Initial characterization of the human central proteome

<p>Abstract</p> <p>Background</p> <p>On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses.</p> <p>Results</p> <p>We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation.</p> <p>Conclusions</p> <p>Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.</p

My contemporaries in 20^<th> century.

Original PCR screens for CD74-ROS1 translocation and BCR-ABL1 reversion (only results for 96 clones are shown for each rearrangement). (A) PCR screen on gDNA for clones harbouring CD74-ROS1 and ROS1-CD74 rearrangements. Asterisk indicates positive clone. (B) PCR screen on gDNA for clones harbouring repaired BCR and ABL1 genes. (PPTX 1609 kb

The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response

TANK-binding kinase 1 (TBK1) is of central importance for the induction of type-I interferon (IFN) in response to pathogens. We identified the DEAD-box helicase DDX3X as an interaction partner of TBK1. TBK1 and DDX3X acted synergistically in their ability to stimulate the IFN promoter, whereas RNAi-mediated reduction of DDX3X expression led to an impairment of IFN production. Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction

Identifizierung zellulärer Zielproteine des Hepatitis C-Virus Nichtstrukturproteins 5A.

Title page and table of contents Chapter 1: Introduction Chapter 2: Aim of the study Chapter 3: Materials Chapter 4: Methods Chapter 5: Results Chapter 6: Discussion Chapter 7: Summary Chapter 8: Zusammenfassung (deutsch) Chapter 9: Literature Chapter 10: AbbreviationsHepatitis C virus (HCV) is a small RNA virus that causes severe liver pathogenesis in humans. It encodes for 3 structural and at least 7 non- structural proteins. The non-structural protein 5A (NS5A) has been implicated in the deregulation of host cell signal transduction, interfering with signaling pathways that regulate cell growth, cell proliferation and cell survival. Modulation of these signaling pathways is of potential interest with regard to viral replication, immune evasion and virus-associated liver pathogenesis. Therefore, this study was focused on the identification of cellular targets of NS5A that might trigger the NS5A-mediated deregulation of host cell signal transduction. Using an affinity chromatography approach, c-Raf1 was identified as a novel binding partner of NS5A. Binding was confirmed by immunoprecipitation using baculovirus-infected Sf21 cells. NS5A and c-Raf1 were found to colocalize in transiently-transfected HuH-7 cells and HCV replicon cells. Furthermore, deletion mutants of NS5A that are localized in the nucleus cause the nuclear translocation of endogenous c-Raf1. This demonstrates that NS5A and c-Raf1 interact in a physiologically relevant model system, ie. in human hepatoma cells. Next, it was analyzed how modulation of c-Raf1 activity affects HCV replication. Inhibition of c-Raf1 by the means of a small-molecule inhibitor (BAY43-9006) lead to a decrease in HCV replication. Reducing c-Raf1 expression by the means of siRNA lead to a similar reduction of HCV replication, arguing that integrity of c-Raf1 is required for viral replication. Disruption of the MAP kinase cascade at the level of MEK (U0126) did not affect viral replication. These data suggest that the c-Raf1-mediated reduction of HCV replication is not due to the disruption of the MAP kinase cascade. Furthermore, these data highlight the exclusive role of c-Raf1 in viral replication. In the following set of experiments, the impact of NS5A on c-Raf1-mediated signal transduction was investigated. Surprisingly, the level of ERK phosphorylation was not affected by the presence of NS5A. Moreover, NS5A did not affect the level of basal activation of SRF, AP-1, NF-κB or STAT3 � either if expressed as an individual protein or if expressed in the HCV polyprotein context. This argues that NS5A does not modulate c-Raf1-mediated signal transduction. Since NS5A is a phosphoprotein and c-Raf1 is a kinase, the impact of c-Raf1 on NS5A phosphorylation was analyzed. To that end, both NS5A and GST-Raf were purified by affinity chromatography. Phosphorylation of NS5A by GST-Raf was reconstituted in vitro using highly purified GST-Raf. An inactive mutant of GST-Raf (K375W) did not phosphorylate NS5A, arguing that phosphorylation of NS5A was specific on behalf of c-Raf1. This suggests that c-Raf1 is an NS5A kinase in vitro. Moreover, c-Raf1 inhibition by the means of