16 results on '"Judith Schlegl"'
Search Results
2. ProteomicsDB
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Tobias Schmidt, Patroklos Samaras, Martin Frejno, Siegfried Gessulat, Maximilian Barnert, Harald Kienegger, Helmut Krcmar, Judith Schlegl, Hans-Christian Ehrlich, Stephan Aiche, Bernhard Kuster, and Mathias Wilhelm
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Proteomics ,0301 basic medicine ,Internet ,Cell Survival ,Proteins ,ddc ,03 medical and health sciences ,ComputingMethodologies_PATTERNRECOGNITION ,030104 developmental biology ,0302 clinical medicine ,Pharmaceutical Preparations ,Tandem Mass Spectrometry ,Data Display ,Genetics ,Database Issue ,Humans ,Protein Interaction Maps ,Databases, Protein ,030217 neurology & neurosurgery - Abstract
ProteomicsDB (https://www.ProteomicsDB.org) is a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. ProteomicsDB was first released in 2014 to enable the interactive exploration of the first draft of the human proteome. To date, it contains quantitative data from 78 projects totalling over 19k LC–MS/MS experiments. A standardized analysis pipeline enables comparisons between multiple datasets to facilitate the exploration of protein expression across hundreds of tissues, body fluids and cell lines. We recently extended the data model to enable the storage and integrated visualization of other quantitative omics data. This includes transcriptomics data from e.g. NCBI GEO, protein–protein interaction information from STRING, functional annotations from KEGG, drug-sensitivity/selectivity data from several public sources and reference mass spectra from the ProteomeTools project. The extended functionality transforms ProteomicsDB into a multi-purpose resource connecting quantification and meta-data for each protein. The rich user interface helps researchers to navigate all data sources in either a protein-centric or multi-protein-centric manner. Several options are available to download data manually, while our application programming interface enables accessing quantitative data systematically.
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- 2017
3. Building ProteomeTools based on a complete synthetic human proteome
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Andreas Huhmer, Tobias Schmidt, Derek J. Bailey, Stephan Aiche, Johannes Zerweck, Daniel P Zolg, Eric W. Deutsch, Bernhard Kuster, Robert L. Moritz, Ruedi Aebersold, Thomas Moehring, Peng Yu, Bernard Delanghe, Tobias Knaute, Judith Schlegl, Siegfried Gessulat, Karsten Schnatbaum, Ulf Reimer, Maximilian Weininger, Hans-Christian Ehrlich, Ulrike Kusebauch, Mathias Wilhelm, Karl Kramer, and Holger Wenschuh
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0301 basic medicine ,030102 biochemistry & molecular biology ,Tryptic peptide ,Cell Biology ,Computational biology ,Biology ,Bioinformatics ,Biochemistry ,Article ,03 medical and health sciences ,030104 developmental biology ,Resource (project management) ,Human proteome project ,Molecular Biology ,Biotechnology - Abstract
We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange.
