+

WO2006048234A2 - Diagnostic et traitement de maladies associees a l'autophagie - Google Patents

Diagnostic et traitement de maladies associees a l'autophagie Download PDF

Info

Publication number
WO2006048234A2
WO2006048234A2 PCT/EP2005/011680 EP2005011680W WO2006048234A2 WO 2006048234 A2 WO2006048234 A2 WO 2006048234A2 EP 2005011680 W EP2005011680 W EP 2005011680W WO 2006048234 A2 WO2006048234 A2 WO 2006048234A2
Authority
WO
WIPO (PCT)
Prior art keywords
wipi
protein
seq
provision
active ingredient
Prior art date
Application number
PCT/EP2005/011680
Other languages
German (de)
English (en)
Other versions
WO2006048234A3 (fr
Inventor
Tassula Proikas-Cezanne
Alfred Nordheim
Original Assignee
Universität Tübingen
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 Universität Tübingen filed Critical Universität Tübingen
Publication of WO2006048234A2 publication Critical patent/WO2006048234A2/fr
Publication of WO2006048234A3 publication Critical patent/WO2006048234A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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 present invention relates to proteins which are crucially involved in the cellularyngic process of autophagy and the corresponding nucleotide sequences. It further comprises the provision of a substance for diagnostic detection of at least one of these proteins, and the provision of an agent for influencing at least one of these proteins for the therapeutic treatment of diseases associated with misdirected autophagy, and methods therefor.
  • the present invention relates to a pharmaceutical composition and a diagnostic kit.
  • Autophagy is a cellular process known for several years that results in the degradation of cellular components, particularly proteins, organelles, infectious agents, etc. that are unusable or undesirable for the maintenance of normal cellular physiology.
  • the process of autophagy is evolutionarily conserved, and the cellular proteins for performing autophagy are homologous in yeasts and mammals (evolutionary preservation).
  • autophagosomes which surround the components to be degraded and, by subsequent membrane fusion with the lysosomes, make them accessible to the enzymes of the lysosomal degradation machinery.
  • Autophagy can be triggered by nutrient deficiency and, under these conditions, represents a process which leads to the provision of amino acids and energy by intracellular removal of already existing cell constituents. Under these conditions, especially in the case of flies and worms, a life-prolonging mechanism may be be tivated. In humans, autophagy is also activated under starvation conditions or in the fasting state ("calorie restriction") as well as in reduced levels of insulin, while in obesity (obesity) disorders of the autophagy process are to be expected.
  • Non-apoptotic programmed cell death occurs naturally in the elimination of unwanted immune cells, for example B cells, as well as in embryonic development Development of mammals (cavitation) and in organogenesis in mammals.
  • a misdirected autophagy may be involved in the development of a variety of diseases, particularly pathological conditions of severe severity.
  • tumor diseases can be triggered by incomplete degradation of oncoproteins or by incomplete elimination of mutant cells.
  • Neurodegenerative diseases for example Huntington's disease, are frequently due to incomplete degradation of neuronal proteins ("plaques") or to the autophagic cell death of neurons
  • plaques neuronal proteins
  • Myopathies in particular cardiomyopathies, are based on an unregulated elimination of muscle cells
  • infectious diseases the infectious agents within an infected cell are often only incompletely eliminated, or se lective autophagic cell death of cell populations of the immune system takes place.
  • Fluther diseases which are caused by a disturbed autophagy, relate to inflammatory diseases as well as disorders of fat metabolism , especially obesity, type II diabetes, cachexia, etc.
  • the invention therefore has as its object to identify at least one protein or the corresponding nucleotide sequence which (s) is involved in the process of autophagy and thus also in the development of autophagy. phagia-associated diseases.
  • the provision of substances for detecting and influencing these novel proteins is a further object of the invention.
  • Claim 8 relates to a substance for detecting the expression and / or function of at least one of these proteins.
  • Claims 9 and 10 are concerned with the active compounds according to the invention. Preferred embodiments are mentioned in the dependent claims 11 to 27.
  • Claims 28 and 33 relate to a method, claim 35 to a pharmaceutical composition, claim 38 to a diagnostic kit and claim 40 to a use.
