Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).
AuthorsSchuman EM
JournalProceedings of the National Academy of Sciences of the United States of America
PubMed ID16769897
In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We ... More
In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.
AuthorsSchuman EM
JournalNature neuroscience
PubMed ID20543841
Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging ... More
Two-color labeling of temporally defined protein populations in mammalian cells.
AuthorsTirrell DA
JournalBioorganic & medicinal chemistry letters
PubMed ID18774715
The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in ... More
Fluorescence visualization of newly synthesized proteins in mammalian cells.
AuthorsTirrell DA
JournalAngewandte Chemie (International ed. in English)
PubMed ID17036290
Noncanonical amino acid tagging enables the selective fluorescent visualization of newly synthesized proteins in mammalian cells (see the picture). Susceptibility to tagging is determined by the spatial and temporal character of the protein synthesis, thus providing a complement to methods which identify relevant members of the proteome. ... More
Spatial coupling of mTOR and autophagy augments secretory phenotypes.
AuthorsNarita M., et al
JournalScience (New York, N.Y.)
PubMed ID21512002
Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the ... More
Protein synthesis in distal axons is not required for growth cone responses to guidance cues.
AuthorsRoche FK, Marsick BM, Letourneau PC,
JournalJ Neurosci
PubMed ID19158291
'Recent evidence suggests that growth cone responses to guidance cues require local protein synthesis. Using chick neurons, we investigated whether protein synthesis is required for growth cones of several types to respond to guidance cues. First, we found that global inhibition of protein synthesis stops axonal elongation after 2 h. ... More
AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.
AuthorsKobayashi M, Aida M, Nagaoka H, Begum NA, Kitawaki Y, Nakata M, Stanlie A, Doi T, Kato L, Okazaki IM, Shinkura R, Muramatsu M, Kinoshita K, Honjo T,
JournalProc Natl Acad Sci U S A
PubMed ID20018730
'To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5'' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. ... More
Two-color labeling of temporally defined protein populations in mammalian cells.
AuthorsBeatty KE, Tirrell DA,
JournalBioorg Med Chem Lett
PubMed ID18774715
'The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in ... More
Azidoalanine mutagenicity in Salmonella: effect of homologation and alpha-methyl substitution.
AuthorsMangold JB, Mischke MR, LaVelle JM,
JournalMutat Res
PubMed ID2645513
'Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of L-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free L-amino acid character, and that bioactivation of azidoalanine to ... More
Mild and chemoselective peptide-bond cleavage of peptides and proteins at azido homoalanine.
AuthorsBack JW, David O, Kramer G, Masson G, Kasper PT, de Koning LJ, de Jong L, van Maarseveen JH, de Koster CG
JournalAngew Chem Int Ed Engl
PubMed ID16281315
The chemical cleavage of amide (peptide) bonds usually requires harsh conditions. As a result, side reactions and the lack of specificity of chemical amide-bond hydrolysis limits its scope in chemical biology and synthetic applications. Herein, we disclose our results on the selective cleavage of amide bonds in peptides and proteins ... More
Dynamic monitoring of newly synthesized proteomes: up-regulation of myristoylated protein kinase A during butyric acid induced apoptosis.
AuthorsLiu K, Yang PY, Na Z, Yao SQ,
JournalAngew Chem Int Ed Engl
PubMed ID21678537
Doubly charged: A double metabolic incorporation approach capable of proteome-wide profiling of post-translational modification dynamics on newly synthesized proteins has been developed (see scheme; blue box: methionine surrogate, orange diamond: PTM probe). This strategy reveals for the first time that up-regulation of myristoylated PKA protein is necessary for the occurrence ... More
Presentation and detection of azide functionality in bacterial cell surface proteins.
AuthorsLink AJ, Vink MK, Tirrell DA
JournalJ Am Chem Soc
PubMed ID15327317
An improved protocol for copper-catalyzed triazole formation on the bacterial cell surface is described. Addition of highly pure CuBr to cells treated with azidohomoalanine (2) leads to ca. 10-fold more extensive cell surface labeling than previously observed. This highly active catalyst allows detection of the methionine analogues azidoalanine (1), azidonorvaline ... More
Selective enrichment of azide-containing peptides from complex mixtures.
