Publications by authors named "Gerhard Dürnberger"

20 Publications

  • Page 1 of 1

A proteomic survey of microtubule-associated proteins in a R402H TUBA1A mutant mouse.

PLoS Genet 2020 11 2;16(11):e1009104. Epub 2020 Nov 2.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

Microtubules play a critical role in multiple aspects of neurodevelopment, including the generation, migration and differentiation of neurons. A recurrent mutation (R402H) in the α-tubulin gene TUBA1A is known to cause lissencephaly with cerebellar and striatal phenotypes. Previous work has shown that this mutation does not perturb the chaperone-mediated folding of tubulin heterodimers, which are able to assemble and incorporate into the microtubule lattice. To explore the molecular mechanisms that cause the disease state we generated a new conditional mouse line that recapitulates the R402H variant. We show that heterozygous mutants present with laminar phenotypes in the cortex and hippocampus, as well as a reduction in striatal size and cerebellar abnormalities. We demonstrate that homozygous expression of the R402H allele causes neuronal death and exacerbates a cell intrinsic defect in cortical neuronal migration. Microtubule sedimentation assays coupled with quantitative mass spectrometry demonstrated that the binding and/or levels of multiple microtubule associated proteins (MAPs) are perturbed by the R402H mutation including VAPB, REEP1, EZRIN, PRNP and DYNC1l1/2. Consistent with these data we show that the R402H mutation impairs dynein-mediated transport which is associated with a decoupling of the nucleus to the microtubule organising center. Our data support a model whereby the R402H variant is able to fold and incorporate into microtubules, but acts as a gain of function by perturbing the binding of MAPs.
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http://dx.doi.org/10.1371/journal.pgen.1009104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660477PMC
November 2020

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress.

Elife 2020 08 27;9. Epub 2020 Aug 27.

Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria.

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.
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http://dx.doi.org/10.7554/eLife.58396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515635PMC
August 2020

The biophysical, molecular, and anatomical landscape of pigeon CRY4: A candidate light-based quantal magnetosensor.

Sci Adv 2020 Aug 12;6(33):eabb9110. Epub 2020 Aug 12.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus-Vienna-Biocenter 1, Vienna 1030, Austria.

The biophysical and molecular mechanisms that enable animals to detect magnetic fields are unknown. It has been proposed that birds have a light-dependent magnetic compass that relies on the formation of radical pairs within cryptochrome molecules. Using spectroscopic methods, we show that pigeon cryptochrome clCRY4 is photoreduced efficiently and forms long-lived spin-correlated radical pairs via a tetrad of tryptophan residues. We report that clCRY4 is broadly and stably expressed within the retina but enriched at synapses in the outer plexiform layer in a repetitive manner. A proteomic survey for retinal-specific clCRY4 interactors identified molecules that are involved in receptor signaling, including glutamate receptor-interacting protein 2, which colocalizes with clCRY4. Our data support a model whereby clCRY4 acts as an ultraviolet-blue photoreceptor and/or a light-dependent magnetosensor by modulating glutamatergic synapses between horizontal cells and cones.
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http://dx.doi.org/10.1126/sciadv.abb9110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423367PMC
August 2020

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesin from WAPL.

Elife 2020 02 17;9. Epub 2020 Feb 17.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesin which binds chromatin for hours, whereas cohesin binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesin complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesin forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesin extrusion activity.
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http://dx.doi.org/10.7554/eLife.52091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054000PMC
February 2020

Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells.

EMBO J 2020 02 13;39(4):e103315. Epub 2020 Jan 13.

Functional Genomic Section, Department of Biology, University of Copenhagen, Copenhagen, Denmark.

Somatic cells acclimate to changes in the environment by temporary reprogramming. Much has been learned about transcription factors that induce these cell-state switches in both plants and animals, but how cells rapidly modulate their proteome remains elusive. Here, we show rapid induction of autophagy during temporary reprogramming in plants triggered by phytohormones, immune, and danger signals. Quantitative proteomics following sequential reprogramming revealed that autophagy is required for timely decay of previous cellular states and for tweaking the proteome to acclimate to the new conditions. Signatures of previous cellular programs thus persist in autophagy-deficient cells, affecting cellular decision-making. Concordantly, autophagy-deficient cells fail to acclimatize to dynamic climate changes. Similarly, they have defects in dedifferentiating into pluripotent stem cells, and redifferentiation during organogenesis. These observations indicate that autophagy mediates cell-state switches that underlie somatic cell reprogramming in plants and possibly other organisms, and thereby promotes phenotypic plasticity.
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http://dx.doi.org/10.15252/embj.2019103315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024839PMC
February 2020

Absolute quantification of cohesin, CTCF and their regulators in human cells.

Elife 2019 06 17;8. Epub 2019 Jun 17.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria.

The organisation of mammalian genomes into loops and topologically associating domains (TADs) contributes to chromatin structure, gene expression and recombination. TADs and many loops are formed by cohesin and positioned by CTCF. In proliferating cells, cohesin also mediates sister chromatid cohesion, which is essential for chromosome segregation. Current models of chromatin folding and cohesion are based on assumptions of how many cohesin and CTCF molecules organise the genome. Here we have measured absolute copy numbers and dynamics of cohesin, CTCF, NIPBL, WAPL and sororin by mass spectrometry, fluorescence-correlation spectroscopy and fluorescence recovery after photobleaching in HeLa cells. In G1-phase, there are ~250,000 nuclear cohesin complexes, of which ~ 160,000 are chromatin-bound. Comparison with chromatin immunoprecipitation-sequencing data implies that some genomic cohesin and CTCF enrichment sites are unoccupied in single cells at any one time. We discuss the implications of these findings for how cohesin can contribute to genome organisation and cohesion.
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http://dx.doi.org/10.7554/eLife.46269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606026PMC
June 2019

Spectral Clustering Improves Label-Free Quantification of Low-Abundant Proteins.

J Proteome Res 2019 04 22;18(4):1477-1485. Epub 2019 Mar 22.

Research Institute of Molecular Pathology (IMP) , Vienna Biocenter (VBC) , Campus-Vienna-Biocenter 1 , 1030 Vienna , Austria.

Label-free quantification has become a common-practice in many mass spectrometry-based proteomics experiments. In recent years, we and others have shown that spectral clustering can considerably improve the analysis of (primarily large-scale) proteomics data sets. Here we show that spectral clustering can be used to infer additional peptide-spectrum matches and improve the quality of label-free quantitative proteomics data in data sets also containing only tens of MS runs. We analyzed four well-known public benchmark data sets that represent different experimental settings using spectral counting and peak intensity based label-free quantification. In both approaches, the additionally inferred peptide-spectrum matches through our spectra-cluster algorithm improved the detectability of low abundant proteins while increasing the accuracy of the derived quantitative data, without increasing the data sets' noise. Additionally, we developed a Proteome Discoverer node for our spectra-cluster algorithm which allows anyone to rebuild our proposed pipeline using the free version of Proteome Discoverer.
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http://dx.doi.org/10.1021/acs.jproteome.8b00377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456873PMC
April 2019

apQuant: Accurate Label-Free Quantification by Quality Filtering.

J Proteome Res 2019 01 2;18(1):535-541. Epub 2018 Nov 2.

Research Institute of Molecular Pathology (IMP) , Vienna Biocenter (VBC) , Campus-Vienna-Biocenter 1 , 1030 Vienna , Austria.

Label-free quantification of shotgun proteomics data is a frequently used strategy, offering high dynamic range, sensitivity, and the ability to compare a high number of samples without additional labeling effort. Here, we present a bioinformatics approach that significantly improves label-free quantification results. We employ Percolator to assess the quality of quantified peptides. This allows to extract accurate and reliable quantitative results based on false discovery rate. Benchmarking our approach on previously published public data shows that it considerably outperforms currently available algorithms. apQuant is available free of charge as a node for Proteome Discoverer.
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http://dx.doi.org/10.1021/acs.jproteome.8b00113DOI Listing
January 2019

Publisher Correction: Mutations in Vps15 perturb neuronal migration in mice and are associated with neurodevelopmental disease in humans.

