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Cells Apr 2020BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the... (Comparative Study)
Comparative Study
BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.
Topics: A549 Cells; Animals; Biotinylation; Genetic Vectors; Humans; Ligases; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Proteomics
PubMed: 32344865
DOI: 10.3390/cells9051070 -
Nature Communications Feb 2022Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein...
Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein interactions for 109 different human TFs through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.
Topics: Biotinylation; Chromatin; Chromatography, Affinity; Gene Expression Regulation; Gene Regulatory Networks; HEK293 Cells; Humans; Mass Spectrometry; NFI Transcription Factors; Protein Interaction Maps; Proteomics; Transcription Factors
PubMed: 35140242
DOI: 10.1038/s41467-022-28341-5 -
Nature Chemical Biology Mar 2021Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular...
Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.
Topics: Animals; Betaine-Homocysteine S-Methyltransferase; Biotin; Biotinylation; Blood Proteins; Gene Expression; HEK293 Cells; Hepatocytes; Humans; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Muscle Cells; Myeloid Cells; Organ Specificity; Pericytes; Proteome; Proteomics; Staining and Labeling
PubMed: 33199915
DOI: 10.1038/s41589-020-00698-y -
The Journal of Nutrition Aug 2017Although frank symptomatic biotin deficiency is rare, some evidence suggests that marginal biotin deficiency occurs spontaneously in a substantial proportion of women... (Review)
Review
Although frank symptomatic biotin deficiency is rare, some evidence suggests that marginal biotin deficiency occurs spontaneously in a substantial proportion of women during normal human pregnancy and might confer an increased risk of birth defects. Herein I review ) advances in assessing biotin status, including the relation between acylcarnitine excretion and biotin status; ) recent studies of biotin status in pregnancy; ) advances in understanding the role of biotin in gene expression and the potential roles of biotinylated proteins that are neither histones nor carboxylases; and ) novel large-dose biotin supplementation as therapy for multiple sclerosis. The review concludes with a summary of recent studies that have reported potentially dangerous erroneous results in individuals consuming large amounts of biotin for measurements of various plasma hormones for common clinical assays that use streptavidin-biotin technology.
Topics: Animals; Biotin; Biotinylation; Carnitine; Female; Gene Expression; Hormones; Humans; Multiple Sclerosis; Nutritional Status; Pregnancy; Pregnancy Complications; Streptavidin; Vitamin B Complex
PubMed: 28701385
DOI: 10.3945/jn.116.238956 -
Nature Apr 2023Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their...
Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive-compulsive disorder (OCD) and repetitive behaviours, was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders.
Topics: Animals; Mice; Astrocytes; Neurons; Obsessive-Compulsive Disorder; Proteome; Biotinylation; Cell Membrane; Signal Transduction; Cytosol; Homeostasis; Phenotype; Actin Cytoskeleton
PubMed: 37046092
DOI: 10.1038/s41586-023-05927-7 -
Proceedings of the National Academy of... Jun 2020Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by...
Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.
Topics: Biotinylation; Endoplasmic Reticulum; HEK293 Cells; Humans; Membrane Proteins; Mitochondria; Mitochondrial Membranes; Proteome; Staining and Labeling
PubMed: 32424107
DOI: 10.1073/pnas.1919528117 -
Current Protocols in Protein Science Feb 2018BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to...
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; Biotinylation; Carbon-Nitrogen Ligases; Escherichia coli; Escherichia coli Proteins; Humans; Recombinant Fusion Proteins; Repressor Proteins
PubMed: 29516480
DOI: 10.1002/cpps.51 -
Nature Methods Feb 2019T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR...
T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide-major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide-MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.
Topics: Adaptive Immunity; Animals; Antigens; Biotinylation; DNA; Epitopes; Gene Library; Genetic Techniques; HEK293 Cells; Humans; Immunotherapy; Jurkat Cells; K562 Cells; Ligands; Mice; Peptides; Phagocytosis; Receptors, Antigen, T-Cell; T-Lymphocytes
PubMed: 30700903
DOI: 10.1038/s41592-018-0305-7 -
ELife May 2020Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a...
Proximity biotinylation based on BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-IκBα indicated biotinylation of MDM2 or RelA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4 complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.
Topics: Biotin; Biotinylation; Carbon-Nitrogen Ligases; Cell Survival; Escherichia coli; Escherichia coli Proteins; HEK293 Cells; Humans; Mutation; Protein Engineering; Protein Interaction Mapping; Protein Interaction Maps; Recombinant Fusion Proteins; Repressor Proteins
PubMed: 32391793
DOI: 10.7554/eLife.54983 -
Methods in Molecular Biology (Clifton,... 2015The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been...
The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.
Topics: Biotin; Biotinylation; Carbon-Nitrogen Ligases; Chromatography, Affinity; Escherichia coli; Escherichia coli Proteins; Glutathione Transferase; Polymerase Chain Reaction; Protein Binding; Protein Engineering; Recombinant Fusion Proteins; Repressor Proteins; Streptavidin
PubMed: 25560075
DOI: 10.1007/978-1-4939-2272-7_12