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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 -
Cell Feb 2022Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid...
Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.
Topics: Alzheimer Disease; Amino Acids; Biotinylation; Brain; Cell Nucleus; Disease Progression; Energy Metabolism; Frontotemporal Dementia; Humans; Induced Pluripotent Stem Cells; Mitochondria; Mutant Proteins; Mutation; Nerve Degeneration; Neurons; Protein Binding; Protein Domains; Protein Interaction Maps; Proteomics; Severity of Illness Index; Subcellular Fractions; Synapses; Tauopathies; tau Proteins
PubMed: 35063084
DOI: 10.1016/j.cell.2021.12.041 -
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 -
Cell Nov 2020Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of...
Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4, CD8 T cells, and TSA-suppressive CD4 T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8 TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.
Topics: Adult; Amino Acid Sequence; Animals; Antigens, Neoplasm; Biomarkers, Tumor; Biotinylation; Bystander Effect; CD8-Positive T-Lymphocytes; Cell Communication; Cell Membrane; Dendritic Cells; Disease Models, Animal; Female; Fucose; Fucosyltransferases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Helicobacter pylori; Humans; Immunity; Lymphocytes, Tumor-Infiltrating; Melanoma, Experimental; Mice, Inbred C57BL; Peptides; Phenotype; Programmed Cell Death 1 Receptor; Spleen; T-Lymphocytes
PubMed: 33096019
DOI: 10.1016/j.cell.2020.09.048 -
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 -
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 -
Proceedings of the National Academy of... Jun 2021Cytosolic DNA activates cGAS (cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (stimulator of interferon genes) signaling, which triggers interferon and inflammatory...
Cytosolic DNA activates cGAS (cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (stimulator of interferon genes) signaling, which triggers interferon and inflammatory responses that help defend against microbial infection and cancer. However, aberrant cytosolic self-DNA in Aicardi-Goutière's syndrome and constituently active gain-of-function mutations in STING in STING-associated vasculopathy with onset in infancy (SAVI) patients lead to excessive type I interferons and proinflammatory cytokines, which cause difficult-to-treat and sometimes fatal autoimmune disease. Here, in silico docking identified a potent STING antagonist SN-011 that binds with higher affinity to the cyclic dinucleotide (CDN)-binding pocket of STING than endogenous 2'3'-cGAMP. SN-011 locks STING in an open inactive conformation, which inhibits interferon and inflammatory cytokine induction activated by 2'3'-cGAMP, herpes simplex virus type 1 infection, deficiency, overexpression of cGAS-STING, or SAVI STING mutants. In mice, SN-011 was well tolerated, strongly inhibited hallmarks of inflammation and autoimmunity disease, and prevented death. Thus, a specific STING inhibitor that binds to the STING CDN-binding pocket is a promising lead compound for STING-driven disease.
Topics: Animals; Binding Sites; Biotinylation; Cell Death; Exodeoxyribonucleases; Humans; Inflammation; Membrane Proteins; Mice; Molecular Docking Simulation; Mutation; Nucleotides, Cyclic; Phosphoproteins; Protein Domains; Signal Transduction
PubMed: 34099558
DOI: 10.1073/pnas.2105465118 -
Cell Metabolism Sep 2020We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human...
We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.
Topics: Biotinylation; Cells, Cultured; HEK293 Cells; Humans; Mitochondria; Mitochondrial Proteins; Protein Interaction Maps
PubMed: 32877691
DOI: 10.1016/j.cmet.2020.07.017