
Nature Communications Aug 2021Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity...
Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.
Topics: Biotin; Biotinylation; DNA-Binding Proteins; Histones; Humans; Mass Spectrometry; Protein Binding; Protein Interaction Mapping; Proteins; Proteomics
PubMed: 34408139
DOI: 10.1038/s41467-021-25338-4 -
Molecular & Cellular Proteomics : MCP Nov 2022Cellular biomolecular complexes including protein-protein, protein-RNA, and protein-DNA interactions regulate and execute most biological functions. In particular in... (Review)
Review
Cellular biomolecular complexes including protein-protein, protein-RNA, and protein-DNA interactions regulate and execute most biological functions. In particular in brain, protein-protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell-cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte-neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.
Topics: Biotinylation; Proteome; Cell Communication; Synapses; Brain
PubMed: 36198386
DOI: 10.1016/j.mcpro.2022.100422 -
Journal of the American Society For... Sep 2021Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications....
Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency, which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intraorganelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.
Topics: Biotin; Biotinylation; HEK293 Cells; HeLa Cells; Humans; Mitochondria; Mitochondrial Proteins; Proteomics; Sulfhydryl Compounds
PubMed: 33909971
DOI: 10.1021/jasms.1c00079 -
Journal of the American Chemical Society Jul 2016Cupredoxins are electron-transfer proteins that have active sites containing a mononuclear Cu center with an unusual trigonal monopyramidal structure (Type 1 Cu). A...
Cupredoxins are electron-transfer proteins that have active sites containing a mononuclear Cu center with an unusual trigonal monopyramidal structure (Type 1 Cu). A single Cu-Scys bond is present within the trigonal plane that is responsible for its unique physical properties. We demonstrate that a cysteine-containing variant of streptavidin (Sav) can serve as a protein host to model the structure and properties of Type 1 Cu sites. A series of artificial Cu proteins are described that rely on Sav and a series of biotinylated synthetic Cu complexes. Optical and EPR measurements highlight the presence of a Cu-Scys bond, and XRD analysis provides structural evidence. We further provide evidence that changes in the linker between the biotin and Cu complex within the synthetic constructs allows for small changes in the placement of Cu centers within Sav that have dramatic effects on the structural and physical properties of the resulting artificial metalloproteins. These findings highlight the utility of the biotin-Sav technology as an approach for simulating active sites of metalloproteins.
Topics: Azurin; Biotinylation; Catalytic Domain; Copper; Cysteine; Ligands; Streptavidin
PubMed: 27385206
DOI: 10.1021/jacs.6b05428 -
Medecine Sciences : M/S Mar 2019The proteome is a dynamic system in which protein-protein interactions play a crucial role to model together the cellular phenotype. However, given the inherent... (Review)
Review
The proteome is a dynamic system in which protein-protein interactions play a crucial role to model together the cellular phenotype. However, given the inherent limitation of the available technologies to depict the dynamic nature of these interactions, identify protein-protein interaction has for a long time represented an important challenge in proteomic. The recent development of BioID and APEX, two proximity-dependent labeling technologies, opens today new perspectives and yet start changing our vision of protein-protein interaction, and more globally our vision of the proteome. In this review, we describe the recent and conventional tools available to study protein-protein interactions, compare the advantages and limitations of these technics, and discuss the recent progress brought by the proximity-dependent labelling to complete our vision of the proteome, and thus better understand cellular mechanisms.
Topics: Animals; Biotinylation; Humans; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Maps; Proteome; Proteomics; Staining and Labeling
PubMed: 30931906
DOI: 10.1051/medsci/2019035