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- 2017
4. The target landscape of clinical kinase drugs
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Stefania Re Depaolini, Tõnu Vooder, Paul-Albert Koenig, Jana Zecha, Mathias Wilhelm, Florian Bassermann, Harald Polzer, Tobias Schmidt, Hannes Hahne, Jan Huenges, Judith Schlegl, Axel Walch, Irmela Jeremias, Stephanie Heinzlmeir, Binje Vick, Bernhard Kuster, Bjoern-Oliver Gohlke, Dominic Helm, Karsten Spiekermann, Karl Kramer, Daniel P Zolg, Neeme Tõnisson, Juergen Ruland, Hans-Christian Ehrlich, Annette Feuchtinger, Wilhelm Becker, Benjamin Ruprecht, Katharina Götze, Svenja Petzoldt, Stephan Aiche, Lars Rueckert, Susan Klaeger, Sabine Schneider, Melanie Schoof, Anne-Kathrin Garz, Maria Reinecke, G. Canevari, Eduard R. Felder, Guillaume Médard, Chen Meng, Philipp A. Greif, Gian Kayser, Elena Casale, Zhixiang Wu, Robert Preissner, Heiner Koch, Huichao Qiao, and Katrin Reiter
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0301 basic medicine ,Drug ,Cabozantinib ,media_common.quotation_subject ,Druggability ,Computational biology ,Article ,Maternal embryonic leucine zipper kinase ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Kinome ,media_common ,Multidisciplinary ,business.industry ,Kinase ,Drug discovery ,Drug Repositioning ,Cancer ,medicine.disease ,3. Good health ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,business ,Protein Kinases - Abstract
An atlas for drug interactions Kinase inhibitors are an important class of drugs that block certain enzymes involved in diseases such as cancer and inflammatory disorders. There are hundreds of kinases within the human body, so knowing the kinase “target” of each drug is essential for developing successful treatment strategies. Sometimes clinical trials can fail because drugs bind more than one target. Yet sometimes off-target effects can be beneficial, and drugs can be repurposed for treatment of additional diseases. Klaeger et al. performed a comprehensive analysis of 243 kinase inhibitors that are either approved for use or in clinical trials. They provide an open-access resource of target summaries that could help researchers develop better drugs, understand how existing drugs work, and design more effective clinical trials. Science , this issue p. eaan4368
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- 2017
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5. Mass-spectrometry-based draft of the human proteome
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Emanuel Ziegler, Harald Marx, Mathias Wilhelm, Holger Wenschuh, Marcus Bantscheff, Toby Mathieson, Amin Moghaddas Gholami, Lars Butzmann, Bernhard Kuster, Siegfried Gessulat, Ulf Reimer, Anja Gerstmair, Simone Lemeer, Marcus Lieberenz, Martin Mollenhauer, Joos-Hendrik Boese, Mikhail M. Savitski, Judith Schlegl, Franz Faerber, Karsten Schnatbaum, Julia Slotta-Huspenina, and Hannes Hahne
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Transcriptome ,Multidisciplinary ,NeXtProt ,Proteome ,Human proteome project ,Genomics ,Human genome ,Computational biology ,Biology ,Proteogenomics ,Bioinformatics ,Proteomics - Abstract
Proteomes are characterized by large protein-abundance differences, cell-type- and time-dependent expression patterns and post-translational modifications, all of which carry biological information that is not accessible by genomics or transcriptomics. Here we present a mass-spectrometry-based draft of the human proteome and a public, high-performance, in-memory database for real-time analysis of terabytes of big data, called ProteomicsDB. The information assembled from human tissues, cell lines and body fluids enabled estimation of the size of the protein-coding genome, and identified organ-specific proteins and a large number of translated lincRNAs (long intergenic non-coding RNAs). Analysis of messenger RNA and protein-expression profiles of human tissues revealed conserved control of protein abundance, and integration of drug-sensitivity data enabled the identification of proteins predicting resistance or sensitivity. The proteome profiles also hold considerable promise for analysing the composition and stoichiometry of protein complexes. ProteomicsDB thus enables navigation of proteomes, provides biological insight and fosters the development of proteomic technology.