  • the proteins according to the invention are what are known as WIPI (WD-repeat proteins interfering with PJiospholipidinosides) proteins. These belong to the WD-repeating proteins which contain a conserved range of about 40 amino acids, which usually ends with tryptophan aspartate, and forms a circular beta-propeller structure.
  • the WD-repeating proteins are involved in many cellular regulatory processes, in particular cell proliferation, apoptosis, signal transduction, RNA metabolism and chromatin condensation. They regulate the assembly of multi-protein complexes by providing a stable platform for simultaneous and reversible protein-protein interactions.
  • the inventors were able to identify new members in the form of splice variants of the human WIPI gene and protein family. These include hWIPM ⁇ , hWIPI-2 ⁇ , hWIPI-2 ⁇ and hWIPI-2 ⁇ . Furthermore, the hWIPI-3-like protein was identified.
  • hWIPM ß, WIPI-2 ⁇ , hWIPI-3 and hWIPI-4 have already been described in the literature [Jeffries, TR, Dove, SK, Michell, RH & Parker, PJ (2004), Mol Biol Cell, 15, 2652-63].
  • the inventors were able to demonstrate the involvement of human WIPI proteins, in particular of WIPI-1 protein, in the cellular process of autophagy.
  • Autophagy in mammalian cells was induced by amino acid withdrawal.
  • autophagy involving WIPI proteins can be triggered by cell- and / or DNA-damaging influences.
  • special forms of cellular particles, so-called autophagosomes are formed intracellularly.
  • WIPI proteins in particular the WIPI-1 protein
  • PAS pre-autophagosomal structure
  • human WIPI-1 may play a role in late stages of autophagia.
  • WIPI-3 antiserum that the WIPI-3 protein co-localized with mitochondria.
  • WIPI proteins may be involved in the formation and transport of so-called synaptic vesicles in neurons. Synapses are the cell-cell transition regions via which stimulus and information transmission takes place between adjacent nerve cells. This WIPI transport function applies both to the cells of the central nervous system (CNS) and to the cells of the peripheral nervous system (PNS), including the neuromuscular end plates. Furthermore WIPI proteins may be involved in the membrane-supported transport of biological macromolecules, in particular mRNA, microRNA, tRNA and / or proteins, and protein complexes, for example ribosomes, within cells, in particular within nerve cells.
  • the protein according to the invention is characterized in that it is at least partially encoded by a nucleotide sequence or parts thereof which are at least 70%, in particular at least 80%, identical to at least one nucleotide sequence from SEQ ID NO: 1 and SEQ ID NO: 3 and SEQ ID NO: 5 and SEQ ID NO: 7 and SEQ ID NO: 11.
  • the protein according to the invention is characterized in that it is at least partially encoded by a nucleotide sequence or parts thereof which are at least 90%, in particular at least 95%, identical to at least one nucleotide sequence from SEQ ID NO: 1 and SEQ ID NO: 3 and SEQ ID NO: 5 and SEQ ID NO: 7 and SEQ ID NO: 11.
  • the proteins according to the invention can be isolated and purified, for example, from an organism. However, it may also be preferable to express the proteins in an experimental system.
  • the invention comprises nucleotide sequences or parts thereof which are at least 70%, in particular at least 80%, identical to at least one nucleotide sequence from SEQ ID NO: 1 and SEQ ID NO: 3 and SEQ ID NO: 5 and SEQ ID NO: 7 and SEQ ID NO: 11.
  • nucleotide sequences or parts thereof are preferred which are at least 90%, preferably at least 95%, identical to at least one nucleotide sequence from SEQ ID NO: 1 and SEQ ID NO: 3 and SEQ ID NO: 5 and SEQ ID NO: 7 and SEQ ID NO: 11.
  • These nucleotide sequences may be in isolated form, preferably as cDNAs.
  • the present invention encompasses the promoters of WIPI genes that regulate gene expression, in particular induced by stress and / or DNA damage.
  • the invention comprises at least one nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 3 and SEQ ID NO: 5 and SEQ ID NO: 7 and SEQ ID NO: 11.
  • This is at least one nucleotide sequence
  • WIPI protein should be understood as meaning both a wild-type protein and a modified protein which is coded for by a modified WIPI gene sequence, including possible splice variants. In a preferred manner, this is a protein according to the invention as claimed in claim 1.