Nat Neurosci 2018 Aug;21(8):1139

Institute of Molecular Pathology (IMP), Vienna Biocentre (VBC), Vienna, Austria.

In the supplementary information PDF originally posted, there were discrepancies from the integrated supplementary information that appeared in the HTML; the former has been corrected as follows. In the legend to Supplementary Fig. 2c, "major organs of the mouse" has been changed to "major organs of the adult mouse." In the legend to Supplementary Fig. 6d,h, "At E14.5 Mbe/Mbe mutants have a smaller percentage of Brdu positive cells in bin 3" has been changed to "At E14.5 Mbe/Mbe mutants have a higher percentage of Brdu positive cells in bin 3."
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http://dx.doi.org/10.1038/s41593-018-0170-9DOI Listing
August 2018

Mutations in Vps15 perturb neuronal migration in mice and are associated with neurodevelopmental disease in humans.

Nat Neurosci 2018 02 8;21(2):207-217. Epub 2018 Jan 8.

Institute of Molecular Pathology (IMP), Vienna Biocentre (VBC), Vienna, Austria.

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.
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http://dx.doi.org/10.1038/s41593-017-0053-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897053PMC
February 2018

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

Plant J 2018 Jan 2;93(1):17-33. Epub 2017 Dec 2.

School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co-immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein-protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis-associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.
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http://dx.doi.org/10.1111/tpj.13752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767750PMC
January 2018

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

Nature 2017 09 20;549(7673):538-542. Epub 2017 Sep 20.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr Gasse 3, A-1030 Vienna, Austria.

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.
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http://dx.doi.org/10.1038/nature24015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003595PMC
September 2017

A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor.

Nat Chem Biol 2017 Jul 22;13(7):771-778. Epub 2017 May 22.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.
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http://dx.doi.org/10.1038/nchembio.2382DOI Listing
July 2017

Phosphoproteomics with Activated Ion Electron Transfer Dissociation.

Anal Chem 2017 06 17;89(12):6367-6376. Epub 2017 Apr 17.

Morgridge Institute for Research , Madison, Wisconsin 53715, United States.

The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript ( Anal. Chem. 2017 , DOI: 10.1021/acs.analchem.7b00213 ), we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90 min analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photoactivation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in α-casein, an ∼23.5 kDa phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics.
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http://dx.doi.org/10.1021/acs.analchem.7b00212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555596PMC
June 2017

Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense.

Nat Genet 2014 Sep 17;46(9):1028-33. Epub 2014 Aug 17.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria.

Neutrophils are key innate immune effector cells that are essential to fighting bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagn1 (encoding Jagunal homolog 1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis identified marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity in Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection and impaired formation of cytotoxic granules, as well as defective myeloperoxidase release and killing of Candida albicans. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Notably, GM-CSF also restored the defective fungicidal activity of bone marrow cells from humans with JAGN1 mutations. These data directly identify Jagn1 (JAGN1 in humans) as a new regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for humans.
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http://dx.doi.org/10.1038/ng.3070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245568PMC
September 2014

Global analysis of muscle-specific kinase signaling by quantitative phosphoproteomics.

Mol Cell Proteomics 2014 Aug 4;13(8):1993-2003. Epub 2014 Jun 4.

‖Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090 Vienna, Austria; ‡‡Institute of Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 Vienna, Austria.

The development of the neuromuscular synapse depends on signaling processes that involve protein phosphorylation as a crucial regulatory event. Muscle-specific kinase (MuSK) is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional synapse. However, the signaling cascade downstream of MuSK and the regulation of the different components are still poorly understood. In this study we used a quantitative phosphoproteomics approach to study the phosphorylation events and their temporal regulation downstream of MuSK. We identified a total of 10,183 phosphopeptides, of which 203 were significantly up- or down-regulated. Regulated phosphopeptides were classified into four different clusters according to their temporal profiles. Within these clusters we found an overrepresentation of specific protein classes associated with different cellular functions. In particular, we found an enrichment of regulated phosphoproteins involved in posttranscriptional mechanisms and in cytoskeletal organization. These findings provide novel insights into the complex signaling network downstream of MuSK and form the basis for future mechanistic studies.
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http://dx.doi.org/10.1074/mcp.M113.036087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125732PMC
August 2014

Experimental characterization of the human non-sequence-specific nucleic acid interactome.

