WO2001019867A1 - Proteine apparentee au prion - Google Patents

Proteine apparentee au prion Download PDF

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Publication number
WO2001019867A1
WO2001019867A1 PCT/EP2000/008937 EP0008937W WO0119867A1 WO 2001019867 A1 WO2001019867 A1 WO 2001019867A1 EP 0008937 W EP0008937 W EP 0008937W WO 0119867 A1 WO0119867 A1 WO 0119867A1
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WO
WIPO (PCT)
Prior art keywords
prion
related protein
nucleic acid
protein
acid according
Prior art date
Application number
PCT/EP2000/008937
Other languages
German (de)
English (en)
Inventor
Kay Hofmann
Original Assignee
Memorec Stoffel Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Memorec Stoffel Gmbh filed Critical Memorec Stoffel Gmbh
Publication of WO2001019867A1 publication Critical patent/WO2001019867A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to nucleic acids encoding the prion-related protein and their use.
  • the prion protein is a surface protein of cells, especially of neurons, which has an important role in the genesis of neurodegenerative diseases such as scrapie, BSE, Creutzfeld-Jakob disease etc.
  • a common factor in these diseases is the presence of an atypically folded form of PrP.
  • the atypical, pathogenic folding of the PrP protein can be triggered by an inheritable mutation in the PrP gene, e.g. in familial fatal insomnia (FFI) and Gerstmann-St syndrome.
  • FFI familial fatal insomnia
  • Gerstmann-St syndrome e.g. in Gerstmann-St syndrome
  • an atypical folding of the PrP protein can also occur sporadically with a genetically unchanged Prp gene.
  • the PrP protein is bound to the outside of the plasma membrane of eukaryotic cells, especially neurons, by means of a GPI anchor (glycosylphosphatidylinositol).
  • GPI anchor glycosylphosphatidylinositol
  • the spatial structure of the non-anchored protein in solution is known. It has a high proportion of alpha helices and is stabilized by a disulfide bridge.
  • the structure of the pathogenic atypically folded form is not known in detail, but it has a high proportion of beta-sheet elements.
  • Atypically folded PrP protein has a protease-resistant portion and shows a tendency to aggregate. Such aggregated PrP protein is found in the brain of individuals suffering from prion disease. The mechanism of how PrP aggregation leads to the demise of neurons and thus the symptoms of the disease is not known.
  • Protein X plays an important role in the conversion of normally folded PrP into the pathogenic form. Protein X binds to the PrP protein and thus accelerates the change in folding.
  • Protein X is incorporated into the PrP aggregates and thus affects the aggregation. If protein X fulfills a physiologically important function and is in deficit compared to PrP, incorporation into the aggregates would result in the cell no longer having enough free protein and being damaged as a result. In this case, the external administration of protein X or a derivative thereof would alleviate the neurodegenerative phenotype of prion diseases.
  • the present invention for the first time makes nucleic acids available which code for a prion-related protein.
  • the prion-related protein is characterized in that it shows significant similarity to known PrP proteins from the human, mouse, sheep, cattle, and chicken species. The similarity can be shown with the help of the "generalized profile" method described in: P. Bucher, K. Karplus, N. Moeri and K. Hofmann Comput. Chem. 20: 3-23 (1996).
  • the prion-related protein is further characterized by the presence of at least one disulfide bridge, the presence of a signal sequence for protein secretion and the presence of a C-terminal hydrophobic sequence which is typically replaced in eukaryotic cells by a glycosylphosphatidylinositol anchor ,
  • FIG. 1 shows the nucleic acid sequence of the mRNA coding for human prion-related protein
  • FIG. 2 shows the nucleic acid sequence of the mRNA coding for murine prion-related protein
  • Figure 3 shows the peptide sequence of the human prion-related protein
  • Figure 4 shows the peptide sequence of the murine prion-related protein
  • the nucleic acid according to the invention is preferably a nucleic acid which codes for the prion-related protein. It is particularly preferably the human and murine prion-related protein.
  • the corresponding nucleic acid sequences are as Seq. ID. No. 1 and Seq. ID. No. 2 disclosed.
  • the corresponding peptide sequences are as Seq. ID. No. 3 and Seq. ID. No. 4 disclosed.
  • the person skilled in the art can easily find the nucleic acids and proteins from other eukaryotes, taking into account the high homology. For this purpose, he can use cross-reacting antibodies for a specific affinity chromatographic purification, or he can synthesize oligonucleotide primers based on the nucleic acid sequence and amplify the nucleic acids sought using the polymerase chain reaction in a cDNA library of the eukaryote.
  • the corresponding cDNA library can be obtained in a manner known per se by isolating mRNA from a tissue sample and subsequent reverse transcription.
  • the amino acid sequence can be derived from the nucleic acid sequence using the genetic code. Alternatively, it is also possible to search for and combine homologous sequences in EST (Expressed Sequence Tags) databases.
  • nucleic acids according to the invention are suitable for the expression of the prion-related protein in pro- or eukaryotic systems.
  • they are also suitable for the expression of the prion-related protein in vivo in the sense of a gene therapy or in particular in the form of fragments, also in a complementary structure, as antisense nucleotides for reducing the expression of the prion-related protein.
  • nucleic acids according to the invention can be produced by chemical synthesis or by duplication in genetically modified organisms by methods known per se to the person skilled in the art.
  • the invention also relates to the prion-related protein obtainable by the expression of the nucleic acids according to the invention.
  • the prion-related protein according to the invention can be produced by expression in genetically modified organisms.
