Kooperativer Bibliotheksverbund

Berlin Brandenburg


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  • 1
    In: Protein Science, October 2015, Vol.24(10), pp.1686-1694
    Description: Protein‐linked glycans play key roles in cell differentiation, cell–cell interactions, cell growth, adhesion and immune response. Aberrant glycosylation is a characteristic feature of tumor cells and is involved in tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. It can serve as cancer biomarker and treatment target. To enable comprehensive screening for the impact of tumor driving mutations in colorectal cancer cells we present a method for specific analysis of tumor driver‐induced glycome changes. The strategy is based on a combination of three technologies, that is recombinase‐mediated cassette exchange (RMCE), Click‐It chemistry and mass spectrometry. The new method is exemplified by the analysis of the impact of inactivating mutations of the TGF‐ß‐receptor type II (TGFBR2) on sialic acid incorporation into protein‐linked glycans of the colon cancer cell line HCT116. Overall, 70 proteins were found to show sialic acid incorporation exclusively upon TGFBR2 expression whereas 7 proteins lost sialylation upon TGFBR2 reconstitution. Validation of detected candidate glycoproteins is demonstrated with the cell surface glycoprotein nectin‐3 known to be involved in metastasis, invasion and prognosis of various cancers. Altogether, our new approach can help to systematically puzzle out the influence of tumor‐specific mutations in a major signaling pathway, as exemplified by the TGFBR2 tumor suppressor, on the tumor glycome. It facilitates the identification of glycan‐based tumor markers that could be used for diagnostic and therapeutic applications. In principle the outlined strategy can be adapted to any cancer cell line, tumor driver mutation and several glycan‐building blocks.
    Keywords: Colorectal Cancer ; Microsatellite Instability ; Sialylation ; Tgfbr2 ; Nectin‐3
    ISSN: 0961-8368
    E-ISSN: 1469-896X
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  • 2
    Language: English
    In: IUBMB life, March 2019, Vol.71(3), pp.364-375
    Description: Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.
    Keywords: Glutamine Transporter ; Colorectal Cancer ; Galectin-4 ; Growth-Inhibition ; Phosphoproteome ; Tumor Suppressor
    ISSN: 15216543
    E-ISSN: 1521-6551
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  • 3
    Language: English
    In: Journal of Zoological Systematics and Evolutionary Research, 05/2011, Vol.49, supplement s1, pp.111-119
    Description: The eutardigrade Milnesium tardigradum can undergo cryptobiosis and can survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood. Heat shock proteins (Hsps) as an important subgroup of chaperones which protect proteins from irreversible aggregation and degradation might play an important role in anhydrobiosis. In this report, we therefore investigated Hsps in tardigrades in the active versus the anhydrobiotic state employing proteomics techniques. Protein lysates from tardigrades in both states were separated by one-dimensional gel electrophoresis, in-gel digested with trypsin and tryptic peptides were analyzed by high sensitive nanoLC-ESI-MS/MS on a LTQ-Orbitrap mass spectrometer. This study indicates the presence of Hsps of the major chaperone families Hsp40, Hsp60, Hsp70, Hsp90, small Hsps and furthermore nucleotide exchange factors and co-chaperones in Milnesium tardigradum. A comparative analysis of the identified Hsps in both states was performed by calculating the exponentially modified protein abundance index.Original Abstract: Tardigraden haben die ausergewohnliche Faehigkeit, extreme Stress-Bedingungen zu ueberleben, indem sie Kryptobiose eingehen. Dabei aendern sie ihre Korperform, ziehen sich zusammen und bilden ein Tonnchen, in dem keinerlei Metabolismus mehr nachweisbar ist. Obwohl Tardigraden seit ca. 300 Jahren bekannt sind, sind die molekularen Mechanismen waehrend der Kryptobiose aufgrund fehlender fundamentaler Untersuchungen noch unklar. Eine wichtige Proteinklasse, die beim Ueberleben der Tardigraden waehrend der Stress-Situation moglicherweise eine grose Rolle spielt, sind die Hitzeschockproteine. Hitzeschockproteine (Stressproteine, Hsps) sind ubiquitaere, hoch konservierte Chaperone, die die Aufrechterhaltung der Proteine in ihrer funktionellen Konfirmation bewirken, indem sie ihre Aggregation verhindern. Waehrend die Anwesenheit der Hitzeschockproteine in der Zelle unter normalen Bedingungen erforderlich ist, ist ihre Aktivitaet bei Hitze oder in Stress-Situationen zum Ueberleben absolut notwendig. In unserer Studie haben wir die Hitzeschockproteine in Milnesium tardigradum unter Verwendung von Proteomics-Techniken identifiziert. Dazu wurde die eindimensionale Gelelektrophorese gefolgt von der hochsensitiven Analyse der nach Trypsin-Verdau erhaltenen Peptide durch nanoLC-ESI-MS/MS auf einem LTQ-Orbitrap-Massenspektrometer eingesetzt. Proteine aus diversen Hitzeschock-Proteinfamilien Hsp40, Hsp60, Hsp70 und Hsp90 sowie kleine Hitzeschockproteine (Hsp20) und zusaetzlich Co-Chaperone wie GrpE konnten durch MS/MS Analyse und Datenbank-Suche gegen die NCBInr- sowie eine neu zusammengestellte Proteindatenbank aus EST-Sequenzen von Milnesium tardigradum nachgewiesen werden. Auserdem konnten durch eine semiquantitative Methode die identifizierten Hitzeschockproteine in Tardigraden im aktiven und anhydrobiotischen Zustand verglichen werden.
    Keywords: Proteins ; Heat Shock ; Peptides ; Environmental Conditions ; Nucleotides ; Milnesium Tardigradum ; Population Structure;
    ISSN: Journal of Zoological Systematics and Evolutionary Research
    E-ISSN: 09475745
    E-ISSN: 14390469
    Source: Wiley (via CrossRef)
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  • 4
    In: Hepatology, November 2018, Vol.68(5), pp.1817-1832
    Description: The identification of viability‐associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability‐associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up‐regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)–mediated and siRNA pool–mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss‐of‐viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9‐depleted and HNRNPL‐depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient‐derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Taken together, we provide a comprehensive list of viability‐associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability‐associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.
    Keywords: Chorioallantoic Membrane ; RNA-Mediated Interference ; Hepatocellular Carcinoma ; L Form ; Crispr ; Liver Cancer ; Apoptosis ; Cell Culture ; RNA-Binding Protein ; Immunoprecipitation ; DNA Damage ; Phenotypes ; Protein Purification ; DNA Damage ; Cell Lines ; Sirna ; Tumor Cell Lines ; Liver Cancer ; Akt Protein ; Xenografts ; Proteins;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
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  • 5
    Language: English
    In: PROTEOMICS, January 2009, Vol.9(2), pp.322-334
    Description: Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction cascade which uses cAMP and Ca for sensory signaling in the ciliary lumen. Although the canonical transduction pathway is well established, molecular components for more complex aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response have not been systematically identified. Moreover, open questions in olfactory physiology include how the cilia exchange solutes with the surrounding mucus, assemble their highly polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary proteome would promote research efforts in all of these fields. We have improved a method to detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched in ciliary membrane proteins. Using LC‐ESI‐MS/MS analysis, we identified 377 proteins which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set for olfactory research since more than 80% can be attributed to the characteristic functions of olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information about the diverse physiological functions of a sensory organelle.
    Keywords: Mass Spectrometry ; Olfactory Receptor Neurons ; Proteomic Analysis ; Sensory Cilia ; Signal Transduction
    ISSN: 1615-9853
    E-ISSN: 1615-9861
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