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  • PROTEINS  (9)
  • 1
    Keywords: CANCER ; tumor ; CELL ; Germany ; human ; SYSTEM ; SYSTEMS ; DISEASE ; liver ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; MOLECULES ; TISSUE ; TISSUES ; BIOLOGY ; MOLECULE ; IDENTIFICATION ; MEMBRANE ; INSTABILITY ; ELECTROPHORESIS ; pancreatic cancer ; SECTIONS ; molecular biology ; pancreas ; PANCREATIC-CANCER ; EXTRACTION ; SEPARATION ; USA ; protein fractionation ; CELL-CELL ; EFFECTOR MOLECULE ; ISLETS ; protein extraction
    Abstract: Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissue, one of the most challenging tissues for protein isolation because of its high protease content. Success of the different procedures varied greatly. Based on a scheme of tissue-slicing and subsequent cell isolation, we established a reliable workflow for the fractional extraction of cytosolic proteins, membrane and organelle proteins, nuclear proteins, and cytoskeletal filaments. The tissue slices also allow for a representative confirmation of individual samples' cellular status by histochemical processes, and a proper separation or mixing of cellular material from across a tumor if required
    Type of Publication: Journal article published
    PubMed ID: 19450236
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  • 2
    Keywords: brain ; COMBINATION ; Germany ; GENERATION ; SYSTEM ; liver ; PROTEIN ; PROTEINS ; HEART ; COMPLEX ; COMPLEXES ; MECHANISM ; RAT ; PHOSPHORYLATION ; TARGET ; RAT-LIVER ; SUBUNIT ; MEMBRANE ; STRESS ; MODULATION ; MITOCHONDRIA ; OXYGEN ; antioxidants ; PROTEOMICS ; reactive oxygen species ; glutathione-S-transferase ; GENE-EXPRESSION PROFILE ; ageing ; GEL-ELECTROPHORESIS ; assembly ; proteome ; REACTIVE OXYGEN ; ROS ; CALORIE RESTRICTION ; SHORT-TERM ; COMPLEX-I ; MEMBRANE-PROTEINS ; CYTOCHROME-C-OXIDASE ; DIGE ; Species ; ROS PRODUCTION ; SHIFT ; RESPIRATORY-CHAIN
    Abstract: Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such 'putatively hormetic' ageing-analogous effects of CR, besides the age-retarding ones
    Type of Publication: Journal article published
    PubMed ID: 19894137
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  • 3
    Keywords: PROTEINS ; EYE ; LARGE GENE LISTS ; POLYACRYLAMIDE GEL-ELECTROPHORESIS ; PROLIFERATIVE DIABETIC-RETINOPATHY ; AQUEOUS-HUMOR ; PANTHER
    Abstract: Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from "surrogate normal patients" with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.
    Type of Publication: Journal article published
    PubMed ID: 23688336
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  • 4
    Keywords: brain ; RECEPTOR ; CELLS ; Germany ; NETWORKS ; SYSTEM ; TOOL ; DISTINCT ; PROTEIN ; PROTEINS ; TRANSDUCTION ; COMPLEX ; MESSENGER-RNA ; RAT ; signal transduction ; MEMBRANE ; SIGNAL-TRANSDUCTION ; mass spectrometry ; MASS-SPECTROMETRY ; CHROMATOGRAPHY ; PROTEOMIC ANALYSIS ; glutathione-S-transferase ; BINDING PROTEIN ; signaling ; molecular ; NEURONS ; analysis ; cilia ; ENGLAND ; XENOBIOTIC-METABOLIZING ENZYMES ; affinity chromatography ; calcium-calmodulin ; CHEMOSENSORY CILIA ; NUCLEOTIDE-GATED CHANNEL ; olfaction ; olfactory receptor neurons ; PHOSPHOLIPID-BINDING ; SENSORY NEURONS
    Abstract: The olfactory neuroepithelium represents a unique interface between the brain and the external environment. Olfactory function comprises a distinct set of molecular tasks: sensory signal transduction, cytoprotection and adult neurogenesis. A multitude of biochemical studies has revealed the central role of Ca2+ signaling in the function of olfactory receptor neurons (ORNs). We set out to establish Ca2+-dependent signaling networks in ORN cilia by proteomic analysis. We subjected a ciliary membrane preparation to Ca2+/calmodulin-affinity chromatography using mild detergent conditions in order to maintain functional protein complexes involved in olfactory Ca2+ signaling. Thus, calmodulin serves as a valuable tool to gain access to novel Ca2+-regulated protein complexes. Tandem mass spectrometry (nanoscale liquid-chromatography-electrospray injection) identified 123 distinct proteins. Ninety-seven proteins (79%) could be assigned to specific olfactory functions, including 32 to sensory signal transduction and 40 to cytoprotection. We point out novel perspectives for research on the Ca2+-signaling networks in the olfactory system of the rat. (C) 2007 IBRO. Published by Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18155848
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  • 5
    Keywords: CELL ; PROTEINS ; EVOLUTION ; WALL ; VENOM ; BIOLOGICAL ROLES ; CNIDARIAN NEMATOCYST ; DISCHARGE ; MINI-COLLAGENS ; MINICOLLAGENS ; RICH PROTEIN
    Abstract: Stinging cells or nematocytes of jellyfish and other cnidarians represent one of the most poisonous and sophisticated cellular inventions in animal evolution. This ancient cell type is unique in containing a giant secretory vesicle derived from the Golgi apparatus. The organelle structure within the vesicle comprises an elastically stretched capsule (nematocyst) to which a long tubule is attached. During exocytosis, the barbed part of the tubule is accelerated with 〉5 million g in 〈700 ns, enabling a harpoon-like discharge (Nuchter, T., Benoit, M., Engel, U., Ozbek, S., and Holstein, T. W. (2006) Curr. Biol. 16, R316-R318). Hitherto, the molecular components responsible for the organelle's biomechanical properties were largely unknown. Here, we describe the proteome of nematocysts from the freshwater polyp Hydra magnipapillata. Our analysis revealed an unexpectedly complex secretome of 410 proteins with venomous and lytic but also adhesive or fibrous properties. In particular, the insoluble fraction of the nematocyst represents a functional extracellular matrix structure of collagenous and elastic nature. This finding suggests an evolutionary scenario in which exocytic vesicles harboring a venomous secretome assembled a sophisticated predatory structure from extracellular matrix motif proteins.
