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WO2002006309A2 - Peptides signaux d'exportation nucleaire (nes), proteines de fusion qui les contiennent, ainsi que leur utilisation - Google Patents

Peptides signaux d'exportation nucleaire (nes), proteines de fusion qui les contiennent, ainsi que leur utilisation Download PDF

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Publication number
WO2002006309A2
WO2002006309A2 PCT/EP2001/008065 EP0108065W WO0206309A2 WO 2002006309 A2 WO2002006309 A2 WO 2002006309A2 EP 0108065 W EP0108065 W EP 0108065W WO 0206309 A2 WO0206309 A2 WO 0206309A2
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WIPO (PCT)
Prior art keywords
acid sequence
antibody
amino acid
fusion protein
general formula
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PCT/EP2001/008065
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German (de)
English (en)
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WO2002006309A3 (fr
Inventor
Uwe Vinkemeier
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Forschungsverbund Berlin E.V.
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Priority to AU2001276400A priority Critical patent/AU2001276400A1/en
Priority to EP01954035A priority patent/EP1301529A2/fr
Publication of WO2002006309A2 publication Critical patent/WO2002006309A2/fr
Publication of WO2002006309A3 publication Critical patent/WO2002006309A3/fr

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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the invention relates to new nucleus export signal peptides (NES) of the general formula (I), fusion proteins containing them and their use for the detection of inflammatory diseases, in functional tests for immunology and for the screening of active substances and for the detection of inhibitors and activators of the nucleus. Exports and related diseases.
  • NES nucleus export signal peptides
  • DNA replication and RNA biogenesis take place in the cell nucleus, whereas protein synthesis essentially takes place in the cytoplasm.
  • the integration of these activities, which take place in different cell compartments, depends on a selective transport of the proteins and the ribonucleoprotein particles between the cell nucleus and the cytoplasm.
  • the proteins, DNA and / or RNA are transported via nuclear pores, which are located in the membrane that surrounds the cell nucleus.
  • the transport through the nuclear pores which is necessary because important cell activities are spatially separated in the cell, is mediated, among other things, by signals for cellular import and export between the nucleus and the cytoplasm. While information and studies on the transport of the proteins into the cell nucleus are available, there is little information available about the export of proteins and / or peptides or other biological structures from the cell nucleus.
  • the transport of the proteins and / or peptides or other structures from the cell nucleus into the cytoplasm is realized, among other things, via nucleus export signal peptides (NES). In many molecular processes between the cell nucleus and the cytoplasm, it is necessary that there be a coupled import and export of compartments between the cell nucleus and the cytoplasm.
  • stat proteins signal transducers and activators of transcription
  • elements between the cell nucleus and the cytoplasm can be regulated in control loops.
  • Stat proteins form a group of transcription factors which, for example, modify and regulate the regulation of the gene expression of interferons and cytokines as well as other growth factors. The activation of the Stat proteins takes place in the cytoplasm of the cell at activated receptors, whereupon several of the Stat molecules are transported into the nucleus in order to activate the transcription of certain genes there (Damell, JE, Jr. (1997) Science 277, 1630 -1635).
  • the stat proteins are accordingly in the cytoplasm before activation.
  • the cytokine signals originate from cell surface receptors.
  • the binding of cytokines or growth factors to their corresponding receptors initiates a series of tyrosine phosphorylations, which are triggered by kinase members of the Janus family (Jaks).
  • This process commonly referred to as stat activation, causes dimerization of the stat proteins by reciprocal phosphotyrosine / SH2 interactions and later the rapid and efficient translocation of cytoplasmic molecules into the nuclear part, where they activate specific genes.
  • Cytokine-induced transcription is a temporary process that can take a few minutes to hours.
  • the nucleus export signal peptides play a crucial role in numerous disease-relevant, pathogenic processes, such as. B. Disorders of cell growth, wound closure, carcinogenesis, septic shock, arthritis, leukemia, and numerous cardiovascular diseases.
  • the nucleus export signal peptides are of great importance for the development of diagnostic and therapeutic agents, the details of the interactions of the nucleus export signal proteins with their receptors or target sites are largely unknown. The lack of more precise information about this mechanism is particularly disadvantageous since DNA replication, RNA biogenesis and protein synthesis, as well as the transport of these biological structures between the nucleus and the cytoplasm are associated with numerous diseases.
  • the integration of the core and cytoplasm The ongoing processes are crucial for cancer, for defects in the immune system, for the metabolic performance of the liver and kidneys and others.
  • the technical problem underlying the present invention therefore consists in the provision of means and methods which modify the transport of proteins, peptides, nucleic acid sequences and other biological structures, in particular transcription factors between cytoplasm and cell nucleus, and the provision of methods for screening active substances, which are suitable for the diagnosis and treatment of diseases associated with signal peptides which induce and regulate transport between the nucleus and the cytoplasm.