  • the term WIPI protein also expressly includes peptide fragments of a WIPI protein. Preferably, it may be a peptide fragment which is present as a MHC (major histo-compatibility complex) -presented peptide on the surface of a cell, in particular in pathological conditions, preferably in tumor diseases.
  • MHC major histo-compatibility complex
  • At least one substance can be provided for detecting the expression and / or the function of at least one WIPI protein in eukaryotic cells.
  • This substance could be a polypeptide or protein, in particular an enzyme or an immunological reagent, for example an antibody.
  • the antibody can be a monoclonal or polyclonal antibody which is present in particular in a corresponding antiserum (monoclonal or polyclonal antiserum).
  • the antibody can be directed, for example, against the WIPI protein and as a so-called anti-WIPI immunological reagent in the context of an immunoassay, for example Western blot, protein chip or ELISA (enzyme linked immunosorbent assay), for the detection of the WIPI protein to be used.
  • an antibody which is specifically directed against an antigen for example against the WIPI protein
  • a solid support preferably cellulose or polystyrene.
  • a labeled antibody is added in a second step.
  • the antibody may be labeled, for example, with an enzyme which permits colorimetric detection after addition of a chromogenic substrate. Therefore, the antigen concentration in the sample can be determined by a colorimetric determination of the immune complex-bound marker enzymes by comparison with standards of known enzyme activity.
  • all immunoassays known from the prior art can be used.
  • the immunological reagent can be a peptide aptamer, ie an artificially produced peptide which is specifically directed against or interacts with the WIPI protein and the detection of the WIPI protein, in particular according to one of the methods described, allowed.
  • all the Expert known reagents which are suitable for the marking of WIPI expression and / or function, for example Green Fluorescent Protein (GFP) or digoxigenin, are used.
  • GFP Green Fluorescent Protein
  • fusion protein for example GFP (Green Fluorescent Protein) WIPI.
  • oligonucleotides preferably cDNAs
  • a differential mRNA fingerprinting assay can be carried out in order to detect the expression intensity of WIPI genes at different pathological states.
  • nucleic acid detection techniques for example Southern blot, Northern blot and all variants of hybridization techniques, including in situ hybridization, RT-PCR (reverse transcription polymerase chain reaction) or gene chip.
  • the invention further provides an active ingredient for influencing, in particular for activating and / or inhibiting the expression and / or function of at least one WIPI protein in eukaryotic cells for the treatment of pathological conditions, in particular auto-phagia-associated diseases.
  • the active ingredient may also be a polypeptide or protein, in particular an enzyme or immunological reagent.
  • an antibody for example a monoclonal or polyclonal antibody, and / or a peptide aptamer. In particular, it may also be a function inhibitor or activator to a dominant negative mutant of the WIPI protein or to act as an antisense molecule.
  • the active ingredient is preferably used for the production of a medicament or a pharmaceutical composition.
  • the active ingredient is directed against the WIPI protein itself.
  • the active substance may be an antisense sequence, but also an already mentioned functional inhibitor or activator or a dominant negative mutant of the WIPI protein.
  • an activator example a genetically engineered mutant of the WIPI protein can be used.
  • the active ingredient influences, preferably reacts with, evolutionarily conserved amino acids of the WIPI protein.
  • the conserved amino acids in WIP1 proteins define the biological function of WIPI proteins, in particular the binding of phospholipids.
  • the inventors succeeded in demonstrating the function of the WIPI-1 protein as an intracellular binding module of the phospholipid PI (3) -P (phosphatidyl-inositol-3-phosphate).
  • the inventors for the first time identified evolutionarily conserved amino acids which are required for correct intracellular binding of the phospholipid.
  • the following evolutionarily conserved amino acids could be identified for the human WIPI-i ⁇ protein (each with position number): N23, Q24, D25, E64, R107, R110, R112, H185, G198, S203, S205, G208, T209, R212 , E224, R226, R227, G228, S251, T255, H257, S259, S335 and G336.
  • the evolutionarily conserved amino acids have a correspondingly different position in the other human WIPI proteins, in the human WIPI splice variants, in orthologous and paralogue WIPI sequences of other organisms.
