Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
Protein Sci ; 26(2): 327-342, 2017 02.
Article in English | MEDLINE | ID: mdl-27863450

ABSTRACT

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.


Subject(s)
Chaetomium/chemistry , Fungal Proteins/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Chaetomium/metabolism , Fungal Proteins/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism
2.
Methods Mol Biol ; 803: 141-55, 2012.
Article in English | MEDLINE | ID: mdl-22065223

ABSTRACT

Chemical proteomics offers a unique approach for target identification of small molecule inhibitors directly from cell extracts, thus enabling characterization of target proteins under close to physiological conditions. Here, we describe a competition binding procedure that is based on affinity enrichment of potential target proteins on a probe matrix in the presence of increasing amounts of free test compound in solution. Reduced binding of target proteins to the probe matrix as a function of test compound concentration can be measured and thus, enables calculation of IC(50) values. The method employs quantitative mass spectrometry using isobaric mass tags which enables determination of potency for a large number of target proteins in a single analysis.


Subject(s)
Binding, Competitive/drug effects , Biological Assay/methods , Cell Extracts/chemistry , Protein Kinase Inhibitors/pharmacology , Staining and Labeling , Cell Fractionation , Chromatography, Affinity , Chromatography, Liquid , Inhibitory Concentration 50 , Mass Spectrometry , Molecular Weight , Proteomics , Statistics as Topic , Staurosporine/pharmacology , Trypsin
3.
Nat Biotechnol ; 25(9): 1035-44, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17721511

ABSTRACT

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Benzamides , Cell Extracts , Chromatography, Affinity , Discoidin Domain Receptor 1 , Enzymes, Immobilized/antagonists & inhibitors , HeLa Cells , Humans , Imatinib Mesylate , Inhibitory Concentration 50 , K562 Cells , Pharmaceutical Preparations , Phosphorylation/drug effects , Piperazines/pharmacology , Pyrimidines/pharmacology , Quinone Reductases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects
4.
Nature ; 440(7084): 631-6, 2006 Mar 30.
Article in English | MEDLINE | ID: mdl-16429126

ABSTRACT

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.


Subject(s)
Proteome/metabolism , Proteomics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Genome, Fungal , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Open Reading Frames/genetics , Phenotype , Proteome/chemistry , Proteome/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
5.
Nature ; 415(6868): 141-7, 2002 Jan 10.
Article in English | MEDLINE | ID: mdl-11805826

ABSTRACT

Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.


Subject(s)
Proteome/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/physiology , Cells, Cultured , Chromatography, Affinity , Gene Targeting , Humans , Macromolecular Substances , Proteome/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sensitivity and Specificity , Species Specificity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
SELECTION OF CITATIONS
SEARCH DETAIL
...