  • the present invention solves this problem by providing an amino acid sequence according to the general formula (I) with an activity which modifies the transport of proteins, peptides, nucleic acid sequences, in particular transcription factors between nucleus and cytoplasm.
  • the amino acid sequence of the general formula (I) is A1-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13 (I) in which
  • A1 is A, V, L, I, D, F, M or W,
  • A2 is D or E
  • A3 is K, R or H
  • A4 is G, N, Q, C, S, T or Y,
  • A5 is A, V, L, I, D, F, M or W,
  • A6 is G, N, Q, C, S, T or Y,
  • A7 is A, V, L, I, D, F, M or W,
  • A8 is A, V, L, I, D, F, M or W,
  • A9 is G, N, Q, C, S, T or Y,
  • A10 is G, N, Q, C, S, T or Y,
  • A11 is A, V, L, I, D, F, M or W,
  • A12 is A, V, L, I, D, F, M or W,
  • A13 is A, V, L, I, D, F, M or W.
  • a "disease related to nucleus export signal peptides (NES)" is one tes / obesity, acute and chronic rejection of allogeneic organ transplants, nerve line degeneration, Parkinson's disease, septic shock, endotoxinemia, hypersensitivity, uveitis, wound closure / cell growth and others.
  • the NES also play a role in the regulation of the immune system, inflammatory and effector mechanisms of cellular and humoral immunity, septic shock, inflammation of the sensory organs, e.g. the eyes, in the regulation of transcription and cell cycles and acute respiratory insufficiency, physiological stress and other disorders important role
  • the term “pharmaceutical or diagnostic composition” means a substance which is suitable for the diagnosis and / or treatment of diseases, in particular diseases related to the NES, preferably in an amount sufficient to to achieve such an effect.
  • treatment means the prophylactic and / or therapeutic effect of a medicinal substance.
  • NES relates to both naturally occurring NES and all modifications, mutants or derivatives of the NES, NES produced by recombination techniques, which contains amino acid modifications such as inversions, deletions, insertions, additions, etc., provided that at least part of the essential functions of the wild-type NES are present, such NES may also include unusual amino acids and / or modifications such as alkylation, oxidation, thiol modification, denaturation and oligomerization, etc.
  • NES may in particular "Peptides, especially a fusion protein and / or fusion peptide which contains, in addition to other proteins, peptides or parts thereof, all or part of NES.
  • the NES are shortened forms of the naturally occurring NES like a small peptide.
  • promoter means a DNA sequence which is usually located upwards (5 ') from the coding sequence of a structural gene and which controls the expression of the coding region by a recognition sequence for an RNA polymerase and / or for other factors, necessary for the transcription to begin at the correct location. Promoter sequences are necessary, but not always sufficient to control the expression of the gene.
  • Nucleic acid means a large molecule that can be single or double-stranded and consists of monomers (nucleotides) that contain a sugar unit, a phosphate unit and either a purine or a pyrimidine residue.
  • the nucleic acid can be cDNA, act genomic DNA or RNA, for example mRNA.
  • nucleic acid sequence means a natural or synthetic polymer of single or double stranded DNA or RNA which alternatively contains synthetic, non-natural or modified nucleotide bases which can be incorporated into DNA or RNA polymers.
  • gene means a DNA sequence encoding a specific protein and regulatory elements that control the expression of this DNA sequence.
  • coding sequence refers to the part of a gene which codes for a protein, a polypeptide or a part thereof, the regulatory sequences and / or elements which control the initiation or termination of the transcription being excluded.
  • the coding sequence and / or the regulatory element may be one that is normally found in the cell, in which case it is referred to as “autologous” or “endogenous”, or one that is not normally found in the cell is localized, in which case it is referred to as “heterologous”.
  • a heterologous gene can also consist of autologous elements that are arranged in an order and / or orientation that is normally not found in the cell to which the gene is transferred.
  • a whole or part of a heterologous gene may come from any source known in the art, including a bacterial or viral genome or episome, eukaryotic core or plasmid DNA, cDNA, or chemically synthesized DNA.
  • the structural gene can encode an uninterrupted form the region or can comprise one or more introns, which are delimited by suitable splice connections.
  • the structural gene can be composed of sections that come from different, naturally occurring or synthetic sources.
  • a “transactivator protein” is a protein that can bind to the operator region of a gene and thereby promote the transcription of the gene.
  • a "DNA binding domain” is an amino acid sequence that can bind to a specific DNA sequence.
  • a “fusion protein” is a protein that consists of amino acid sequences that come from at least two different sources.
  • a “heterologous” amino acid sequence is a sequence that comes from a different source than the other parts of the fusion protein.
  • a “detectable gene product” is a nucleotide or amino acid sequence that can be detected by means of a test.