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- 2014
6. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling
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Aleem Fazal, Marc Hild, Christine D. Wilson, Shih Min A Huang, Stephen Fawell, Feng Cong, John A. Tallarico, Michael Shultz, Hong Cheng, Andreas Bauer, Christoph Lengauer, Chris Lu, Christina Rau, Judith Schlegl, Jeffery A. Porter, Daniel Curtis, Fabrizio C. Serluca, Stephanie Wiessner, Ronald Tomlinson, Sonja Ghidelli, Marc W. Kirschner, Xiaoying Shi, Elizabeth Wiellette, Vic E. Myer, Markus Schirle, Frank Stegmeier, Yue Zhang, Craig Mickanin, Atwood K. Cheung, Yuji Mishina, Wenlin Shao, Olga Charlat, Gregory A. Michaud, Shanming Liu, Peter Finan, and Tewis Bouwmeester
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Proteomics ,Proteasome Endopeptidase Complex ,Beta-catenin ,Transcription, Genetic ,Biology ,Cell Line ,Axin Protein ,Cell Line, Tumor ,AXIN1 ,Humans ,Transcription factor ,beta Catenin ,Tankyrases ,Multidisciplinary ,Ubiquitin ,Ubiquitination ,Wnt signaling pathway ,LRP6 ,LRP5 ,Cell biology ,Repressor Proteins ,Wnt Proteins ,Catenin ,biology.protein ,Colorectal Neoplasms ,Heterocyclic Compounds, 3-Ring ,Cell Division ,Protein Binding ,Signal Transduction - Abstract
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
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- 2009
7. Precursor of C4 antisense RNA of bacteriophages P1 and P7 is a substrate for RNase P of Escherichia coli
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Heinz Schuster, Judith Schlegl, Roland K. Hartmann, and Jochen Heinrich
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Transcription, Genetic ,RNase P ,Genes, Fungal ,Molecular Sequence Data ,RNA-dependent RNA polymerase ,Biology ,Coliphages ,Polymerase Chain Reaction ,Ribonuclease P ,Endoribonucleases ,Escherichia coli ,RNA Precursors ,RNA, Antisense ,RNA, Catalytic ,RNA, Messenger ,Bacteriophage P1 ,DNA Primers ,Multidisciplinary ,Base Sequence ,Escherichia coli Proteins ,Intron ,RNA ,RNA, Transfer, Gly ,Non-coding RNA ,Molecular biology ,Antisense RNA ,Repressor Proteins ,RNA silencing ,Genes, Bacterial ,RNA editing ,Nucleic Acid Conformation ,RNA, Viral ,Research Article - Abstract
The C4 repressor of the temperate bacteriophages P1 and P7 inhibits antirepressor (Ant) synthesis and is essential for establishment and maintenance of lysogeny. C4 is an antisense RNA acting on a target, Ant mRNA, which is transcribed from the same promoter. The antisense-target RNA interaction requires processing of C4 RNA from a precursor RNA. Here we show that 5' maturation of C4 RNA in vivo depends on RNase P. In vitro, Escherichia coli RNase P and its catalytic RNA subunit (M1 RNA) can generate the mature 5' end of C4 RNA from P1 by a single endonucleolytic cut, whereas RNase P from the E. coli rnpA49 mutant, carrying a missense mutation in the RNase P protein subunit, is defective in the 5' maturation of C4 RNA. Primer extension analysis of RNA transcribed in vivo from a plasmid carrying the P1 c4 gene revealed that 5'-mature C4 RNA was the predominant species in rnpA+ bacteria, whereas virtually no mature C4 RNA was found in the temperature-sensitive rnpA49 strain at the restrictive temperature. Instead, C4 RNA molecules carrying up to five extra nucleotides beyond the 5' end accumulated. The same phenotype was observed in rnpA+ bacteria which harbored a plasmid carrying a P7 c4 mutant gene with a single C-->G base substitution in the structural homologue to the CCA 3' end of tRNAs. Implications of C4 RNA processing for the lysis/lysogeny decision process of bacteriophages P1 and P7 are discussed.
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- 1995
8. Kinetics and Thermodynamics of the RNase P RNA Cleavage Reaction:Analysis of tRNA 3'-end Variants
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Volker A. Erdmann, Wolf-Dietrich Hardt, Roland K. Hartmann, and Judith Schlegl
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Models, Molecular ,RNase P ,Molecular Sequence Data ,Thermodynamics ,Biology ,RNase PH ,Ribonuclease P ,Substrate Specificity ,Structure-Activity Relationship ,Species Specificity ,Structural Biology ,Endoribonucleases ,Escherichia coli ,Magnesium ,RNA, Catalytic ,RNA Processing, Post-Transcriptional ,Binding site ,Molecular Biology ,Base Sequence ,Escherichia coli Proteins ,Hydrolysis ,Thermus thermophilus ,Genetic Variation ,RNA ,RNA, Transfer, Gly ,TRNA binding ,Kinetics ,RNA, Bacterial ,RNase MRP ,Lead ,Biochemistry ,Transfer RNA ,Nucleic Acid Conformation ,RNA Cleavage - Abstract
We have studied the interaction of 3′-end variants of a (pre-)tRNA Gly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus . To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNA Gly variants to RNase P RNAs was studied bu gel retardation. A newly developed assay, based on the reduction of Pb 2+ -hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNA Gly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNA Gly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3′-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNS (possibly a conformational rearrangement. For T. thermo philus RNase P RNA, product release of wild-type tRNA Gly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3′-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.