Genome Biol 2013 Jul 31;14(7):R81. Epub 2013 Jul 31.

Background: The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.

Results: We designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function.

Conclusions: The dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.
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http://dx.doi.org/10.1186/gb-2013-14-7-r81DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053969PMC
July 2013

The TLR-independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature.

Eur J Immunol 2009 Jul;39(7):1929-36

Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Recognition of foreign DNA by cytosolic innate immune receptors triggers the production of IFN-beta. However, it is unclear whether different types of DNA ligands are recognized by similar receptors and whether the resulting response is distinct from the endosomal TLR response. To address these questions, we compared the two most commonly used types of DNA ligands (IFN-stimulatory DNA (ISD) and poly(dAdT)) and assessed the minimal structural requirements for stimulatory capacity in RAW264.7 cells. Gene expression signatures and competition experiments suggest that ISD and poly(dAdT) are qualitatively indistinguishable and differ from the CpG-containing oligonucleotides triggering the TLR9 pathway. Structure - activity relationship analyses revealed that a minimal length of two helical turns is sufficient for ISD-mediated IFN-beta induction, while phosphorylation at the 5'-end is dispensable. Altogether, our data suggest that, in murine macrophages, only one major cytosolic DNA recognition pathway is operational.
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http://dx.doi.org/10.1002/eji.200939344DOI Listing
July 2009

An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome.

Nat Immunol 2009 Mar 21;10(3):266-72. Epub 2009 Jan 21.

Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

Cytoplasmic DNA triggers activation of the innate immune system. Although 'downstream' signaling components have been characterized, the DNA-sensing components remain elusive. Here we present a systematic proteomics screen for proteins that associate with DNA, 'crossed' to a screen for transcripts induced by interferon-beta, which identified AIM2 as a candidate cytoplasmic DNA sensor. AIM2 showed specificity for double-stranded DNA. It also recruited the inflammasome adaptor ASC and localized to ASC 'speckles'. A decrease in AIM2 expression produced by RNA-mediated interference impaired DNA-induced maturation of interleukin 1beta in THP-1 human monocytic cells, which indicated that endogenous AIM2 is required for DNA recognition. Reconstitution of unresponsive HEK293 cells with AIM2, ASC, caspase-1 and interleukin 1beta showed that AIM2 was sufficient for inflammasome activation. Our data suggest that AIM2 is a cytoplasmic DNA sensor for the inflammasome.
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http://dx.doi.org/10.1038/ni.1702DOI Listing
March 2009

Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets.

Blood 2007 Dec 24;110(12):4055-63. Epub 2007 Aug 24.

Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna.

The BCR-ABL tyrosine kinase inhibitor imatinib represents the current frontline therapy in chronic myeloid leukemia. Because many patients develop imatinib resistance, 2 second-generation drugs, nilotinib and dasatinib, displaying increased potency against BCR-ABL were developed. To predict potential side effects and novel medical uses, we generated comprehensive drug-protein interaction profiles by chemical proteomics for all 3 drugs. Our studies yielded 4 major findings: (1) The interaction profiles of the 3 drugs displayed strong differences and only a small overlap covering the ABL kinases. (2) Dasatinib bound in excess of 30 Tyr and Ser/Thr kinases, including major regulators of the immune system, suggesting that dasatinib might have a particular impact on immune function. (3) Despite the high specificity of nilotinib, the receptor tyrosine kinase DDR1 was identified and validated as an additional major target. (4) The oxidoreductase NQO2 was bound and inhibited by imatinib and nilotinib at physiologically relevant drug concentrations, representing the first nonkinase target of these drugs.
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http://dx.doi.org/10.1182/blood-2007-07-102061DOI Listing
December 2007