  • the expression of a detectable gene product gives the cell a feature that allows the cell to be easily selected from other cells that do not express the detectable gene product ,
  • association means any type of interaction between the NES and the targets, in particular a covalent or non-covalent bond or association, such as a covalent bond, hydrophobic / hydrophilic interaction, Van-der-Waalsche Forces, ion pairs, ligand-receptor interaction, interaction between epitope and the antibody binding site, enzyme-substrate interaction, interaction between liposomes and hydrophobic molecules, nucleotide base pairing, interaction between membranes and hydrophobic molecules and the like, without, however, to be restricted.
  • Such an association can also include the presence of further molecules, such as peptides, proteins or further nucleotide sequences.
  • vector means a recombinant DNA construct, which can be a plasmid, virus or an autonomously replicating sequence, a phage or a nucleotide sequence, which is linear or circular, consisting of single or double stranded DNA or RNA in which a series of nucleotide sequences have been linked or recombined into a unique construction and which can introduce a promoter fragment and a DNA sequence of a selected gene product in sense or antisense orientation together with suitable untranslated 3 'sequences into a cell.
  • Plasmids are genetic elements that are stably inherited without being part of the chromosome of their host cell. They can include DNA or RNA and be linear and circular. Plasmids encode molecules that ensure their replication and stable inheritance during cell replication, and can encode products of considerable importance to medicine, agriculture and the environment. For example, they encode toxins that greatly increase the virulence of pathogenic bacteria. They can also encode genes that confer resistance to antibiotics. In molecular biology, plasmids are generally used as vectors for cloning and expression According to the rules of the standard designation familiar to the person skilled in the art, plasmids are generally designated with the lowercase letter p, which is preceded or followed by capital letters and / or digits.
  • plasmids disclosed in the present description are either commercially available, available to the public or can be constructed from available plasmids using routine, well-known, published methods. Many plasmids and other cloning and expression vectors that can be used in the present invention are well known and readily available to those skilled in the art. In addition, those skilled in the art can readily construct any number of other plasmids suitable for use in the invention. From the present disclosure, the properties, construction and use of such plasmids as well as other vectors are readily apparent to the person skilled in the art.
  • expression used in the present description is intended to describe the transcription and / or coding of the sequence of the gene product.
  • a DNA chain encoding the sequence of a gene product is first transcribed into a complementary RNA, which is often an mRNA, and then the mRNA so transcribed is translated into the gene product mentioned above, if it is the gene product is a protein.
  • the expression also includes the transcription of a DNA which has been inserted with respect to its regulatory elements in the antisense direction. Expression that is constitutive and that may possibly be further enhanced by an externally controlled promoter fragment, producing multiple mRNA copies and large amounts of the selected gene product, can also include the overproduction of a gene product.
  • host cell means a cell that has been genetically modified by the transmission of a chimeric, heterologous or autologous nucleic acid sequence or its descendants which still contain this sequence. These cells are also referred to as "transgenic cells". In the case of the transmission of an autologous nucleic acid sequence, the sequence in the host cell is present in a higher copy number than the naturally occurring sequences.
  • the peptides or proteins according to the invention which do not occur in their natural (cellular) environment are isolated.
  • isolated means, in connection with proteins, a polypeptide which is present without the material with which it is associated in its natural state or at most with a part thereof. Based on the weight of the total protein in a particular one Sample makes up the isolated protein at least 0.5%, preferably at least 5%, more preferably at least 25% and even more preferably at least 50%. Most preferably the "isolated” protein is essentially free of other proteins, lipids, carbohydrates or other substances, with which it is naturally associated and forms a single main band on a polyacrylamide gel. "Substantially free” means that the protein is at least 75%, preferably at least 85%, more preferably at least 95% and most preferably at least 99% free from other proteins, lipids, carbohydrates or other substances with which it is natural is associated.
  • Antibody means a polypeptide that is essentially encoded by an immunoglobulin gene or immunoglobin genes, or fragments thereof of that specifically binds and recognizes an analyte (antigen).
  • Known immunoglobin genes include both the kappa, lambda, alpha, gamma, delta, epsilon and mu genes for the constant region and the innumerable genes for the variable immunoglobulin region.
  • Antibodies occur, for example, as intact immunoglobulins or as a series of well-characterized fragments that are generated by cleavage with various peptidases.
  • Antibody also means modified antibodies (for example oligomeric, reduced, oxidized and labeled antibodies).
  • antibody used in the present description also includes antibody fragments that either by means of modification of whole antibodies or by means of de novo synthesis using Recombinant DNA techniques have been generated.