  • the active substance influences, in particular activates or inhibits, the intracellular availability of phospholipids, in particular of Pl (3) -P, for interaction with WIPI proteins.
  • the intracellular availability of phospholipids depends, in particular, on whether or to what extent the phospholipids are present in front of an interaction with the WIPI proteins in bound form, in particular with proteins as binding partner.
  • the active ingredient provided according to the invention preferably influences, in particular activates and / or inhibits, the cellular functions of mitochondria, in particular the various forms of programmed cell death, for example autophagosomal cell death and / or apoptotic cell death, and / or energy balance regulation Cell.
  • the active ingredient influences, in particular activates and / or inhibits, the expression and / or function of the WIPI-3 protein.
  • a low molecular weight molecule in particular one with a molecular weight (MW) ⁇ 1000, possible.
  • a low molecular weight molecule can be, for example, a functional inhibitor or activator. It is also advantageous to use a polynucleotide which codes for a peptide which influences, preferably activates or inhibits, the expression of the WIPI protein.
  • the active ingredient is directed against at least one regulator, activator, inhibitor and / or against a biological precursor of the WIPI protein.
  • This regulator, activator, inhibitor and / or biological precursor can be, for example, an up- and / or downstream transcrip- be responsible for the level of expression of the WlPI protein ver ⁇ .
  • These are preferably sulfatase.3 and / or the ILF (interleukin enhancer-binding factor) -1-like protein, where sulfatase.3 is the anti-sense transcript to WIPI-1 and the ILF-1-like protein is anti-sense Transcript to WIPI-3, which can therefore influence the gene regulation of WIPI-1 and WIPI-3, in particular activate or inhibit.
  • the regulator may be the so-called TOR (Target Of Rapamycin) protein.
  • TOR Target Of Rapamycin
  • it may also be a protease which is responsible for the proteolytic degradation of a regulator, activator, inhibitor and / or biological precursor.
  • the active ingredient may be a polynucleotide which codes for a polypeptide which influences the expression and / or function of at least one regulator, activator, inhibitor and / or biological precursor of the WIPI protein, in particular activated or inhibited. This includes sequences which completely or partially encode these proteins, both as sense and as antisense sequences.
  • the active substance can modulate, in particular activate or inhibit, post-translational modifications of the WIPI protein.
  • modifications of phosphorylation and sumoylation This can take place, for example, by influencing, in particular activating or inhibiting, an enzyme, preferably a kinase and / or sumo ligase, etc., which carries out the post-translational modification of the WIPI protein.
  • an enzyme preferably a kinase and / or sumo ligase, etc.
  • any other form of post-translational modification is encompassed by the present invention.
  • the active ingredient it is likewise possible for the active ingredient to modulate, in particular activate or inhibit, the effector function of the WIPI protein by influencing the assembly with at least one binding partner, preferably a protein.
  • WIPI partner proteins preferably include the tumor suppressor proteins p53 and Rb, the cell-division cyclin D regulatory regulator, and nuclear receptors of steroid hormones, in particular estrogen receptors, retinoic acid receptors and PPAR (peroxisome proliferator-activated receptor), as well as all future identified partner proteins ,
  • WIPI proteins are also important targets for the correction of any cellular miscarriage involving WIPI proteins in the development of human disease.
  • the invention can be used to treat all forms of disorders associated with misdirected autophagy. These are in particular tumor diseases, neurodegenerative diseases, myopathies, infectious diseases, inflammatory diseases and / or aging diseases.
  • the invention can be used for the treatment of diseases which are based on a disturbed peroxisome biogenesis.
  • Peroxisomes are cell organelles that carry out the breakdown of fatty acids and amino acids. The number of peroxisomes in a cell undergoes a dynamic equilibrium (synthesis / synthesis versus degradation of peroxisomes). Disturbed peroxisome biogenesis can lead to pathological conditions, in particular to hyperoxuria syndrome, Refsum's syndrome, ALDP (adreno-leuco-dystrophy), RCDP (rhizomelic chondrodysplasia punctata), Zellweger syndrome and all PBDs (peroxisome biogenesis disorder).