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- 1995
9. Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function
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Volker A. Erdmann, Wolf-Dietrich Hardt, Judith Schlegl, and Roland K. Hartmann
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RNase P ,Protein subunit ,Molecular Sequence Data ,Catalysis ,Ribonuclease P ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,chemistry.chemical_compound ,Endoribonucleases ,RNA, Catalytic ,Nucleotide ,Ribonuclease ,Molecular Biology ,chemistry.chemical_classification ,Base Sequence ,General Immunology and Microbiology ,biology ,Escherichia coli Proteins ,Thermus thermophilus ,General Neuroscience ,RNA ,Cytidine ,biology.organism_classification ,RNA, Bacterial ,chemistry ,Biochemistry ,Mutation ,biology.protein ,Nucleic Acid Conformation ,Pseudoknot ,Research Article - Abstract
We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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- 1994
10. Lead-ion-induced cleavage of RNase P RNA
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Volker A. Erdmann, Judith Schlegl, Roland K. Hartmann, Jerzy Ciesiołka, and Wolf-Dietrich Hardt
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Transcription, Genetic ,RNase P ,Molecular Sequence Data ,Biochemistry ,RNase PH ,Ribonuclease P ,Endoribonucleases ,Escherichia coli ,RNA, Catalytic ,RNase H ,Binding Sites ,Base Sequence ,biology ,Escherichia coli Proteins ,Hydrolysis ,Thermus thermophilus ,RNA ,Non-coding RNA ,biology.organism_classification ,Molecular biology ,Kinetics ,RNase MRP ,Lead ,Transfer RNA ,biology.protein ,Nucleic Acid Conformation ,Thermodynamics - Abstract
Pb(2+)-induced hydrolysis of RNase P RNAs from Escherichia coli and the thermophilic eubacterium Thermus thermophilus HB8 revealed one prominent site-specific cleavage in the two RNAs and several minor cleavage sites in structurally corresponding regions of both RNAs. Data presented here and in a previous study [Kazakov, S.Altman, S. (1991) Proc. Natl Acad. Sci. USA 88, 9193-9197] provide evidence for several ubiquitous metal-ion-binding sites in eubacterial RNase P RNA subunits. With the T. thermophilus RNase P RNA, susceptibility to Pb(2+)-induced strand scission at the most prominent site was hypersensitive at the temperature of highest enzyme activity (55 degrees C). Pb2+ hydrolysis at this site was strongly reduced at a temperature of 37 degrees C, where processing is also inefficient. For E. coli RNase P RNA, specific changes in the lead hydrolysis pattern were observed due to the presence of excess tRNA. Thus, Pb(2+)-induced hydrolysis seems suitable to sense different conformations of RNase P RNAs. The T. thermophilus RNase P RNA, in particular, displayed significant processing activity after severe fragmentation by Pb2+, and therefore appears to be suited for reconstituting an active enzyme from RNA subfragments.