  • Eukaryotic expression systems are particularly suitable.
  • Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene). Purification from genetically modified organisms, particularly in the case of overexpression, provides easy and direct access to the protein according to the invention and also allows isolation in large amounts. Variants of the prion-related protein are also claimed.
  • variants includes both naturally occurring allelic variations of the prion-related protein and recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and subsequent expression of proteins which are biological and / or immunological Activity correspond to the prion-related protein.
  • Amino acids can be deleted, inserted or exchanged conservatively. Conservative exchange means that an amino acid is replaced by an amino acid that has similar physicochemical properties.
  • amino acids are interchangeable: serine for / against alanine, alanine for / against glycine, methionine for / against serine, lysine for / against arginine, lysine for / against serine.
  • variants also encompasses N- and / or C-terminal truncated proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives.
  • Compounds in which the prion-related protein or its variants are coupled to further molecules such as dyes, radionuclides or affinity components also represent variants according to the invention.
  • nucleic acids which code for the prion-related protein or which are complementary to these nucleic acids.
  • the nucleic acids can be, for example, DNA, RNA, PNA or nuclease-resistant analogs.
  • Nuclease-resistant analogs are, in particular, those compounds in which the phosphodiester bond is modified by hydrolysis-stable compounds, for example phosphothioates, methylphosphonates or the like.
  • Short fragments of the nucleic acids are particularly suitable for antisense nucleotides. For reasons of specificity, these should preferably be more than 6, more preferably more than 8 and most preferably more than 12 nucleotides exhibit. For diffusion and cost reasons, they are typically less than 30 nucleotides in length, preferably 24 or less, and even more preferably 18 or fewer nucleotides in length.
  • the invention also relates to derivatives of nucleic acids which are coupled with other molecules for diagnostic or therapeutic purposes, for example with fluorescent dyes, radioactive markers or affinity components, and fragments of the nucleic acids according to the invention and of the nucleic acids complementary to these nucleic acids, and variants of the nucleic acids.
  • Fragments refer to nucleic acids that are shortened on the 5 'or 3' or on both sides.
  • variants is understood to mean that these nucleic acids hybridize under stringent conditions with the nucleic acid according to the invention or nucleic acids complementary thereto.
  • stringent conditions is understood to mean that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (under otherwise identical conditions) under which exactly complementary nucleic acids would just just hybridize. For example, if a precisely hybridizing nucleic acid hybridizes under given conditions up to a temperature of approx. 55 ° C, then stringent conditions are temperatures equal to or higher than 45 ° C.
  • the preferred temperature range for stringent conditions is 5 ° C, more preferably 3 ° C.
  • the invention further relates to antibodies which are directed against the prion-related protein according to the invention or the nucleic acids according to the invention. These substances are particularly suitable for use in diagnostics, immunoassays known to those skilled in the art, for histological examination and as a medicament for the treatment of conditions which are associated with overexpression of the prion-related protein.
  • Such antibodies according to the invention can be obtained by methods known per se to those skilled in the art by immunization with prion-related protein, ß nucleic acids or peptide and nucleic acid fragments are obtained in the presence of auxiliary reagents.
  • the invention furthermore relates to cell lines which overexpress the prion-related protein according to the invention.
  • Such cell lines are obtainable by transfection with vectors which contain the nucleic acids according to the invention which code for the prion-related protein.
  • the transfection can be carried out, for example, by electroporation.
  • the cell lines are preferably stably transfected.
  • the prion-related protein according to the invention, the nucleic acids according to the invention and the antibodies according to the invention can optionally be contained in medicaments and diagnostic agents together with further auxiliaries.
  • These medicinal and diagnostic agents are suitable for the diagnosis and treatment of diseases which are based on over- or under-expression and / or an increased or reduced activity of the prion-related protein and / or on disorders of protein aggregation, the copper balance of the cell and / or those associated with increased levels of neuronal cell death.
  • a pharmaceutical screening method according to the invention is based on the observation of protein aggregates in cell lines which overexpress the prion-related protein according to the invention, possibly in combination with PrP, at least one potentially pharmaceutically active substance being added. Such cell lines are therefore particularly suitable for the development and testing of pharmaceutical lead structures.
  • a pharmaceutical screening method according to the invention is based on measuring the cell survivability of the cell lines mentioned under the influence of at least one potentially pharmaceutically active substance.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Toxicology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Psychiatry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hospice & Palliative Care (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne la protéine apparentée au prion et son utilisation.
PCT/EP2000/008937 1999-09-14 2000-09-13 Proteine apparentee au prion WO2001019867A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19943888.9 1999-09-14
DE19943888 1999-09-14
EP00103631 2000-02-21
EP00103631.8 2000-02-21