AuthorsNessen MA, Kramer G, Back J, Baskin JM, Smeenk LE, de Koning LJ, van Maarseveen JH, de Jong L, Bertozzi CR, Hiemstra H, de Koster CG,
JournalJ Proteome Res
PubMed ID19402736
A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different ... More
Processing of N-terminal unnatural amino acids in recombinant human interferon-beta in Escherichia coli.
AuthorsWang A, Winblade Nairn N, Johnson RS, Tirrell DA, Grabstein K,
JournalChembiochem
PubMed ID18098265
Incorporation of unnatural amino acids into recombinant proteins represents a powerful tool for protein engineering and protein therapeutic development. While the processing of the N-terminal methionine (Met) residues in proteins is well studied, the processing of unnatural amino acids used for replacing the N-terminal Met remains largely unknown. Here we ... More
Identification and quantitation of newly synthesized proteins in Escherichia coli by enrichment of azidohomoalanine-labeled peptides with diagonal chromatography.
AuthorsKramer G, Sprenger RR, Back J, Dekker HL, Nessen MA, van Maarseveen JH, de Koning LJ, Hellingwerf KJ, de Jong L, de Koster CG,
JournalMol Cell Proteomics
PubMed ID19321432
A method is presented to identify and quantify several hundreds of newly synthesized proteins in Escherichia coli upon pulse labeling cells with the methionine analogue azidohomoalanine (azhal). For the first 30 min after inoculation, a methionine-auxotrophic strain grows equally well on azhal as on methionine. Upon a pulse of 15 ... More
Respiratory syncytial virus limits alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) phosphorylation to maintain translation and viral replication.
AuthorsGroskreutz DJ, Babor EC, Monick MM, Varga SM, Hunninghake GW,
JournalJ Biol Chem
PubMed ID20519500
The impact of respiratory syncytial virus (RSV) on morbidity and mortality is significant in that it causes bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and pneumonia in immunocompromised hosts. RSV activates protein kinase R (PKR), a cellular kinase relevant to limiting viral replication (Groskreutz, D. J., Monick, ... More
Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules.
AuthorsBest MD,
JournalBiochemistry
PubMed ID19485420
In recent years, a number of bioorthogonal reactions have been developed, exemplified by click chemistry, that enable the efficient formation of a specific product, even within a highly complex chemical environment. While the exquisite selectivity and reliability of these transformations have led to their broad application in diverse research areas, ... More
In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.
AuthorsDieterich DC, Hodas JJ, Gouzer G, Shadrin IY, Ngo JT, Triller A, Tirrell DA, Schuman EM,
JournalNat Neurosci
PubMed ID20543841
Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging ... More
Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation.
AuthorsKiick KL, Saxon E, Tirrell DA, Bertozzi CR
JournalProc Natl Acad Sci U S A
PubMed ID11752401
The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, ... More
Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging.
AuthorsDieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM
JournalNat Protoc
PubMed ID17406607
A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized ... More
Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).
AuthorsDieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM
JournalProc Natl Acad Sci U S A
PubMed ID16769897
In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We ... More
Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis.
Authors
JournalMol Cell
PubMed ID33606974
RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.
Authors
JournalNature
PubMed ID25079319
Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay.
Authors
JournalNucleic Acids Res
PubMed ID32941650
Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation.
Authors
JournalMol Cell
PubMed ID33156996
PINK1 Inhibits Local Protein Synthesis to Limit Transmission of Deleterious Mitochondrial DNA Mutations.
Authors
JournalMol Cell
PubMed ID30772175
Mechanical regulation of glycolysis via cytoskeleton architecture.
Authors
JournalNature
PubMed ID32051585
Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors.
Authors
JournalNat Commun
PubMed ID35764642
Electrophilic properties of itaconate and derivatives regulate the IκBζ-ATF3 inflammatory axis.
Authors
JournalNature
PubMed ID29670287
Altered translation of GATA1 in Diamond-Blackfan anemia.