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- 1994
11. Gel retardation analysis ofE.coliM1 RNA-tRNA complexes
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Judith Schlegl, Roland K. Hartmann, Wolf-Dietrich Hardt, and Volker A. Erdmann
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RNase P ,Protein subunit ,Molecular Sequence Data ,Biology ,Binding, Competitive ,Ribonuclease P ,RNA, Transfer ,Endoribonucleases ,Escherichia coli ,Genetics ,Magnesium ,RNA, Catalytic ,Nucleotide ,RNA, Transfer, Ile ,chemistry.chemical_classification ,Base Sequence ,Escherichia coli Proteins ,Intron ,RNA ,RNA, Transfer, Gly ,TRNA binding ,Introns ,Kinetics ,RNA, Bacterial ,Enzyme ,chemistry ,Biochemistry ,Transfer RNA ,Nucleic Acid Conformation ,Calcium - Abstract
We have analyzed complexes between tRNA and E. coli M1 RNA by electrophoresis in non-denaturing polyacrylamide gels. The RNA subunit of E. coli RNase P formed a specific complex with mature tRNA molecules. A derivative of the tRNA(Gly), endowed with the intron of yeast tRNA(ile) (60 nt), was employed to improve separation of complexed and unbound M1 RNA. Binding assays with tRNA(Gly) and intron-tRNA(Gly) as well as analysis of intron-tRNA/M1 RNA complexes on denaturing gels showed that one tRNA is bound per molecule of M1 RNA. A tRNA carrying a truncation as small as the 5'-nucleotide had a strongly reduced affinity to M1 RNA and was also a weak competitor in the cleavage reaction, suggesting that nucleotide +1 is a major determinant of tRNA recognition and that the thermodynamically stable tRNA-M1 RNA complex is relevant for enzyme function. Binding was shown to be dependent on the M1 RNA concentration in a cooperative fashion. Only a fraction of M1 RNAs (50-60%) readily formed a complex with intron-tRNA(Gly), indicating that distinct conformational subpopulations of M1 RNA may exist. Formation of the M1 RNA-tRNA(Gly), complex was very similar at 100 mM Mg++ and Ca++, corroborating earlier data that Ca++ is competent in promoting M1 RNA folding and tRNA binding. Determination of apparent equilibrium constants (app Kd) for tRNA(Gly) as a function of the Mg++ concentration supports an uptake of at least two additional Mg++ ions upon complex formation. At 20-30 mM Mg++, highest cleavage rates but strongly reduced complex formation were observed. This indicates that tight binding of the tRNA to the catalytic RNA at higher magnesium concentrations retards product release and therefore substrate turnover.
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- 1993
12. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes
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Isabelle Becher, Valerie Reader, Giovanna Bergamini, Gavain Sweetman, Carola Huthmacher, Nigel Ramsden, Katja Strunk, Dirk Eberhard, Markus Boesche, Antje Dittmann, Marcus Bantscheff, Mikhail M. Savitski, Ulrich Kruse, Daniel Poeckel, Birgit Dümpelfeld, Gitte Neubauer, Judith Schlegl, Anne-Marie Michon, Gerard Drewes, Paola Grandi, Toby Mathieson, Carsten Hopf, Yann Abraham, and Manja Delling
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Proteomics ,SIN3 Complex ,medicine.drug_class ,Chemistry ,HDAC10 ,Histone deacetylase inhibitor ,Biomedical Engineering ,Bioengineering ,HDAC6 ,Applied Microbiology and Biotechnology ,Small molecule ,Peptide Mapping ,Histone Deacetylases ,Mass Spectrometry ,Biochemistry ,Protein Interaction Mapping ,medicine ,Molecular Medicine ,Chemoproteomics ,Histone deacetylase ,Mitosis ,Biotechnology - Abstract
The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.
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- 2010
13. Cleavage efficiencies of model substrates for ribonuclease P fromEscherichia coliandThermus thermophilus
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Judith Schlegl, Volker A. Erdmann, Jens P. Fürste, Roland K. Hartmann, and Rolf Bald
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DNA, Bacterial ,Transcription, Genetic ,Base pair ,RNase P ,Molecular Sequence Data ,medicine.disease_cause ,Cleavage (embryo) ,Ribonuclease P ,Substrate Specificity ,Endoribonucleases ,Escherichia coli ,Genetics ,medicine ,RNA, Catalytic ,Base Sequence ,biology ,Escherichia coli Proteins ,Thermus thermophilus ,RNA ,RNA, Transfer, Gly ,biology.organism_classification ,Kinetics ,RNA, Bacterial ,Biochemistry ,Transfer RNA ,Nucleic Acid Conformation ,T arm - Abstract
We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing.