  • antibody includes both intact molecules and fragments thereof, such as Fab, F (ab ') 2 and Fv, which can bind the epitope determinant. With these fragments, the ability of the antibody to selectively bind its antigen or receptor is partial has been preserved, the fragments being defined as follows:
  • Fab the fragment containing a monovalent antigen binding fragment of an antibody molecule, can be produced by cleavage of an entire antibody with the enzyme papain, whereby an intact light chain and part of a heavy chain are obtained;
  • the Fab 'fragment of an antibody molecule can be obtained by treating an entire antibody with pepsin and then reducing it, whereby an intact light chain and part of the heavy chain are obtained; two Fab 'fragments are obtained per antibody molecule;
  • F (ab ') 2 the fragment of the antibody which can be obtained by treating an entire antibody with the enzyme pepsin without subsequent reduction;
  • f (ab ') 2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • Fv defined as a genetically engineered fragment that contains the light chain variable region and the heavy chain variable region and is expressed in the form of two chains
  • Single chain antibody defined as a genetically engineered molecule that encompasses the variable region of the light chain and the variable loading. contains the heavy chain, which are linked by a suitable polypeptide linker to a genetically fused single chain molecule.
  • epitope determinants used in the present invention means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have both specific features of the three-dimensional structure and specific charge features.
  • the person skilled in the art can easily produce “monoclonal antibodies” for the purposes of the invention against the proteins according to the invention and the fragments thereof or other biological structures.
  • the general methods for producing monoclonal antibodies using hybridoma techniques are well known.
  • Immortalized, antibody-producing cell lines can be by means of Cell fusion and also by means of other methods such as direct transformation of B lymphocytes with oncogenic DNA or transfection with the Epstein-Barr virus. See, for example, M.
  • genes encoding monoclonal antibodies of interest can be isolated from hybridomas using PCR techniques known in the art and cloned and expressed in suitable vectors.
  • monoclonal antibodies are suitable for purifying the individual proteins against which they are directed.
  • the antibodies according to the invention have an additional benefit in that they can be used as reagents in immunoassays, such as RIA, ELISA and the like. You can also they are used to isolate the NES or NES domains from cells or other biological samples.
  • the antibodies could e.g. B. to establish a test based on a tissue culture to find, isolate or modify novel NES or novel compounds that modify the interaction of NES and receptors and / or target sites.
  • the humanized or chimeric antibodies can include parts derived from two different types (e.g. human constant region and mouse binding region).
  • the parts originating from two different types can be chemically combined using conventional methods or can be produced as a single fusion protein using genetic engineering methods.
  • a DNA encoding the proteins of the two parts of the chimeric antibody can be expressed as a single fusion protein.
  • an antibody “specifically binds" to a protein, for example a different biological structure or thus shows "a specific immunoreactivity” if the antibody performs its function in a binding reaction in the presence of a heterogeneous population of proteins and other biological substances, on the basis of which it can be decided that whether the protein or other biological structure is present.
  • the specified antibodies preferably bind to a specific protein, while there is no significant binding to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that has been selected for a specific protein because of its specificity.
  • Various immunoassay embodiments can be used to select antibodies which show a specific immunoreactivity with a special protein.
  • solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that show specific immunoreactivity with a protein. Harlow and Lane, Antibodies, A Laboratory Manual, (1988), Cold Spring Harbor Publications, New York, describe immunoassay embodiments and conditions that can be used to determine a specific immunoreactivity.
  • Immunoassay refers to a test in which an antibody is used to specifically bind an analyte.
  • the immunoassay is characterized in that specific binding properties of a specific antibody are used. in order to isolate the analyte, to test it in a targeted manner and / or to determine it quantitatively.
  • the amino acid sequence of the general formula (I) at positions A7 and / or A11 comprises the amino acids leucine, isoleucine and / or valine, preferably leucine.
  • positions A9, A10 and / or A13 comprise the amino acids glutamine, asparagine and / or tyrosine.
  • positions A1, A5 and / or A12 comprise the amino acids trytophan, phenylalanine and / or isoleucine.
  • positions A2, A3, A4 and / or A8 comprise the amino acids aspartic acid, arginine, threonine and / or phenylalanine.
  • the invention preferably relates to an amino acid sequence according to the general formula (I), where A1 W, A2 D, A3 R, A4 T, A5 F, A6 S, A7 L, A8 F, A9 Q, A10 Q, A11 L, A12 I and A13 Q means.
  • the amino acid sequence according to the invention can also be, for example, a helical segment of the Stat1 protein, namely amino acids 302 to 314, which are located in Helix-4 of the "wound spiral domain" of the Statl protein.
  • This peptide containing this amino acid and at least two leucine residues which can also be called a leucine-rich peptide, is advantageously responsible for the efficient nuclear export of the stat protein.
  • the leucine residues in the amino acid sequence according to the invention can be replaced by other, in particular hydrophobic, amino acids, such as isoleucine, valine, cysteine and others. There are advantageously three other amino acids between each of the leucine building blocks.
  • the teaching according to the invention advantageously also includes homologs of the helical element of the stat protein, for example Drosophila melanogaster Stat (Dm), Anopheles gambia Stat (Ag), human CAMP-dependent protein kinase inhibitor alpha (PKI), human IKB alpha and others.