  • the cellular degradation of peroxisomes proceeds with the aid of autophagosomal proteins, in particular with the aid of WIPI proteins. It is therefore possible, by influencing, in particular activation and / or inhibition of expressions sion and / or function of WIP1 proteins to treat the diseases just mentioned.
  • diseases of the fatty substance metabolism can be treated by the invention.
  • the fat-degrading function of the hepatocyte essentially restores the function of the peroxisomes of liver cells.
  • the WlPI proteins can be used as target structures for the therapy of disorders of lipid metabolism, in particular of the liver.
  • the provision of an active substance in the manner described for the stimulation of lipid metabolism and improved regeneration of the liver can lead to infections, for example hepatitis, cancers, for example hepatocellular carcinoma, and fibroses (alcoholic liver).
  • Cachexia is an emaciation syndrome that affects 50% of cancer patients.
  • fat and muscle tissue are broken down.
  • the proteolytic process is of great importance, even with the involvement of autophagy.
  • the present invention is also suitable for the therapy of Cache ⁇ xie.
  • the invention furthermore relates to a method for detecting and / or investigating the autophagy status in eukaryotic cells, encompassing the method steps
  • the detection method mitotic, d. H. in the growth phase, cells provided.
  • the mitotic cells are G361 cells, as these express a high concentration of WIPI proteins and perform autophagy efficiently.
  • the WIPI protein is coupled with a labeling reagent, in particular a fluorescent labeling reagent the WIPI protein is coupled with a fluorescent immunological anti-WIPI reagent, preferably with a fluorescent antibody and / or a fluorescing peptide aptamer.
  • the WIPI protein is expressed as a WIPI fusion protein, in particular as a fluorescent WIPI fusion protein.
  • the WIPI protein is expressed as GFP-WIPI fusion protein. In this way, a labeling step required after the expression of the WIPI protein is omitted.
  • the expression and / or distribution and / or function of the WIPI protein can be detected and / or examined by visualization methods, in particular fluorescence microscopic, fluorimetric, fluorescence-cytometric and / or immunological methods, in particular according to one of the methods already described.
  • the invention also includes a method for assaying the activity of at least one substance for influencing the autophagy process, in particular by activating and / or inhibiting the expression and / or function of at least one WIPI protein in eukaryotic cells for the treatment of pathological conditions
  • the disorders associated with autophagy include the process steps
  • mitotic cells preferably G361 cells
  • the substance are incubated with the substance, and subsequently the influence of this substance on the formation of autophagosomes having WIPI proteins is examined.
  • activators of autophagy can be recognized by the induction of autophageal particles.
  • the substance can be administered in combination with an inducer of autophagy, for example EBSS (Earle's buffered salt solution) medium, whose positive effect on autophagy by the desired inhibitor, for example PI-3 kinase inhibitor word- mannin, is stopped.
  • EBSS Aral buffered salt solution
  • PI-3 kinase inhibitor word- mannin an inducer of autophagy
  • this process is suitable for the identification of active substances, in particular activators or inhibitors. ren, the car dealership.
  • Such agents may either affect, in particular activate and / or inhibit, the expression and / or function of WIPI proteins or their regulators, activators, inhibitors and / or biological precursors.
  • these active ingredients could influence, in particular activate and / or inhibit, the process of autophagy via other, in particular still unknown, cellular target structures.
  • the invention further encompasses a pharmaceutical composition which comprises at least one active substance which influences the expression and / or function of at least one WIPI protein, in particular activates or inhibits it, and optionally a pharmaceutical carrier material.
  • the active ingredient may be a polypeptide or protein, in particular an enzyme or antibody. Preferably, as already mentioned above, it may be a function inhibitor or activator or a dominant negative mutant of the WIPI protein.
  • the active ingredient may be a poly nucleotide, in particular a cDNA.
  • the active agent may be a polynucleotide encoding a polypeptide that affects, preferably activates or inhibits, the expression of the WIPI protein.
  • the active ingredient according to the invention may, moreover, be a low molecular weight compound, in particular a low molecular weight molecule having a molecular weight (MW) ⁇ 1,000.
  • the active ingredient may also be a so-called antisense sequence. This is understood as meaning a sequence which is able to form a double strand with the mRNA, for example of the WIPI protein, in order to prevent the translation of a target peptide or protein in this way. It is also possible to use the sequence of the WlPI protein itself in order to avoid overexpression. sion, for example by incorporation into a vector or a plasmid.