    Type of Publication: Journal article published
    PubMed ID: 22291027
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  • 6
    Keywords: RECEPTOR ; EXPRESSION ; Germany ; INFORMATION ; SUPPORT ; TOOL ; SITE ; PROTEIN ; PROTEINS ; TRANSDUCTION ; MECHANISM ; primary ; RAT ; mechanisms ; signal transduction ; MEMBRANE ; SIGNAL-TRANSDUCTION ; mass spectrometry ; MASS-SPECTROMETRY ; LOCALIZATION ; RECEPTORS ; FOOD ; protein expression ; PROTEOMIC ANALYSIS ; POLYACRYLAMIDE GELS ; NEURONS ; analysis ; TECHNOLOGY ; EPITHELIUM ; ENGLAND ; enzymatic ; XENOBIOTIC-METABOLIZING ENZYMES ; CHEMOSENSORY CILIA ; NUCLEOTIDE-GATED CHANNEL ; olfactory receptor neurons ; ODORANT RECEPTORS ; SENSITIVE ADENYLATE-CYCLASE ; sensory cilia
    Abstract: The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research
    Type of Publication: Journal article published
    PubMed ID: 18032372
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  • 7
    Keywords: RECEPTOR ; Germany ; PATHWAY ; PROTEIN ; PROTEINS ; COMPLEX ; RAT ; signal transduction ; SIGNAL ; MOUSE ; MEMBRANE ; SIGNAL-TRANSDUCTION ; mass spectrometry ; PROTEOMIC ANALYSIS ; signaling ; NEURONS ; EPITHELIUM ; NUCLEOTIDE-GATED CHANNEL ; olfactory receptor neurons ; sensory cilia ; ADENYLYL-CYCLASE ; CHLORIDE ACCUMULATION ; MAMMALIAN ODORANT RECEPTORS ; ORGANELLAR PROTEOMICS
    Abstract: 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 Ca2+ 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
    Type of Publication: Journal article published
    PubMed ID: 19086097
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  • 8
    Keywords: EXPRESSION ; Germany ; MODEL ; INFORMATION ; SYSTEM ; GENE ; GENE-EXPRESSION ; GENOME ; PROTEIN ; PROTEINS ; RESOLUTION ; MECHANISM ; FAMILY ; DOMAIN ; mechanisms ; TOLERANCE ; CYCLE ; SEQUENCE ; IDENTIFICATION ; gene expression ; HEAT-SHOCK ; mass spectrometry ; SPECTROMETRY ; DATABASE ; MASS-SPECTROMETRY ; PROJECT ; PROTEOMICS ; PROTEIN IDENTIFICATION ; ARABIDOPSIS-THALIANA ; HIGH-RESOLUTION ; ANNOTATION ; SCIENCE ; LIFE ; MOLECULAR-MECHANISMS ; GLUTATHIONE S-TRANSFERASES ; Genetic ; protein extraction ; MILNESIUM-TARDIGRADUM ; RICHTERSIUS-CORONIFER ; ARTEMIA-FRANCISCANA ; DESICCATION TOLERANCE ; EST ; Sequence information ; Molecular mechanisms ; BRINE SHRIMP ; TREHALOSE
    Abstract: Background: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. Principal Findings: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. Conclusions: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades
    Type of Publication: Journal article published
    PubMed ID: 20224743
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  • 9
    Keywords: EXPRESSION ; PROTEINS ; BIOMARKERS ; Drosophila ; TUMOR-SUPPRESSOR GENE ; SIGNALING PATHWAY ; beta-catenin ; ADHESION MOLECULE ; SERUM ; PROTOCADHERIN FAT1
    Abstract: In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.
    Type of Publication: Journal article published
    PubMed ID: 24625754
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