  • Dm Drosophila melanogaster Stat
  • Ag Anopheles gambia Stat
  • PKI human CAMP-dependent protein kinase inhibitor alpha
  • IKB alpha human IKB alpha
  • the present invention also relates to a fusion protein comprising the amino acid sequence of the formula (I) or a part thereof and at least one marker structure.
  • the marker structure can be, for example, a reporter gene.
  • a reporter gene is a protein or peptide that can be detected using various biological, chemical and / or physical methods.
  • the reporter gene according to the invention can be both the entire construct and the protein to be detected, but it is also possible that this is only the part of the protein which has an activity to be detected.
  • any DNA structure that codes for an area that can be detected using various detection methods can serve as a reporter gene. Expression constructs which code for proteins which can be detected easily and with high sensitivity are preferred.
  • the marker structure according to the invention in the fusion protein can be, for example, chloramphenicol acetyl transferase (CAT).
  • CAT catalyzes in particular the transfer of an acetyl residue from acetyl-CoA to chloramphenicol.
  • One of the advantages of CAT is that it is stable in the cell with a half-life of approximately 50 hours. This is particularly advantageous if, for example, the highest possible yields of the reporter gene are to be achieved.
  • the CAT advantageously has a good signal-to-background ratio.
  • luciferase Another advantageous marker structure in the sense of the invention is luciferase.
  • the luciferase advantageously has a high sensitivity and, because of its relatively short half-lives of approximately 3 hours, is well suited for induction studies.
  • a marker structure which comprises two luciferases with different properties is particularly advantageous, one luciferase, for example from Phottinus pyralis, detecting the activity of its own promoter, while the cotransfected luciferase, for example from Renilla reniformis, is under the control of the constitutive promoter and is thus advantageously used for standardization can be.
  • marker structures in the sense of the invention are beta-galactosidase, human growth hormone and secreted alkaline phosphorase.
  • the marker structures according to the invention can also advantageously be used as reporter genes for qualitative detection. With the qualitative proof it can be determined, for example, whether an selected cell is transfected or is not transfected, or it can be determined in the entire organism in which tissues a promoter is active. Reporter genes for qualitative detection are, for example, luciferase or beta-galactosidase.
  • the marker structure is a green fluorescent protein GFP, the glutathione S transferase and / or a fragment thereof.
  • the green fluorescent protein advantageously has little or no cytotoxic activity.
  • the absorption and emission maxima of the green fluorescent protein are advantageously similar to those of fluorescin, so that, for example, a GFP-positive cell can be detected using the same methods as those cells which have been stained with fluorescin-coupled antibodies, that is to say, for example, in the UV -Light, under the fluorescence microscope or in the cell sorter (FACS).
  • marker proteins are understood to be those proteins which are coupled to NES and enable the detection of the NES.
  • Preferred marker proteins that can be fused with the NES are also: glutathione-S-transferase (GST), but also other fluorescent proteins, such as the red or yellow, and the maltose-binding protein (MBP) and others.
  • the fusion protein contains the marker structure at the NH end and / or the COOH end.
  • the invention also relates to a nucleic acid sequence encoding the amino acid sequence of formula (I) or the fusion protein, or a complementary strand thereof.
  • nucleic acid refers to a natural or synthetic polymer of DNA or RNA that can be single or double-stranded and, in another embodiment, can contain a synthetic, non-natural or modified nucleotide base that can be incorporated into DNA or RNA polymers.
  • the nucleic acid molecule can be cDNA, genomic DNA or RNA, e.g. B. mRNA.
  • the present invention also relates to a vector Tor which comprises the nucleic acid sequence described above, in particular a bacterial vector such as a plasmid or a virus.
  • the present invention relates to host cells which have been transformed with a vector according to the invention, in particular procariotic or eukaryotic cells.
  • the present invention also relates to cell cultures, tissues, organs and the like which comprise a cell which contain a plasmid or a vector as described above.
  • the present invention also relates to an antibody or a fragment thereof which has a specific reactivity with the amino acid sequence according to formula (I) or with the fusion protein according to the invention and / or the nucleic acid sequence according to the invention or a single domain thereof.
  • These antibodies can be used to screen expression libraries, to identify clones which produce the complexes according to the invention and also for therapeutic purposes.
  • the term “relates to an antibody” used in the present description relates to the detection, activation or inhibition of molecular or cellular metabolic pathways which are induced by the interaction of the amino acid sequences according to formula (I) or the fusion protein according to the invention.
  • antibody relates to bivalent and monovalent molecules which have the property of having a complex or having the isolated structure of the fusion protein of the invention or of the invention
  • antibody used in the present description means a protein which consists of one or more polypeptides which are essentially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • Light chains of the antibodies according to the invention are expressed either in kappa or lambda
  • Heavy chains of the antibodies according to the invention are divided into gamma, mu, alpha, delta or epsilon chains, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD or IgE (for details see the definition of the terms in the front part of the description).