  • the active ingredient of the pharmaceutical composition influences, in particular activates or inhibits, the expression and / or function of a regulator, activator, inhibitor and / or biological precursor of the WIPI protein.
  • This regulator, activator, inhibitor and / or biological precursor can be, for example, a kinase and / or a ligase which is involved in the regulation of the activity of the WIPI protein.
  • this is a transcription factor which plays a role in the level of expression of the WIPI protein.
  • a polynucleotide which codes for a polypeptide which influences, in particular activates or inhibits, the expression of a regulator, activator, inhibitor and / or biological precursor of the WIPI protein can also be present in such a composition.
  • the active substance is a low molecular weight molecule which preferably has a molecular weight (MW) ⁇ 1,000, and which influences the expression and / or function of the WIPI protein, in particular activates or inhibits it.
  • MW molecular weight
  • the invention further relates to a diagnostic kit.
  • This comprises at least one substance which is suitable for detecting the expression and / or distribution and / or function of at least one WIPI protein in eukaryotic cells, for the diagnosis of pathological conditions, in particular of autophagy-associated diseases.
  • kits diseases can be diagnosed which are associated with an over- or under-expression or with an over- or under function of the WIPI protein.
  • the detection of the diseases can be carried out by detecting a disturbed expression and / or function of the WIPI protein.
  • the substance may be one which provides this detection as a poly-nucleotide and / or polypeptide.
  • the protein equivalents are proteins from other organisms, in particular yeast, fruit fly and / or plants.
  • WIPI proteins are evolutionarily highly conserved and exist in all organisms, from unicellular organisms, for example yeasts, to humans, including plants.
  • the modulation of WIPI protein functions can be usefully employed in many organisms.
  • the present invention therefore also encompasses the use of an active ingredient according to the above description in biotechnology, in particular for improving the properties of useful plants and / or microorganisms.
  • biotechnological use of crops for example in terms of defense against plant infections, improved viability of plants under stress conditions, etc., be optimized by modulating agents of WIPI proteins.
  • the yields of agricultural crops (food technology) can be improved.
  • the biotechnological use of microorganisms can be optimized by influencing the WIPI protein functions. It may further be preferred to use WIPI genes or WIPI proteins in biosensing, preferably in unicellular organisms, in order to reflect environmental conditions by inducible autophagy. It may also be preferred to establish a biosensor in yeast cells or in suitable multicellular organisms in order to detect chemical and biological substances by receptor-coupled, inducible autophagy and the associated accumulation of WIPI proteins, preferably as a fluorescent fusion protein. Particularly preferably, the WIPI genes or WIPI proteins can be used in the biosynthetic productivity and / or in the aging behavior.
  • SEQ ID NO: 2 hWIPI-1a amino acid sequence
  • SEQ ID NO: 4 hWIPI-2 ⁇ amino acid sequence
  • SEQ ID NO: 6 hWIPI-2 ⁇ amino acid sequence
  • SEQ ID NO: 7 hWIPI-2 ⁇ cDNA
  • SEQ ID NO: 8 hWIPI-2 ⁇ amino acid sequence
  • SEQ ID NO: 12 hWIPI-3-like amino acid sequence
  • SEQ ID NO: 15-22 primer for preparation of specific 32 P-labeled cDNA samples for the human WIPI proteins 1-4 (Example I)
  • SEQ ID NO: 23-34 primer for the isolation of WIPI genes (Example II)
  • FIG. 1 Analysis of hWIPI-1 mRNA expression in human melanoma cell lines and hWIPI protein expression in G361 and He-La cells
  • the oligonucleotide sequences SEQ ID NO: 15-22 were amplified in standard PCR amplifications (20 cycles: 94 ° C. 30 s, 55 ° C. 30 s, 72 ° C. 1 min, 1 cycle: 72 0 C 2 min; REDTAG TM, polymerase, SIGMA) were used: human WIPI-1 SEQ ID NO: 15, SEQ ID NO: 16, human WIPI-2 SEQ ID NO: 17, SEQ ID NO : 18, human WIPI-3 SEQ ID NO: 19, SEQ ID NO: 20, and human WIPI-4 SEQ ID NO: 21, SEQ ID NO: 22.