Publications (1)

Publication Number Publication Date
WO2001019867A1 true WO2001019867A1 (fr) 2001-03-22

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001046419A2 (fr) * 1999-12-23 2001-06-28 Schering Corporation Proteines mammaliennnes, reactifs et procedes associes
US20140206021A1 (en) * 2002-12-03 2014-07-24 North Carolina State University Prion protein ligands and methods of use

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010505A1 (fr) * 1995-09-14 1997-03-20 The Regents Of The University Of California ANTICORPS SPECIFIQUE DU PrPSc NATIF
WO1997037227A1 (fr) * 1996-04-03 1997-10-09 Stichting Instituut Voor Dierhouderij En Diergezondheid Procede de detection de maladies a prions
US5792901A (en) * 1994-05-13 1998-08-11 The Regents Of The University Of California Detecting prions in a sample and prion preparation and transgenic animal used for same
US5908969A (en) * 1994-05-13 1999-06-01 The Regents Of The University Of California Method of detecting prions in a sample and transgenic animal used for same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5792901A (en) * 1994-05-13 1998-08-11 The Regents Of The University Of California Detecting prions in a sample and prion preparation and transgenic animal used for same
US5908969A (en) * 1994-05-13 1999-06-01 The Regents Of The University Of California Method of detecting prions in a sample and transgenic animal used for same
WO1997010505A1 (fr) * 1995-09-14 1997-03-20 The Regents Of The University Of California ANTICORPS SPECIFIQUE DU PrPSc NATIF
WO1997037227A1 (fr) * 1996-04-03 1997-10-09 Stichting Instituut Voor Dierhouderij En Diergezondheid Procede de detection de maladies a prions

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE GENEMBL 1 July 1999 (1999-07-01), LEE ET AL.: "Mus musculus long incubation prion protein (Prnpb) and prion-like protein (Prnd) genes, complete cds.", XP002143522 *
DATABASE GENEMBL 18 October 1999 (1999-10-18), MOORE ET AL.: "Homo sapiens BAC RG73E2 prion-like protein (PRND) gene, complete cds.", XP002143523 *
DATABASE GENEMBL 18 October 1999 (1999-10-18), MOORE ET AL.: "Mus musculus clone 9 prion protein-like protein Dpl, alternatively spliced mRNA, complete cds.", XP002143524 *
DATABASE GENEMBL 22 November 1999 (1999-11-22), LI ET AL.: "Mus musculus prion protein-like, long mRNA, complete cds.", XP002143525 *
LEE ET AL.: "Complete genomic sequence and analysis of the prion protein gene region from three mammalian species", GENOME RESEARCH, vol. 8, no. 10, October 1998 (1998-10-01), pages 1022 - 1037, XP002143521 *
LIAO Y-C ET AL: "CLONING OF RAT PRION-RELATED PROTEIN COMPLEMENTARY DNA", LABORATORY INVESTIGATION, vol. 57, no. 4, 1987, pages 370 - 374, XP000925801, ISSN: 0023-6837 *
MOORE ET AL: "Ataxia in prion protein (PrP)-deficient mice is associated with upregultaion of the novel PrP-like protein doppel", JOURNAL OF MOLECULAR BIOLOGY, vol. 292, no. 4, 1 October 1999 (1999-10-01), pages 797 - 817, XP000925693 *
S PRUSINER ET AL: "Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA,US,NATIONAL ACADEMY OF SCIENCE. WASHINGTON, vol. 90, no. 22, 15 November 1993 (1993-11-15), pages 10608 - 10612-10612, XP002127973, ISSN: 0027-8424 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001046419A2 (fr) * 1999-12-23 2001-06-28 Schering Corporation Proteines mammaliennnes, reactifs et procedes associes
WO2001046419A3 (fr) * 1999-12-23 2002-02-14 Schering Corp Proteines mammaliennnes, reactifs et procedes associes
US20140206021A1 (en) * 2002-12-03 2014-07-24 North Carolina State University Prion protein ligands and methods of use
US9678085B2 (en) * 2002-12-03 2017-06-13 Pathogen Removal And Diagnostic Technologies Inc. Prion protein ligands and methods of use

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