  • the antibodies according to the invention therefore bind to specific domains, whereas the binding to other proteins and / or peptides or other structures according to the invention present in the sample is not significant.
  • a specific binding to the domain under such conditions may require an antibody which, due to its specificity for the amino acid sequence according to the invention according to formula (I), the fusion proteins according to the invention and / or the nucleic acid sequences according to the invention, for the selection of antibodies which have a specific immunoreactivity with the structure according to the invention
  • Various immunoassay embodiments can be used, such as the amino acid sequence according to formula (I), the fusion proteins and / or the nucleic acid sequences.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that show specific immunoreactivity with the domains mentioned.
  • the immunoassays that can be used include, but are not limited to, competitive and non-competitive test systems using techniques such as Western blot analysis, radioimmunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion-precipitin reaction, Immunodiffusion tests, agglutination tests,
  • the antibodies according to the invention bind specifically to one or more epitopes on a domain which is involved with the biological structures according to the invention such as, for example, the amino acid sequences according to formula (I), the fusion protein and / or the nucleic acid sequences or structures which interact with these domains.
  • “Epitope” means the region of the biological structures according to the invention or the domains, proteins or nucleotide acid sequences which interact with these structures and are bound by an antibody, the binding preventing the association of a second antibody with the structures according to the invention and the antibody binding other proteins prevented.
  • the antibodies are polyclonal, monoclonal antibodies or fragments thereof.
  • Antibody fragments include those fragments which interact with the structures according to the invention. They can also be humanized antibodies, which are typically produced by means of recombination methods, the human sequences partially or entirely comprising an antibody which interacts with the structures according to the invention.
  • humanized antibodies include heavy antibodies or antibodies with a built-in CDR. It can also be completely human antibodies against the structures according to the invention, such as the nucleic acid sequences according to the invention, the fusion proteins according to the invention and / or the amino acids according to the invention, which are produced in genetically modified mice.
  • Antibodies according to the invention can also have a detectable label attached to them. Such a label can be a fluorescence label, for example with fluorescin isocyanate, an enzymatic label, for example with horseradish oxidase, an affinity label, for example with biotin and / or an isotope label with 125 l.
  • the invention also encompasses hybridoma cell lines which produce a monoclonal antibody which interacts with the structures according to the invention, such as, for example, the amino acid sequences, fusion protein and / or nucleic acid sequences according to the invention.
  • the antibodies according to the invention are suitable for the diagnosis and therapy of diseases which are related to the nucleus export signal peptides and the amino acid sequences according to the invention, the fusion proteins according to the invention and / or the nucleic acid sequences according to the invention.
  • the antibodies can be used as part of a diagnostic kit to prove whether a pathological change or a disease-inducing modification of the amino acid sequences according to the invention, of the fusion proteins according to the invention, of the nucleic acid sequences according to the invention and / or of the nucleus export signal peptides according to the invention has taken place or in a biological sample. is present.
  • the biological samples include tissues, test samples and intact cells or extracts thereof.
  • kits use antibodies that have an attached label that allows detection.
  • the antibodies are suitable to identify normal domains of the biological structures according to the invention, or with domains and areas that interact with these structures.
  • the antibodies themselves are also suitable for diagnosis and therapy.
  • the present invention also relates to a kit comprising the amino acid sequence of the formula (I), the fusion protein according to the invention, the nucleic acid sequences according to the invention, the vector according to the invention, the host cell according to the invention and / or the antibodies according to the invention.
  • the amino acids according to the invention, the fusion proteins according to the invention, the nucleic acid sequences according to the invention, the vectors according to the invention, the host cells according to the invention and / or the antibodies according to the invention can be contained in liposomes with pure lipid membranes or with biological membranes.
  • the nucleic acid sequence according to the invention can be used as an adjuvant or as a substance for immunization.
  • the liposomes or the adjuvant are suitable for the diagnosis and therapy of diseases which are related to the amino acids according to the invention, the nucleic acid sequences according to the invention, the vectors according to the invention, the host cells according to the invention, the antibodies according to the invention and / or the nucleus export signal peptides according to the invention.
  • the present invention therefore also relates to the use of the nucleotide sequence encoding the amino acid sequence according to the invention and / or the nucleotide sequence encoding the fusion protein for the production of a medicament, drug or therapeutic agent for the treatment of diseases which are associated with the nucleus export signal peptides.
  • the present invention also relates to the use of a fusion protein for the manufacture of a medicament for the diagnosis and / or treatment of diseases associated with the nucleus export signal peptides.
  • the present invention also relates to non-human mammalian cells which show expression of the amino acid sequence of formula (I).
  • the invention further relates to cells which contain a gene for expressing the amino acid sequence of the formula (1) which have at least one mutation.
  • the cell according to the invention in particular a non-human mammalian cell, has at least one antisense construct which counteracts the expression of the amino acid sequence according to formula (I).
