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  • 1
    Keywords: SIGNAL-TRANSDUCTION ; MAMMALIAN-CELLS ; LIPID RAFTS ; SINGLE-CELL ; reverse transfection ; PROTEIN PALMITOYLATION ; FAMILY KINASES ; CONTENT SCREENING MICROSCOPY ; AUTOMATIC IDENTIFICATION ; COATOMER COMPLEXES
    Abstract: SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.
    Type of Publication: Journal article published
    PubMed ID: 21795383
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  • 2
    Keywords: CELLS ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; PROTECTION ; CELL ; Germany ; PATHWAY ; VITRO ; SYSTEM ; DISTINCT ; PROTEIN ; PROTEINS ; CONTRAST ; BIOLOGY ; PLASMA ; MEMBRANE ; FIBROBLAST-GROWTH-FACTOR ; EXTRACELLULAR-MATRIX ; ATP ; MAMMALIAN-CELLS ; MIGRATION ; MEMBRANE PROTEIN ; ENDOPLASMIC-RETICULUM ; structural protein ; MEMBRANE-PROTEIN ; BINDING CASSETTE TRANSPORTER ; COPI-COATED VESICLES ; INNER MEMBRANES ; INTERCELLULAR TRAFFICKING ; MIGRATION INHIBITORY FACTOR ; RETICULUM-GOLGI-COMPLEX ; SIGNAL SEQUENCE
    Abstract: Fibroblast growth factor 2 (FGF-2) is a pro-angiogenic mediator that is secreted by both normal and neoplastic cells. Intriguingly, FGF-2 has been shown to be exported by an endoplasmic reticulum/Golgi-independent pathway; however, the molecular machinery mediating this process has remained elusive. Here we introduce a novel in vitro system that functionally reconstitutes FGF-2 secretion. Based on affinity-purified plasma membrane inside-out vesicles, we demonstrate post-translational membrane translocation of FGF-2 as shown by protease protection experiments. This process is blocked at low temperature but apparently does not appear to be driven by ATP hydrolysis. FGF-2 membrane translocation occurs in a unidirectional fashion requiring both integral and peripheral membrane proteins. These findings provide direct evidence that FGF-2 secretion is based on its direct translocation across the plasma membrane of mammalian cells. When galectin-1 and macrophage migration inhibitory factor, other proteins exported by unconventional means, were analyzed for translocation into plasma membrane inside-out vesicles, galectin-1 was found to be transported as efficiently as FGF-2. By contrast, migration inhibitory factor failed to traverse the membrane of inside-out vesicles. These findings establish the existence of multiple distinct secretory routes that are operational in the absence of a functional endoplasmic reticulum/Golgi system
    Type of Publication: Journal article published
    PubMed ID: 14645213
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  • 3
    Keywords: CELL-PROLIFERATION ; OXIDATIVE STRESS ; RAT-BRAIN ; MEMBRANE-FUSION ; TRANSMITTER RELEASE ; unfolded protein response ; GUINEA-PIG ; GLUTAMATE RELEASE ; PEROXISOMAL DISORDERS ; RAPID METHOD
    Abstract: Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet., 12, 18811895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca-2-dependent exocytosis and Ca-2-independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na, K and Ca-2 homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca-2-independent efflux of NTs increases while the Ca-2-dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum
    Type of Publication: Journal article published
    PubMed ID: 22403185
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  • 4
    Abstract: Congenital disorders of glycosylation (CDG) are genetic defects in the glycoconjugate biosynthesis. 〉100 types of CDG are known, most of them cause multi-organ diseases. Here we describe a boy whose leading symptoms comprise cutis laxa, pancreatic insufficiency and hepatosplenomegaly. Whole exome sequencing identified the novel hemizygous mutation c.542T〉G (p.L181R) in the X-linked ATP6AP1, an accessory protein of the mammalian vacuolar H(+)-ATPase, which led to a general N-glycosylation deficiency. Studies of serum N-glycans revealed reduction of complex sialylated and appearance of truncated diantennary structures. Proliferation of the patient's fibroblasts was significantly reduced and doubling time prolonged. Additionally, there were alterations in the fibroblasts' amino acid levels and the acylcarnitine composition. Especially, short-chain species were reduced, whereas several medium- to long-chain acylcarnitines (C14-OH to C18) were elevated. Investigation of the main lipid classes revealed that total cholesterol was significantly enriched in the patient's fibroblasts at the expense of phophatidylcholine and phosphatidylethanolamine. Within the minor lipid species, hexosylceramide was reduced, while its immediate precursor ceramide was increased. Since catalase activity and ACOX3 expression in peroxisomes were reduced, we assume an ATP6AP1-dependent impact on the beta-oxidation of fatty acids. These results help to understand the complex clinical characteristics of this new patient.
    Type of Publication: Journal article published
    PubMed ID: 29396028
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  • 5
    Publication Date: 2012-01-11
    Description: Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Contreras, F-Xabier -- Ernst, Andreas M -- Haberkant, Per -- Bjorkholm, Patrik -- Lindahl, Erik -- Gonen, Basak -- Tischer, Christian -- Elofsson, Arne -- von Heijne, Gunnar -- Thiele, Christoph -- Pepperkok, Rainer -- Wieland, Felix -- Brugger, Britta -- 232648/European Research Council/International -- England -- Nature. 2012 Jan 9;481(7382):525-9. doi: 10.1038/nature10742.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/22230960" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Binding Sites ; CHO Cells ; COP-Coated Vesicles/metabolism ; Cell Membrane/*metabolism ; Computational Biology ; Conserved Sequence ; Cricetinae ; Membrane Proteins/*chemistry/*metabolism ; Models, Molecular ; Molecular Sequence Data ; Protein Binding ; Protein Multimerization ; Protein Structure, Tertiary ; Protein Transport ; Second Messenger Systems/physiology ; Sphingolipids/*metabolism ; Sphingomyelins/metabolism ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 6
    Publication Date: 2012-05-26
    Description: Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Faini, Marco -- Prinz, Simone -- Beck, Rainer -- Schorb, Martin -- Riches, James D -- Bacia, Kirsten -- Brugger, Britta -- Wieland, Felix T -- Briggs, John A G -- New York, N.Y. -- Science. 2012 Jun 15;336(6087):1451-4. doi: 10.1126/science.1221443. Epub 2012 May 24.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/22628556" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; COP-Coated Vesicles/*chemistry/*ultrastructure ; Coat Protein Complex I/*chemistry ; Coatomer Protein/*chemistry ; Cryoelectron Microscopy ; Electron Microscope Tomography ; Image Processing, Computer-Assisted ; Mice ; Models, Molecular ; Protein Conformation
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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