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- 1992
14. A physical and functional map of the human TNF-α/NF-κB signal transduction pathway
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Bettina Huhse, Heinz Ruffner, Judith Schlegl, Karen Croughton, Anne-Claude Gavin, Martin Stein, Jens Rick, Georg Casari, Markus Schirle, Tewis Bouwmeester, Sonja Ghidelli, Bernhard Kuster, David B. Jackson, Gitte Neubauer, Giulio Superti-Furga, Raffaella Mangano, Giovanna Bergamini, Gerard Joberty, Dirk Eberhard, Markus Schwab, Angela Bauch, Pierre-Olivier Angrand, Julien Gagneur, Cristina Cruciat, Carsten Hopf, Anne-Marie Michon, Gerard Drewes, and Andreas Bauer
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Chaperonins ,Proteome ,Macromolecular Substances ,medicine.medical_treatment ,Cell Cycle Proteins ,MAP Kinase Kinase Kinase 3 ,Biology ,Models, Biological ,Chromatography, Affinity ,Mass Spectrometry ,Receptors, Tumor Necrosis Factor ,Cell Line ,Tacrolimus Binding Proteins ,Enzyme activator ,RNA interference ,Interaction network ,medicine ,Animals ,Drosophila Proteins ,Humans ,HSP90 Heat-Shock Proteins ,ddc:612 ,Transcription factor ,Tandem affinity purification ,Tumor Necrosis Factor-alpha ,Tumor Suppressor Proteins ,NF-kappa B ,Cell Biology ,NFKB1 ,MAP Kinase Kinase Kinases ,Cell biology ,Enzyme Activation ,Cytokine ,I-kappa B Proteins ,RNA Interference ,Signal transduction ,Carrier Proteins ,Molecular Chaperones ,Signal Transduction - Abstract
Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.
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- 2004
15. Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting
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Joseph Reinbolt, Judith Schlegl, Chantal Ehresmann, Britta Schläger, Bernard Ehresmann, Valérie Gegout, Pascale Romby, and Matthias W. Hentze
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Iron-Sulfur Proteins ,Molecular Sequence Data ,DNA Footprinting ,Transferrin receptor ,Biology ,Ligands ,Biochemistry ,Aconitase ,Amino Acid Sequence ,Iron Regulatory Protein 1 ,RNA, Messenger ,Binding site ,Molecular Biology ,chemistry.chemical_classification ,Messenger RNA ,Protein footprinting ,RNA ,Iron-Regulatory Proteins ,RNA-Binding Proteins ,Cell Biology ,Amino acid ,Ferritin ,chemistry ,biology.protein ,Apoproteins - Abstract
Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.
- Published
- 1999
16. Role of the D arm and the anticodon arm in tRNA recognition by eubacterial and eukaryotic RNase P enzymes
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Judith Schlegl, Volker A. Erdmann, Roland K. Hartmann, and Wolf-Dietrich Hardt
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RNase P ,Molecular Sequence Data ,D arm ,Biology ,Biochemistry ,RNase PH ,Ribonuclease P ,Substrate Specificity ,Structure-Activity Relationship ,Endoribonucleases ,Anticodon ,Escherichia coli ,Humans ,RNA, Catalytic ,RNA Processing, Post-Transcriptional ,Sequence Deletion ,Binding Sites ,Base Sequence ,Escherichia coli Proteins ,Thermus thermophilus ,RNA ,Nucleic Acid Precursors ,Hydrogen Bonding ,RNA, Transfer, Gly ,Cations, Monovalent ,biology.organism_classification ,Molecular biology ,RNase MRP ,RNA, Bacterial ,Lead ,Transfer RNA ,bacteria ,Nucleic Acid Conformation ,T arm ,HeLa Cells - Abstract
Truncated precursor tRNAs lacking the D arm or anticodon arm were studied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm still allowed 5'-processing by E. coli RNase P, but strongly impaired maturation by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activities from all three organisms. Inhibition kinetics and gel retardation studies showed that deletion of the D arm leads to low-affinity binding to E. coli RNase P RNA (M1 RNA). However, the E. coli enzyme appears to form sufficiently strong contacts in the region of the T arm, acceptor stem, and CCA terminus to still allow productive enzyme-substrate interaction even in the absence of the structural contribution provided by the D arm. Pb(2+)-induced hydrolysis of a tRNAGly from T. thermophilus gave identical cleavage patterns in the D arm and anticodon loop in the absence and presence of E. coli M1 RNA, whereas lead hydrolysis was strongly reduced at the CUCCAA 3'-terminus due to the presence of the enzyme.
- Published
- 1993
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