  • the present invention also relates to non-human mammals which contain at least one of the cells according to the invention.
  • the non-human mammals can be mammals that are common in research and development or for different laboratory test series, such as rats, mice, primates, hamsters, rabbits, goats and the like. a.
  • the present invention also relates to the use of the amino acid sequence according to formula (I), the fusion protein according to the invention, the nucleic acid sequences according to the invention, the vectors according to the invention, the host cells according to the invention and / or the antibodies according to the invention for the production of a medicament for the diagnosis and / or treatment of with nucleus -Export signal peptides related diseases.
  • the invention also relates to the use of amino acid sequences according to the invention, the fusion protein according to the invention, the nucleic acid sequences according to the invention, the vectors according to the invention, the host cells according to the invention and / or the antibodies according to the invention for the screening of active substances which modify the nucleus export activity.
  • Modifying in the sense of the invention is understood to mean any influence on the nucleus export activity which leads to an increase or decrease in the activity which does not interact with the amino acid sequences according to the invention, the fusion protein according to the invention, or the inventive proteins
  • Nucleic acid sequences, the vectors according to the invention, the host cells according to the invention and / or the antibodies according to the invention can be determined using biological, chemical or physical measurement methods.
  • nucleus export signal peptides and fusion proteins according to the invention can be used in functional tests in immunology, for example for Identification of specific antibodies, in particular against the amino acid sequence according to formula (I).
  • They can also be used to screen drugs that inhibit or activate nucleus export activity. In particular for screening for substances that have the functions of regulating cytokines and growth factors.
  • the amino-terminal third of the Statl protein contains a number of helices that house amino acids.
  • the drug leptomycin B (LMB), which is known as a specific inhibitor of the export receptor CRM1 (also called exportin 1), was used to identify peptides with an NES function. The export activity of a stat-nuclear export signal can therefore be blocked by LMB.
  • Human HeLa S3 cells and 293T cells were cultivated at 37 ° C. and 7% CO 2 atmosphere in Dulbecco's modified Eagle medium. The growth medium also included streptomycin, penicillin and amphotericin (Biochrom, Berlin). The 293T cells were cultivated on poly-L-lysine-coated culture vessels and transfected with lipofectamine plus (Gibco) in 12-well plates in accordance with the manufacturer's instructions. 20 hours after the transfection, the cells were grown for 30 minutes with growth medium comprising human interferon- ⁇ (IFN ⁇ ; 5 ng / ml; Gibco) and cycloheximidine (CHX; 10 ⁇ g / ml).
  • IFN ⁇ human interferon- ⁇
  • CHX cycloheximidine
  • Helix-1 (amino acids 127-188),
  • Helix-2 (amino acids 183-254)
  • Helix-4 (amino acids 289-314) using the following primer sets:
  • N domain 5 '-atataaggatccccatgtctcagtggtacgaacttcagc-3 ' and
  • the plasmids for generating the GST-GFP fusion proteins for microinjection were prepared as follows:
  • a section of the vector pEGFPNI (from Clontech), which codes for the GFP (green fluorescent protein) protein, was amplified by PCR (primer 5'-atatatagaattcagatggtgagcaaggggcgaggagctg; 5'-gattatgatctagagtcgcggccgc), and with the enzymes Eco, with N enzymes Receiving of the reading frame in the vector pGEX5X-2 (from Pharmacia) alloyed. Genes and gene fragments can now be cloned into this vector. Restriction interfaces are available in the vector EcoRI and BamHI. The molecular biological manipulations were carried out according to standard methods (alleys and shrinking; genetic engineering methods, Spektrum Akademischer Verlag, Heidelberg).
  • the PCR products were ligated into BglII / EcoRI areas of pEGFPC2 (Clontech) for the transfection of the 293T cells, alternatively it was also possible to transfer the PCR products into the BamHI / EcoRI- Cloning areas of the bacterial expression vector (pGST-FFP) . alloy, which are between the cDNAs for the glutathione S-transferase (GST) and the green fluorescent protein (GFP) for the microinjection of the fluorescence fusion protein with StatI fragments (GST-Stat-GFP) in HeLa S3 cells.
  • Bacteria of the E. coli BL21 pLysS strain were transformed with plasmids for the expression of the GST-GFP fusion proteins by means of electroporation. Positive clones were selected using antibiotic resistance.
  • An overnight culture of the corresponding clones was diluted 1: 100 with LB medium (total volume 1 liter) and incubated until an optical density (600 nm) of 0.7 at 37 ° C. with shaking. The culture was then cooled to 15-18 ° C. in ice water, 1 ml of PIG was added and the mixture was incubated for a further 20 hours at 18 ° C. while shaking. The cells were then centrifuged and by freezing three times (on dry ice) / thawing (at 37 ° C.
  • the extraction buffer PBS with 2mM EDTA, 2mM EGTA, 1% Triton X-100, 1mM DTT, complete protease inhibitor, Röche.
