14 results on '"Martin Vey"'
Search Results
2. Assembly of SIV Virus-like Particles Containing Envelope Proteins Using a Baculovirus Expression System
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Richard W. Compans, Martin Vey, Galina V. Yamshchikov, and G. Douglas Ritter
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viruses ,Genetic Vectors ,Molecular Sequence Data ,Gene Products, gag ,Sf9 ,HIV Envelope Protein gp120 ,Spodoptera ,Biology ,Gp41 ,Cleavage (embryo) ,Virus ,Cell Line ,law.invention ,Viral Envelope Proteins ,law ,Virology ,Polyhedrin ,Animals ,Subtilisins ,Protein Precursors ,Furin ,DNA Primers ,Membrane Glycoproteins ,Base Sequence ,Virus Assembly ,Virion ,Gene Products, env ,virus diseases ,Molecular biology ,Nucleopolyhedroviruses ,Capsid ,Recombinant DNA ,biology.protein ,Simian Immunodeficiency Virus - Abstract
The requirements for SIV particle assembly and envelope incorporation were investigated using a baculovirus expression system. The Pr56gagprecursor protein expressed under control of the polyhedrin promoter (pPolh) produced high levels of immature retrovirus-like particles (VLP) upon expression in Sf9 insect cells. To determine the optimal conditions for envelope protein (Env) incorporation into VLP, two recombinant baculoviruses expressing the SIV envelope protein under control of a very late pPolh or a hybrid late/very late capsid/polyhedrin (Pcap/polh) promoter and a recombinant expressing a truncated form of the SIV envelope protein (Envt) under the hybrid Pcap/polh promoter were compared. We have observed that utilization of the earlier hybrid promoter resulted in higher levels of Env expression on the cell surface and its incorporation into budding virus particles. We have also found that the Envtprotein is transported to the cell surface of insect cells and incorporated into VLP more efficiently than full-length Env. In addition, we examined the effect of coexpression of the protease furin, which has been implicated in the proteolytic cleavage of the Env precursor gp160 in mammalian cells. Coexpression of furin in insect cells resulted in more efficient proteolytic cleavage into gp120 and gp41, and the cleaved proteins were incorporated into VLP.
- Published
- 1995
3. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin
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J. Seiler, Hans-Dieter Klenk, Martin Vey, Susanne Berghöfer, Annemarie Stroh, M.L. Kruse, Wolfram Schäfer, Wolfgang Garten, and H.F. Kern
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Signal peptide ,Cytoplasm ,animal structures ,Endosome ,Recombinant Fusion Proteins ,viruses ,Molecular Sequence Data ,DNA, Recombinant ,Golgi Apparatus ,Hemagglutinins, Viral ,Hemagglutinin Glycoproteins, Influenza Virus ,Endosomes ,Biology ,Transfection ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,symbols.namesake ,Animals ,Amino Acid Sequence ,Subtilisins ,Microscopy, Immunoelectron ,Molecular Biology ,Integral membrane protein ,Peptide sequence ,Furin ,Base Sequence ,General Immunology and Microbiology ,General Neuroscience ,Constitutive secretory pathway ,Golgi apparatus ,Proprotein convertase ,Rats ,Biochemistry ,Mutation ,embryonic structures ,symbols ,biology.protein ,Cattle ,Research Article ,Signal Transduction ,Subcellular Fractions - Abstract
Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.
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- 1995
4. Endoproteolytic Cleavage of Its Propeptide Is a Prerequisite for Efficient Transport of Furin Out of the Endoplasmic Reticulum
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Hans-Dieter Klenk, Wolfram Schäfer, John W.M. Creemers, Anton J.M. Roebroek, Martin Vey, Wolfgang Garten, Torik A.Y. Ayoubi, and Wim J.M. Van de Ven
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Glycosylation ,animal structures ,Swine ,viruses ,Molecular Sequence Data ,Endoplasmic Reticulum ,Cleavage (embryo) ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,symbols.namesake ,Animals ,Humans ,Amino Acid Sequence ,Subtilisins ,Protein Precursors ,Protein precursor ,Molecular Biology ,Furin ,DNA Primers ,Binding Sites ,Base Sequence ,biology ,Hydrolysis ,Endoplasmic reticulum ,Biological Transport ,ER retention ,Cell Biology ,Golgi apparatus ,Proprotein convertase ,chemistry ,Mutation ,embryonic structures ,symbols ,biology.protein ,Cattle ,Peptides ,Protein Processing, Post-Translational - Abstract
The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1 + P4 + P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.