BAY43-9006 lead to a decrease in NS5A hyperphosphorylation in HCV replicon cells, arguing that c-Raf1 contributes to NS5A phosphorylation in vivo. In conclusion, this study describes a novel cellular binding partner of HCV NS5A that is essential for NS5A phosphorylation and HCV replication. Among the multitude of binding partners described for NS5A, there are few binding partners that fit these criteria and provide an impact on understanding this exciting, though enigmatic protein.Das Hepatitis-C Virus (HCV) ist ein kleines RNA-Virus, das beim Menschen schwere Lebererkrankungen hervorruft. Es kodiert für 3 Struktur- und mindestens 7 Nichtstrukturproteine. Das Nichtstrukturprotein 5A (NS5A) moduliert verschiedene Signaltransduktionswege der Wirtszelle, die für die Regulation von Zellwachstum und Zellproliferation von zentraler Bedeutung sind. Die Deregulation dieser Signalwege ist potenziell relevant im Hinblick auf die Virusreplikation, die Immunantwort des Wirts sowie die HCV-assoziierte Leberpathogenese. Aus diesem Grund war das Ziel der vorliegenden Arbeit, zelluläre Faktoren zu identifizieren, die für die NS5A-vermittelte Modulation zellulärer Signalwege verantwortlich sind. Mit Hilfe eines affinitätschromatographischen Ansatzes wurde c-Raf1 als neuer Bindungspartner von NS5A identifiziert. Die Interaktion wurde in Immunpräzipitationsexperimenten aus Baculovirus-infizierten Sf21-Zellen bestätigt. Des Weiteren wurde beobachtet, dass NS5A und c-Raf1 in transient- transfizierten HuH-7-Zellen sowie in HCV-Replikon-Zellen kolokalisiert sind. N-terminale Deletionsmutanten von NS5A, die im Zellkern lokalisiert sind, bewirken eine Translokation von endogenem c-Raf1 in den Kern. Das zeigt, dass NS5A und c-Raf1 in einem physiologisch-relevanten Modellsystem interagieren. In der Folge wurde untersucht, wie sich eine Modulation der c-Raf1-Aktivität auf die Virusreplikation auswirkt. Die Hemmung von c-Raf1 mittels eines synthetischen Inhibitors (BAY43-9006) führte zu einer Verminderung der HCV- Replikation. Dasselbe wurde beobachtet, wenn die Expression von c-Raf1 durch siRNAs reduziert wurde. Das legt den Schluss nahe, dass die Integrität von c-Raf1 eine Voraussetzung für die Virusreplikation darstellt. Die Hemmung der MAP-Kinase-Kaskade auf der Ebene von MEK (U0126) hatte keinen Einfluss auf die Virusreplikation. Das bedeutet, dass der Effekt von c-Raf1 auf die Virusreplikation nicht durch eine Hemmung der MAP-Kinase-Kaskade bedingt ist. Im Übrigen unterstreicht dieses Experiment die besondere Bedeutung von c-Raf1 für die Virusreplikation. Außerdem wurde analysiert, inwieweit NS5A einen Einfluss auf die c-Raf1-vermittelte Signaltransduktion hat. Dabei wurde beobachtet, dass die Phosphorylierung von ERK, einem zentralen Effektor von c-Raf1, in Gegenwart von NS5A unverändert ist. Des Weiteren trägt NS5A nicht zur basalen Aktivierung der Transkriptionsfaktoren SRF, AP-1, NF-κB oder STAT3 bei. Dies gilt sowohl für NS5A als isoliertes Protein als auch für NS5A im Kontext des HCV-Polyproteins. Das legt den Schluss nahe, dass NS5A keinen Einfluss auf die c-Raf1-vermittelte Signaltransduktion hat. Da NS5A ein Phosphoprotein und c-Raf1 eine Kinase ist, war es nahe liegend zu untersuchen, inwieweit NS5A durch c-Raf1 phosphoryliert werden kann. Zu diesem Zweck wurden NS5A und GST-Raf durch Affinitätschromatographie gereinigt. Die gereinigten Proteine wurden in einem Kinase-Assay umgesetzt. Dabei konnte gezeigt werden, dass hoch-reines GST-Raf NS5A phosphoryliert. Eine inaktive Mutante von GST- Raf (K375W) war nicht in der Lage, NS5A zu phosphorylieren. Das bedeutet, dass die Phosphorylierung im Hinblick auf c-Raf1 spezifisch war und lässt den Schluss zu, dass c-Raf1 eine NS5A-Kinase ist. In der Folge wurde untersucht, wie sich eine Inhibition von c-Raf1 (durch BAY43-9006) auf die Hyperphosphorylierung von NS5A auswirkt. Es wurde beobachtet, dass die Inhibition von c-Raf1 den Verlust der Hyperphosphorylierung von NS5A zur Folge hat. Das bedeutet, dass c-Raf1 die NS5A-Phosphorylierung auch in vivo beeinflusst. Zusammenfassend kann gesagt werden, dass in dieser Arbeit ein neuer zellulärer Bindungspartner von NS5A identifiziert wurde, der von zentraler Bedeutung für die Phosphorylierung von NS5A und die Virusreplikation ist. Unter den zahlreichen Bindungspartnern von NS5A gibt es nur wenige, die diese Kriterien erfüllen und die zum Verständnis dieses spannenden und zugleich rätselhaften Proteins beitragen