  • the extract was centrifuged (40 min at 20,000 g) and the supernatant with 1.5 ml glutathione-Sepharose 4B (Pharmacia) added. After rotating for 2 hours at 4 C, the Sepharose was sedimented (10 min. 10,000 g) and the supernatant was discarded. The Sepharose was washed three times with extraction buffer, once with extraction buffer without Triton X-100, and then eluted (shake gently for 5 min at RT, centrifuge).
  • the elution buffer consisted of PBS with 5 mM reduced glutathione.
  • the eluted protein was then brought to a concentration of approximately 1 mg / ml by ultrafiltration (Centriprep 50 device, from Amicon) (measured using the Biorad protein assay, from Biorad) and then for 15 hours against injection buffer (20 mM HEPES, 110 mM k-acetate, 2 mM Mg acetate, 0.5 mM EDTA, 0.5 mM DTT, pH 7.5) dialyzed.
  • injection buffer (20 mM HEPES, 110 mM k-acetate, 2 mM Mg acetate, 0.5 mM EDTA, 0.5 mM DTT, pH 7.5) dialyzed.
  • the protein could now be injected directly or stored at -80 ° C. Freezing was done on dry ice.
  • Statl redistribution following IFN gamma stimulation follows a typical schedule. 10 minutes after half an hour of stimulation with IFN-gamma, the entire statl is nuclear. After another 3 hours, approximately the same fluorescence is found in the cytoplasmic and nuclear compartments, and after a further five hours the nucleus appears to contain less statl than the surrounding cytoplasm.
  • the helix-4 of the "wound spiral domain” (amino acids 289-314 from Statl) showed translocation activity when the recombinant proteins were injected into the nucleus. After 3 hours of incubation, the fusion protein was located in the cytoplasm and the cell nucleus was largely free of it ,
  • GST-GFP fusion proteins were also formed with the amino- or carboxy-terminal half of helix-4 (GST-h4N (> AS 289-301) lh4C (AS 302 - 314) -GFP). Only the carboxy-terminal half, which contained amino acids 302 to 314 (GST-h4C-GFP), had nuclear export capability. However, a further reduction to 5 residues at the amino terminus of the helix-4C ended the activity of the peptide.
  • a fusion gene was encoded, which encodes Helix-4 (amino acids 292-314 of the Statl protein) and the green fluorescence protein (GFP). Since the molecular mass of the expressed fusion protein ( ⁇ 30 kDa) is small enough to allow entry into the nucleus by diffusion, the GFP fusion protein can be used as a marker for nuclear export. The fusion protein of Helix-4 with GFP was no longer located in the nucleus but clearly in the cytoplasm. This proves that the linear sequence of residues 302 to 314 of Helix-4 can be regarded as a transferable nuclear export signal.

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Abstract

La présente invention concerne des peptides signaux d'exportation nucléaire ainsi que des protéines de fusion qui les contiennent et que leur utilisation. Les peptides signaux de l'invention permettent de modifier le transport de structures biologiques entre le noyau et le cytoplasme.
PCT/EP2001/008065 2000-07-14 2001-07-12 Peptides signaux d'exportation nucleaire (nes), proteines de fusion qui les contiennent, ainsi que leur utilisation WO2002006309A2 (fr)

Priority Applications (2)

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AU2001276400A AU2001276400A1 (en) 2000-07-14 2001-07-12 Novel nucleus export signal peptides (nes), fusion proteins containing the same,and the use thereof
EP01954035A EP1301529A2 (fr) 2000-07-14 2001-07-12 Peptides signaux d'exportation nucleaire (nes), proteines de fusion qui les contiennent, ainsi que leur utilisation

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DE2000135867 DE10035867A1 (de) 2000-07-14 2000-07-14 Neue Nucleus-Export-Signalpeptide (NES), sie enthaltende Fusionsproteine sowie deren Verwendung
DE10035867.5 2000-07-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117304282A (zh) * 2023-10-23 2023-12-29 安徽大学 一种核仁定位信号肽及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
OLAF ROSORIUS ET AL.: "Human Ribosomal Protein L5 Contains Defined Nuclear Localization and Export Signals" THE JOURNAL OF BIOLOGICAL CHEMISTRY, Bd. 275, Nr. 16, 21. April 2000 (2000-04-21), Seiten 12061-12068, XP002186817 *
TONY T. HUANG ET AL.: "A nuclear export signal in the N-terminal regulatory domain of I(kappa)B(alpha) controls cytoplasmic localization of inactive NF-(kappa)B/I(kappa)B(alpha) complexes" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, Bd. 97, Nr. 3, 1. Februar 2000 (2000-02-01), Seiten 1014-1019, XP002186818 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117304282A (zh) * 2023-10-23 2023-12-29 安徽大学 一种核仁定位信号肽及其应用

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