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- 1995
5. Proteolytic processing of human cytomegalovirus glycoprotein B (gpUL55) is mediatedby the human endoprotease furin
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H.-D. Klenk, R. Ohuchi, W. Schäfer, K. Radsak, W. Garten, Martin Vey, William J. Britt, and B. Reis
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Human cytomegalovirus ,Cytomegalovirus ,Biology ,Cleavage (embryo) ,law.invention ,chemistry.chemical_compound ,Endoglycosidase H ,Viral Envelope Proteins ,law ,Virology ,medicine ,Humans ,Subtilisins ,Furin ,Cells, Cultured ,chemistry.chemical_classification ,Hydrolysis ,Fibroblasts ,medicine.disease ,Molecular biology ,Amino acid ,chemistry ,Human Cytomegalovirus Glycoprotein B ,biology.protein ,Recombinant DNA ,Vaccinia ,Protein Processing, Post-Translational - Abstract
Inhibition of endoproteolytic cleavage of glycoprotein B (gB; gpUL55) of human cytomegalovirus was achieved by treatmentof infected fibroblasts with decanoyl peptidyl chloromethyl ketone (decRVKR-CMK), which inhibits the action of cellular subtilisin-like endoproteases with the amino acid recognition motif R × K/R R. Uncleaved gB precusor molecules of 160 kDa that were accumulated were endoglycosidase H resistant, suggesting that correct cellular transport occurred in the presence of the drug. The inhibitor also prevented endoproteolytic gB processing in CV-1 cells infected with a recombinant vaccinia virus-gB construct (VVgB). Evidence for direct involvement of the ubiquitous subtilisin-like endoprotease furin in gB cleavage was obtained from the observation that coinfection of CV-1 cells with WgB and a recombinant vaccinia-human furin construct reestablished endoproteolytic activity which was normally absent late after infection with WgB alone.
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- 1995
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6. Rescue of vector-expressed fowl plague virus hemagglutinin in biologically active form by acidotropic agents and coexpressed M2 protein
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A Cramer, Wolfgang Garten, Hans-Dieter Klenk, Martin Vey, Reiko Ohuchi, and Masanobu Ohuchi
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Molecular Sequence Data ,Immunology ,Orthomyxoviridae ,Fluorescent Antibody Technique ,Hemagglutinins, Viral ,Hemagglutinin Glycoproteins, Influenza Virus ,Simian virus 40 ,medicine.disease_cause ,Microbiology ,Ammonium Chloride ,Virus ,Cell Fusion ,Viral Matrix Proteins ,Virology ,Chlorocebus aethiops ,Influenza A virus ,medicine ,Animals ,Cells, Cultured ,Cell fusion ,Viral matrix protein ,Base Sequence ,biology ,Membrane Proteins ,Biological Transport ,Biological activity ,Hydrogen-Ion Concentration ,biology.organism_classification ,Trypsin ,Recombinant Proteins ,Cell biology ,Biochemistry ,Insect Science ,Viral Fusion Proteins ,Intracellular ,Research Article ,medicine.drug - Abstract
The hemagglutinin of the Rostock strain of fowl plague virus was expressed in CV-1 cells by a simian virus 40 vector, and its stability in the exocytotic transport process was examined by a fusion assay. A 50-fold increase in the fusion activity of the hemagglutinin was observed when expression occurred in the presence of ammonium chloride, Tris-HCl, or high doses of amantadine. When chloroquine, another acidotropic agent, was used, the hemagglutinin exposed at the cell surface had to be activated by trypsin, because intracellular cleavage was inhibited by this compound. Hemagglutinin mutants resistant to intracellular cleavage did not require acidotropic agents for full expression of fusion activity, when treated with trypsin after arrival at the cell surface. These results indicate that fowl plague virus hemagglutinin expressed by a simian virus 40 vector is denatured in the acidic milieu of the exocytotic pathway and that cleavage is a major factor responsible for the pH instability. Coexpression with the M2 protein also markedly enhanced the fusion activity of the hemagglutinin, and this effect was inhibited by low doses of amantadine. These results support the concept that M2, known to have ion channel function, protects the hemagglutinin from denaturation by raising the pH in the exocytotic transport system. The data also stress the importance of acidotropic agents or coexpressed M2 for the structural and functional integrity of vector-expressed hemagglutinin.
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- 1994
7. Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation
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Hans-Dieter Klenk, Martin Vey, Wolfgang Garten, Wolfram Schäfer, and Susanne Berghöfer
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animal structures ,Time Factors ,medicine.medical_treatment ,viruses ,Molecular Sequence Data ,Golgi Apparatus ,Cleavage (embryo) ,Endoplasmic Reticulum ,Models, Biological ,Endoglycosidase H ,symbols.namesake ,chemistry.chemical_compound ,Species Specificity ,medicine ,Animals ,Humans ,Secretion ,Amino Acid Sequence ,Disulfides ,Subtilisins ,Protein Precursors ,Protein precursor ,Furin ,Cells, Cultured ,Protease ,biology ,Base Sequence ,Biological Transport ,Cell Biology ,Articles ,Golgi apparatus ,Brefeldin A ,Hydrogen-Ion Concentration ,N-Acetylneuraminic Acid ,Cell Compartmentation ,Biochemistry ,chemistry ,embryonic structures ,biology.protein ,symbols ,Sialic Acids ,Calcium ,Cattle ,Protein Processing, Post-Translational - Abstract
We have cloned a bovine cDNA encoding the trans-Golgi network (TGN) protease furin and expressed it via recombinant vaccinia viruses to investigate intracellular maturation. Pulse-chase labeling reveals that the 104-kD pro-furin bearing high mannose N-glycans is rapidly processed into the 98-kD protease whose N-glycans remain sensitive to endoglycosidase H for a certain period of time. Furthermore, in the presence of brefeldin A, pro-furin cleavage occurs. From these data we conclude that the ER is the compartment of propeptide removal. Studies employing the ionophore A23187 and DTT show that autocatalysis is Ca2+ dependent and that it does not occur under reducing conditions. Pro-furin produced under these conditions never gains endo H resistance indicating that it is retained in the ER. Coexpression of furin with the fowl plague virus hemagglutinin in the presence of brefeldin A and monensin reveals that furin has to enter the Golgi region to gain substrate cleaving activity. N-glycans of furin are sialylated proving its transit through the trans-Golgi network. A truncated form of furin is found in supernatants of cells. Truncation is inhibited in the absence of Ca2+ ions and in the presence of acidotropic agents indicating that it takes place in an acidic compartment of cells. Comparative analysis with furin expressed from cDNA reveals that the truncated form prevails in preparations of biologically active, endogenous furin obtained from MDBK cells. This observation supports the concept that secretion of truncated furin is a physiological event that may have important implications for the processing of extracellular substrates.
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- 1994
8. The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes
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Bill Yajima, Roman Jerala, Iva Hafner-Bratkovič, Graciela Garen, Adriano Aguzzi, Simone Hornemann, Christina J. Sigurdson, Yong-zhong Shi, Nat N. V. Kav, Rita Moos, Martin Vey, Anne Bellon, Michael N.G. James, Kurt Wüthrich, Magdalini Polymenidou, Mike Scott, University of Zurich, and Polymenidou, M
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animal diseases ,Antibody Affinity ,Immunoglobulin Variable Region ,lcsh:Medicine ,Epitope ,law.invention ,Epitopes ,Mice ,law ,lcsh:Science ,Biochemistry/Biomacromolecule-Ligand Interactions ,Multidisciplinary ,biology ,Antibodies, Monoclonal ,Flow Cytometry ,Immunohistochemistry ,Immunoglobulin Isotypes ,Neurological Disorders/Prion Diseases ,Recombinant DNA ,Antibody ,Research Article ,Immunoprecipitation ,medicine.drug_class ,Prions ,Blotting, Western ,10208 Institute of Neuropathology ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,1100 General Agricultural and Biological Sciences ,Cross Reactions ,Monoclonal antibody ,Peptide Mapping ,1300 General Biochemistry, Genetics and Molecular Biology ,Infectious Diseases/Prion Diseases ,mental disorders ,medicine ,Animals ,Humans ,Avidity ,1000 Multidisciplinary ,Molecular mass ,lcsh:R ,Surface Plasmon Resonance ,Molecular biology ,nervous system diseases ,Epitope mapping ,biology.protein ,570 Life sciences ,lcsh:Q ,U7 Systems Biology / Functional Genomics ,Epitope Mapping - Abstract
PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays., PLoS ONE, 3 (12), ISSN:1932-6203
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- 2008
9. Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease
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Magdalini Polymenidou, Markus Glatzel, Anne Bellon, Katharina Stoeck, Martin Vey, and Adriano Aguzzi
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Male ,PrPSc Proteins ,medicine.drug_class ,animal diseases ,Blotting, Western ,Enzyme-Linked Immunosorbent Assay ,Monoclonal antibody ,Epitope ,Creutzfeldt-Jakob Syndrome ,PRNP ,Epitopes ,mental disorders ,medicine ,Humans ,chemistry.chemical_classification ,biology ,Dose-Response Relationship, Drug ,Haplotype ,Antibodies, Monoclonal ,Brain ,Hydrogen-Ion Concentration ,Surface Plasmon Resonance ,Proteinase K ,Virology ,Immunohistochemistry ,nervous system diseases ,Amino acid ,Protein Structure, Tertiary ,Blot ,chemistry ,biology.protein ,Female ,Neurology (clinical) ,Antibody ,Endopeptidase K ,Epitope Mapping ,Densitometry - Abstract
Summary Background The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrP Sc ), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrP Sc display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively. Methods We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrP Sc . Findings We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrP Sc -rich brain areas, with a typical PrP Sc type 1 migration pattern. Interpretation The regular coexistence of multiple PrP Sc types in patients with CJD casts doubts on the validity of electrophoretic PrP Sc mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications.
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- 2005
10. Diagnosis of human prion disease
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Mamta Sattavat, Bruce L. Miller, Henry Sanchez, Stephen J. DeArmond, Jiri G. Safar, Camille Deering, Michael D. Geschwind, Martin Vey, Henry Baron, Stanley B. Prusiner, Ana Serban, Svetlana Didorenko, and Kurt Giles
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genetic structures ,PrPSc Proteins ,animal diseases ,Biopsy ,detection ,Disease ,Biology ,Sensitivity and Specificity ,Creutzfeldt-Jakob Syndrome ,mental disorders ,medicine ,Humans ,immunoassay ,Codon ,Multidisciplinary ,Polymorphism, Genetic ,medicine.diagnostic_test ,Brain biopsy ,neurodegeneration ,Brain ,Histology ,Middle Aged ,Biological Sciences ,Virology ,Creutzfeldt-Jakob disease ,nervous system diseases ,endpoint titration ,Immunoassay ,Immunohistochemistry ,Female - Abstract
With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt–Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP Sc ) used limited proteolysis to digest the precursor cellular PrP (PrP C ). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP Sc , we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP Sc in all 10–24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, ≈90% of the total PrP Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP C . Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.
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- 2005
11. Molecular Biology of Prion Propagation
- Author
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Glenn C. Telling, Kiyotoshi Kaneko, A. Taraboulos, Stanley B. Prusiner, Fred E. Cohen, Stephen J. De Armond, Ruth Gabizon, Michael Scott, and Martin Vey
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Gene isoform ,chemistry.chemical_classification ,Conformational change ,biology ,animal diseases ,Transgene ,Scrapie ,Clathrin ,Transmembrane protein ,nervous system diseases ,Cell biology ,Amino acid ,chemistry ,mental disorders ,biology.protein ,Gene - Abstract
Prions are novel pathogens and are distinct from both viroids and viruses. They are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrP) designated PrPSc. Prions cause neurodegenerative diseases including scrapie of sheep, mad cow disease, and Creutzfeldt-Jakob disease (CJD) of humans. That prion diseases are manifest as genetic, infectious and sporadic illnesses is unprecedented. The conversion of PrPC into PrPSc is a post-translational process involving a profound conformational change which is the fundamental event underlying the propagation of prions. PrPC contains −40% α-helix and virtually no β-sheet; in contrast, PrPSc has −30% α-helix and −40% β-sheet. These data argue that the conversion of α-helices into β-sheets underlies the formation of PrPSc. Efficient formation of PrPSc requires targeting PrPC by glycosylphosphatidyl inositol (GPI) anchor to a caveolae-like membrane domain (CLD) which is detergent insoluble and enriched for cholesterol and glycosphingolipids. Redirecting PrPC to clathrin-coated pits by creating chimeric PrP molecules with four different C-terminal targeting domains prevented the formation of PrPSc. To determine if these C-terminal transmembrane segments prevented PrPC from refolding into PrPSc by altering the structure of the polypeptide, we fused the 28 amino acid C-termini from the Qa protein. Two C-terminal Qa segments differing by a single residue direct the trans-membrane protein to clathrin coated pits or the BPI form to CLDs. The CLD targeted PrPC was converted into PrPSc while the transmembrane PrPC was not. Transgenic (Tg) mice expressing human (Hu) prion protein (PrP) and chimeric Hu/mouse (Mo) PrP genes were inoculated with brain extracts from humans with inherited or sporadic prion disease.
- Published
- 1998
12. COOH-terminal sequence of the cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform
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Kiyotoshi Kaneko, Susanne Pilkuhn, Martin Vey, Frederick Cohen, Stanley B. Prusiner, and Michael J. Scott
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Gene isoform ,Glycosylphosphatidylinositols ,Prions ,animal diseases ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Scrapie ,Biology ,Transfection ,chemistry.chemical_compound ,Mice ,Neuroblastoma ,Tumor Cells, Cultured ,Animals ,Point Mutation ,Inositol ,Multidisciplinary ,Base Sequence ,Point mutation ,Compartment (chemistry) ,Biological Sciences ,Transmembrane protein ,Peptide Fragments ,nervous system diseases ,Clone Cells ,Transmembrane domain ,chemistry ,Biochemistry ,nervous system ,Oligodeoxyribonucleotides ,Mutagenesis, Site-Directed - Abstract
Efficient formation of scrapie isoform of prion protein (PrPSc) requires targeting PrPScby glycophosphatidyl inositol (GPI) anchors to caveolae-like domains (CLDs). Redirecting the cellular isoform of prion protein (PrPC) to clathrin-coated pits by creating chimeric PrP molecules with four different COOH-terminal transmembrane domains prevented the formation of PrPSc. To determine if these COOH-terminal transmembrane segments prevented PrPCfrom refolding into PrPScby altering the structure of the polypeptide, we fused the 28-aa COOH termini from the Qa protein. Two COOH-terminal Qa segments differing by a single residue direct the transmembrane protein to clathrin-coated pits or the GPI form to CLDs; PrPScwas formed from GPI-anchored PrPCbut not from transmembrane PrPC. Our findings argue that PrPScformation is restricted to a specific subcellular compartment and as such, it is likely to involve auxiliary macromolecules found within CLDs.
- Published
- 1997
13. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease
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E. Shaw, Martin Vey, C. Roberts, Wolfgang Garten, H. Angliker, G. Thomas, Hans-Dieter Klenk, and A. Stieneke-Gröber
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Proteases ,animal structures ,medicine.medical_treatment ,viruses ,Blotting, Western ,Molecular Sequence Data ,Hemagglutinins, Viral ,Hemagglutinin Glycoproteins, Influenza Virus ,Vaccinia virus ,Chick Embryo ,Cleavage (embryo) ,General Biochemistry, Genetics and Molecular Biology ,Virus ,Amino Acid Chloromethyl Ketones ,Dogs ,Viral Envelope Proteins ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Subtilisins ,Molecular Biology ,Furin ,Cells, Cultured ,chemistry.chemical_classification ,Protease ,General Immunology and Microbiology ,biology ,General Neuroscience ,Subtilisin ,Membrane Proteins ,Affinity Labels ,DNA ,Hydrogen-Ion Concentration ,Molecular biology ,Membrane glycoproteins ,Biochemistry ,chemistry ,Influenza A virus ,biology.protein ,Cattle ,Glycoprotein ,Chromatography, Liquid ,Research Article - Abstract
Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.
- Published
- 1992
14. Proteolytic Activation of Influenza Viruses: Substrates and Proteases
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Hans-Dieter Klenk, Reiko Ohuchi, Masanobu Ohuchi, Martin Vey, Wolfgang Garten, and Andrea Stieneke-Gröber
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chemistry.chemical_classification ,Infectivity ,Proteases ,Host (biology) ,viruses ,Cell ,Biology ,medicine.disease_cause ,Virology ,Influenza A virus subtype H5N1 ,Virus ,medicine.anatomical_structure ,Enzyme ,chemistry ,medicine ,Glycoprotein - Abstract
Like many other viral glycoproteins, the hemagglutinin (HA) of influenza viruses is activated by proteolytic cleavage. Cleavage which is necessary for the fusion activity of the hemagglutinin and thus for the infectivity of the virus is exerted by host cell proteases, and the presence of an appropriate enzyme determines whether infectious virus is made in a given cell. Proteolytic activation is therefore indispensable for effective virus spread in the infected host and has been found to be a prime determinant for virus pathogenicity. This concept has been derived mainly from studies on avian influenza viruses. The pathogenic strains of these viruses are activated by ubiquitous proteases and cause therefore systemic infection mostly leading to rapid death of the animal, whereas activation of the apathogenic strains occurs only in epithelial cells of the respiratory or the enteric tract resulting in local infection of these organs. The mammalian influenza viruses, including the human ones, resemble the apathogenic avian strains in possessing also hemagglutinins of restricted cleavability and in causing usually local infection of the respiratory tract.1
- Published
- 1992
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