  • PCR fragments were labeled using the random primer DNA labeling system (Fig. Invitrogen) and Chroma Spin TM columns (BD Biosciences) according to the manufacturer's protocols cleaned.
  • the instructions from the following arrays or blots were followed: Cancer Profiling Array II, RNA Master Blot, Human MTN Blot 1 / ll (BD Biosciences).
  • the RNeasy kit (Qiagen) was used for the isolation of the entire RNA and the standardized Northern blotting and hybridization methods were carried out by using the ExpressHyb solution (BD Biosciences).
  • RNA was stained with ethidium bromide, and the arrays or blots were hybridized with 32 P labeled actin or ubiquitin (BD Biosciences) hybri ⁇ .
  • a partial hWIPI-1 cDNA fragment (without nucleotides 1-152) was isolated from a human liver cDNA library (clone 78) in a p53 inhibitory capacity assay.
  • the missing 5 'DNA sequence was cloned by RT-PCR (advantage of one-step RT-PCR, BD Biosciences) from mRNA of human testes (BD Biosciences).
  • the 3'-oligonucleotide having the sequence SEQ ID NO: 23 was designed so that it coincides with a single BglII site (nucleotides 277/281) within the ORF of hWIPI-1.
  • the 5'-oligonucleotide having the sequence SEQ ID NO: 24 was designed in such a way that it agrees with the sequence of the EST sequences obtained from NCBI (AA482531, Z24843, AA043660, AK079986). This RT-PCR fragment was fused to the initial cDNA clone isolate (clone 78) to obtain pAR31CD-hWIPI-1 ⁇ .
  • the full hWIPM ⁇ cDNA by PCR was amplificate sheet (15 cycles; 1 min 96 0 C, 53 0 C 1, 5 min, 72 0 C for 2.5 min; Pfu Turbo polymerase / Stratagene) using the pAR31CD-hWIPI-1 ⁇ as a template with oligonucleotide sequences SEQ ID NO: 25-26.
  • the resulting PCR fragment was subcloned into pEGFP.CI (BD Biociences); Cut EcoRI / Xhol).
  • RT-PCR TITANUM one-step RT-PCR, BD Biosciences
  • the mRNA of human testes BD Biosciences
  • the oligonucleotides according to the sequences 27- 34 which were prepared according to the BLAST search results at NCBI, were used: hWIPI-2 ⁇ SEQ ID NO: 27-28, hWIPI-2 ⁇ SEQ ID NO: 29-30, hWIPI-3 SEQ ID NO: 31-32 , hWIPI-4 SEQ ID NO: 33-34.
  • the resulting PCR fragments were cloned into pEGFP.CI.
  • Genbank accession numbers were assigned to our WIPI isolates: AY691424 (hWIPM ⁇ ), AY691425 (hWIPI-2 ⁇ ), AY691426 (hWIPI-2 ⁇ ), AY691427 (hWIPI-3), AY691428 (hWIPI-4).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Physiology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne de nouveaux membres de la famille des protéines WIPI, ces membres constituant des structures cibles intéressantes pour la modulation de l'autophagie cellulaire et donc pour le diagnostic et le traitement de maladies associées à l'autophagie. L'invention concerne en outre des séquences nucléotidiques codant pour ces protéines. L'invention concerne par ailleurs un procédé permettant de mettre en évidence et/ou d'analyser une autophagie et d'identifier des matières actives qui influent sur l'autophagie, notamment qui l'activent et/ou l'inhibent.