Additional file 5: Figure S5. of Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology

Sequences of gDNA junctions. (A) gDNA sequence of CD74-ROS1 fusion junction in 1G13 clone. Red rectangle indicates deletion. (B) gDNA sequence of ROS1-CD74 fusion junction in 1G13 clone. Red rectangle indicates insertion. (C) Repaired genomic sequence of BCR gene in 4 L20 clone. (D) Repaired genomic sequences of ABL1 gene in 4 L20 clone. (PPTX 314 kb

Additional file 3: Figure S3. of Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology

Primers used for gDNA and cDNA amplification. (PPTX 38 kb

Additional file 2: Figure S2. of Efficient generation and reversion of chromosomal translocations using CRISPR/Cas technology

Original PCR screens for CD74-ROS1 translocation and BCR-ABL1 reversion (only results for 96 clones are shown for each rearrangement). (A) PCR screen on gDNA for clones harbouring CD74-ROS1 and ROS1-CD74 rearrangements. Asterisk indicates positive clone. (B) PCR screen on gDNA for clones harbouring repaired BCR and ABL1 genes. (PPTX 1609 kb

Distinct interaction of human and guinea pig histamine H_2-Receptor with guanidine-type agonists,

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H2-receptor (H2R) agonists are lower at the H2R of human neutrophils than at the H2R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H2R (hH2R) and guinea pig H2R (gpH2R), respectively, and the short splice variant of Gsalpha (Gsalpha S) expressed in Sf9 cells. The potencies and efficacies of small H2R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH2R-Gsalpha S and gpH2R-Gsalpha S were similar. In contrast, the potencies and efficacies of guanidines were lower at hH2R-Gsalpha S than at gpH2R-Gsalpha S. Guanidines bound to hH2R-Gsalpha S with lower affinity than to gpH2R-Gsalpha S, and high-affinity binding of guanidines at gpH2R-Gsalpha S was more resistant to disruption by GTPgamma S than binding at hH2R-Gsalpha S. Molecular modeling suggested that the nonconserved Asp-271 in transmembrane domain 7 of gpH2R (Ala-271 in hH2R) confers high potency to guanidines. This hypothesis was confirmed by Ala-271right-arrowAsp-271 mutation in hH2R-Gsalpha S. Intriguingly, the efficacies of guanidines at the Ala-271right-arrowAsp-271 mutant and at hH2R/gpH2R chimeras were lower than at gpH2R. Our model suggests that a Tyr-17/Asp-271 H-bond, present only in gpH2R-Gsalpha S but not the other constructs studied, stabilizes the active guanidine-H2R state. Collectively, our data show 1) distinct interaction of H2R species isoforms with guanidines, 2) that a single amino acid in transmembrane domain 7 critically determines guanidine potency, and 3) that an interaction between transmembrane domains 1 and 7 is important for guanidine efficacy

An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors