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Molecular & Cellular Proteomics : MCP Jun 2023Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific...
Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).
Topics: Animals; Mice; Microglia; Proteome; Biotin; Proteomics; Lipopolysaccharides; Cell Line; Neurons; Biotinylation
PubMed: 37061046
DOI: 10.1016/j.mcpro.2023.100546 -
Journal of Proteome Research Sep 2021Nonreceptor tyrosine kinases (NRTKs) represent an important class of signaling molecules driving diverse cellular pathways. Aberrant expression and hyperphosphorylation...
Nonreceptor tyrosine kinases (NRTKs) represent an important class of signaling molecules driving diverse cellular pathways. Aberrant expression and hyperphosphorylation of TNK2, an NRTK, have been implicated in multiple cancers. However, the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent biotinylation methods, such as BioID, are being increasingly used to map protein-protein interactions, as they provide increased sensitivity in discovering interaction partners. In this study, we employed stable isotope labeling with amino acids in cell culture and BioID coupled to the biotinylation site identification technology (BioSITe) method that we recently developed to quantitatively explore the interactome of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were able to not only identify site-level biotinylation of previously well-established TNK2 binders and substrates including NCK1, NCK2, CTTN, and STAT3, but also discover several novel TNK2 interacting partners. We also performed co-immunoprecipitation and immunofluorescence analysis to validate the interaction between TNK2 and CLINT1, a novel TNK2 interacting protein. Overall, this work reveals the power of the BioSITe method coupled to BioID and highlights several molecules that warrant further exploration to assess their functional significance in TNK2-mediated signaling.
Topics: Biotinylation; Protein Binding; Protein-Tyrosine Kinases; Signal Transduction
PubMed: 34428048
DOI: 10.1021/acs.jproteome.1c00551 -
Bioorganic & Medicinal Chemistry Jan 2016Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased...
Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signaling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.
Topics: Biotin; Biotinylation; Humans; Kinetics; Molecular Docking Simulation; Phosphorylation; Protein Kinases
PubMed: 26672511
DOI: 10.1016/j.bmc.2015.11.029 -
Proteomics Oct 2016Proximity-dependent biotin identification (BioID) is a recently developed method that allows the identification of proteins in the close vicinity of a protein of... (Review)
Review
Proximity-dependent biotin identification (BioID) is a recently developed method that allows the identification of proteins in the close vicinity of a protein of interest in living cells. BioID relies on fusion of the protein of interest with a mutant form of the biotin ligase enzyme BirA (BirA*) that is capable of promiscuously biotinylating proximal proteins irrespective of whether these interact directly or indirectly with the fusion protein or are merely located in the same subcellular neighborhood. The covalent addition of biotin allows the labeled proteins to be purified from cell extracts on the basis of their affinity for streptavidin and identified by mass spectrometry. To date, BioID has been successfully applied to study a variety of proteins and processes in mammalian cells and unicellular eukaryotes and has been shown to be particularly suited to the study of insoluble or inaccessible cellular structures and for detecting weak or transient protein associations. Here, we provide an introduction to BioID, together with a detailed summary of where and how the method has been applied to date, and briefly discuss technical aspects involved in the planning and execution of a BioID study.
Topics: Animals; Biotin; Biotinylation; Humans; Protein Binding; Protein Interaction Mapping
PubMed: 27329485
DOI: 10.1002/pmic.201600123 -
Current Protocols in Cytometry Apr 2017Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and...
Fluorescent proteins have become standard tools for cell and molecular biologists. The color palette of fluorescent proteins spans the ultraviolet, visible, and near-infrared spectrum. Utility of fluorescent proteins has been greatly facilitated by the availability of compact and affordable solid state lasers capable of providing various excitation wavelengths. In theory, the plethora of fluorescent proteins and lasers make it easy to detect multiple fluorescent proteins simultaneously. However, in practice, heavy spectral overlap due to broad excitation and emission spectra presents a challenge. In conventional flow cytometry, careful selection of excitation wavelengths and detection filters is necessary. Spectral flow cytometry, an emerging methodology that is not confined by the "one color, one detector" paradigm, shows promise in the facile detection of multiple fluorescent proteins. This chapter provides a synopsis of fluorescent protein development, a list of commonly used fluorescent proteins, some practical considerations and strategies for detection, and examples of applications. © 2017 by John Wiley & Sons, Inc.
Topics: Biosensing Techniques; Biotinylation; Flow Cytometry; Genetic Vectors; Lasers; Luminescent Proteins; Protein Interaction Mapping
PubMed: 28369764
DOI: 10.1002/cpcy.17 -
Science Translational Medicine Apr 2018Successful drug treatment for tuberculosis (TB) depends on the unique contributions of its component drugs. Drug resistance poses a threat to the efficacy of individual...
Successful drug treatment for tuberculosis (TB) depends on the unique contributions of its component drugs. Drug resistance poses a threat to the efficacy of individual drugs and the regimens to which they contribute. Biologically and chemically validated targets capable of replacing individual components of current TB chemotherapy are a major unmet need in TB drug development. We demonstrate that chemical inhibition of the bacterial biotin protein ligase (BPL) with the inhibitor Bio-AMS (5'-[-(d-biotinoyl)sulfamoyl]amino-5'-deoxyadenosine) killed (), the bacterial pathogen causing TB. We also show that genetic silencing of BPL eliminated the pathogen efficiently from mice during acute and chronic infection with Partial chemical inactivation of BPL increased the potency of two first-line drugs, rifampicin and ethambutol, and genetic interference with protein biotinylation accelerated clearance of from mouse lungs and spleens by rifampicin. These studies validate BPL as a potential drug target that could serve as an alternate frontline target in the development of new drugs against .
Topics: Animals; Antitubercular Agents; Bacterial Proteins; Biotinylation; Female; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Sulfurtransferases; Tuberculosis
PubMed: 29695454
DOI: 10.1126/scitranslmed.aal1803 -
Nutrition Reviews Aug 2008Histones H2A, H3, and H4 are modified by covalent binding of the vitamin biotin to distinct lysine residues. Binding of biotin to histones is mediated by holocarboxylase... (Review)
Review
Histones H2A, H3, and H4 are modified by covalent binding of the vitamin biotin to distinct lysine residues. Binding of biotin to histones is mediated by holocarboxylase synthetase (HCS) and perhaps biotinidase. Biotinylation of lysine- 12 in histone H4 (K12BioH4) plays roles in gene repression, stability of repeat regions and transposable elements, and regulation of biotin transporter expression in eukaryotes. Decreased biotinylation of histones in biotin-deficient and HCS-deficient human cells and impairs stress resistance, life span, and biotin homeostasis.
Topics: Animals; Biotin; Biotinylation; Carbon-Nitrogen Ligases; Chromatin; Dietary Supplements; Epigenesis, Genetic; Female; Histones; Humans; Pregnancy
PubMed: 18673490
DOI: 10.1111/j.1753-4887.2008.00073.x -
Methods in Enzymology 2016A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins...
A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The identification of proteins associated with lamins is likely to provide fundamental insight into these mechanisms. BioID (proximity-dependent biotin identification) is a unique and powerful method for identifying protein-protein and proximity-based interactions in living cells. BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID-fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry. BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. BioID is applicable to insoluble proteins such as lamins that are often refractory to study by other methods and can identify weak and/or transient interactions. We discuss the use of BioID to elucidate novel lamin-interacting proteins and its applications in a broad range of biological systems, and provide detailed protocols to guide new applications.
Topics: Animals; Biotinylation; Cell Line; Humans; Lamins; Protein Binding; Protein Interaction Mapping; Staining and Labeling
PubMed: 26778550
DOI: 10.1016/bs.mie.2015.08.008 -
Methods in Molecular Biology (Clifton,... 2021Identifying the proteins that associate with RAS oncoproteins has great potential, not only to elucidate how these mutant proteins are regulated and signal but also to...
Identifying the proteins that associate with RAS oncoproteins has great potential, not only to elucidate how these mutant proteins are regulated and signal but also to identify potential therapeutic targets. Here we describe a detailed protocol to employ proximity labeling by the BioID methodology, which has the advantage of capturing weak or transient interactions, to identify in an unbiased manner those proteins within the immediate vicinity of oncogenic RAS proteins.
Topics: Biotin; Biotinylation; Humans; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; ras Proteins
PubMed: 33977483
DOI: 10.1007/978-1-0716-1190-6_16 -
Acta Biochimica Polonica Dec 2019Two biotinylated derivatives of the main hop chalcone xanthohumol (1) were prepared by a one-step synthesis via esterification using biotin and...
Two biotinylated derivatives of the main hop chalcone xanthohumol (1) were prepared by a one-step synthesis via esterification using biotin and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC×HCl) and 4-dimethylaminopyridine (DMAP) as coupling reagents. The products were characterized spectroscopically and their antiproliferative activity toward MCF-7, MCF-10A, HepG2, MDA-MB-231, 4T1 and Balb/3T3 cell lines was investigated using the SRB assay. For all three tested compounds the best activity was noted in the case of human (MCF-7) and mice (4T1) breast cancer cell lines (IC50 values < 9 μM). Both biotinylated derivatives showed slightly higher anticancer activity than xanthohumol (1) towards all types of tested breast cancer cells. Double biotinylated xanthohumol (3) proved to be the most active in inhibiting cell growth, with IC50 values equal to 5.35 ± 1.5 μM for 4T1 and 8.03 ± 0.53 µM for MCF-7 cell lines. Compound 3 was also more active than 1 and 2 against liver cancer cells HepG2 (IC50 = 17.37 ± 5.1 μM), while the IC50 values for 1 and 2 were equal to 21.5 ± 2.7 and 22.1 ± 3.9 µM, respectively. 4‑O‑biotinylxanthohumol (2) was the second most active growth inhibitor, particularly with respect to MCF-7 (IC50 = 6.19 ± 1.7 μM) and 4T1 (IC50 = 6.64 ± 0.4 μM) cell lines. Our preliminary study on biotinylated xanthohumol (1) have shown that this type of functionalization is an effective method for the production of active biomolecules and study on this area should be continued thereby extending their applications.
Topics: Animals; Antineoplastic Agents; BALB 3T3 Cells; Biotinylation; Breast Neoplasms; Cell Proliferation; Cell Survival; Female; Flavonoids; Hep G2 Cells; Humans; Liver Neoplasms; MCF-7 Cells; Mice; Propiophenones
PubMed: 31820895
DOI: 10.18388/abp.2019_2876