PCT/EP2005/011680 2004-11-02 2005-11-02 Diagnostic et traitement de maladies associees a l'autophagie WO2006048234A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE102004053508 2004-11-02
DE102004053508.6 2004-11-02
DE102005028117.6 2005-06-10
DE102005028117A DE102005028117A1 (de) 2004-11-02 2005-06-10 Diagnostik und Therapie von Autophagie-assoziierten Erkrankungen

Publications (2)

Publication Number Publication Date
WO2006048234A2 true WO2006048234A2 (fr) 2006-05-11
WO2006048234A3 WO2006048234A3 (fr) 2007-01-18

Family

ID=36123068

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/011680 WO2006048234A2 (fr) 2004-11-02 2005-11-02 Diagnostic et traitement de maladies associees a l'autophagie

Country Status (2)

Country Link
DE (1) DE102005028117A1 (fr)
WO (1) WO2006048234A2 (fr)

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BARTH HENNING ET AL: "Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p" FEBS LETTERS, Bd. 508, Nr. 1, 9. November 2001 (2001-11-09), Seiten 23-28, XP004322270 ISSN: 0014-5793 *
DATABASE EMBL [Online] 1. März 2001 (2001-03-01), "602436137F1 NIH_MGC_46 Homo sapiens cDNA clone IMAGE:4553960 5', mRNA sequence." XP002394169 gefunden im EBI accession no. EM_EST:BG338356 Database accession no. BG338356 *
DATABASE EMBL [Online] 12. Oktober 2001 (2001-10-12), "603390123F1 NIH_MGC_87 Homo sapiens cDNA clone IMAGE:5399237 5', mRNA sequence." XP002377077 gefunden im EBI accession no. EM_PRO:BI860703 Database accession no. BI860703 *
DATABASE EMBL [Online] 22. Februar 2002 (2002-02-22), "ih21b03.x1 Human insulinoma Homo sapiens cDNA clone IMAGE: 3' similar to TR:Q9Y4P8 Q9Y4P8 HYPOTHETICAL 49.4 KD PROTEIN. ;, mRNA sequence." XP002394168 gefunden im EBI accession no. EM_EST:BM504100 Database accession no. BM504100 *
DATABASE EMBL [Online] 4. Januar 2002 (2002-01-04), "Homo sapiens WD repeat domain, phosphoinositide interacting 2, mRNA (cDNA clone IMAGE:2966543)." XP002394166 gefunden im EBI accession no. EM_HTG:BC021068 Database accession no. BC021068 *
DATABASE EMBL [Online] 6. April 1998 (1998-04-06), "aj90h11.s1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone IMAGE:1403781 3', mRNA sequence." XP002377076 gefunden im EBI accession no. EM_PRO:AA890032 Database accession no. AA890032 *
DATABASE EMBL [Online] 6. September 2001 (2001-09-06), "603241648F1 NIH_MGC_95 Homo sapiens cDNA clone IMAGE:5284450 5', mRNA sequence." XP002394170 gefunden im EBI accession no. EM_EST:BI544273 Database accession no. BI544273 *
DATABASE GENBANK-SVA [Online] 10. Dezember 2001 (2001-12-10), "Homo sapiens hypothetical protein 628 (LOC56270), mRNA." XP002394171 gefunden im NCBI accession no. gi:9625015 Database accession no. NM_019613 *
DATABASE GENBANK-SVA [Online] 7. April 2003 (2003-04-07), "Homo sapiens hypothetical protein FLJ10055 (FLJ10055), mRNA." XP002377075 gefunden im NCBI accession no. gi:8922207 Database accession no. NM_017983 *
DATABASE UNIPROTKB-SVA [Online] 1. Juni 2001 (2001-06-01), "WD40-like containing protein, full insert sequence" XP002394172 gefunden im EBI accession no. Q9CR39 Database accession no. Q9CR39 *
DATABASE UNIPROTKB-SVA [Online] 1. November 1999 (1999-11-01), "Hypothetical protein DKFZp434J154." XP002394167 gefunden im EBI accession no. Q9Y4P8 Database accession no. Q9Y4P8 *
DOVE STEPHEN K ET AL: "Svp1p defines a family of phosphatidylinositol 3,5- bisphosphate effectors" EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, Bd. 23, Nr. 9, 5. Mai 2004 (2004-05-05), Seiten 1922-1933, XP002377034 ISSN: 0261-4189 *
JEFFRIES TIM R ET AL: "PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49" MOLECULAR BIOLOGY OF THE CELL, Bd. 15, Nr. 6, Juni 2004 (2004-06), Seiten 2652-2663, XP002377032 ISSN: 1059-1524 in der Anmeldung erw{hnt *
PROIKAS-CEZANNE TASSULA ET AL: "WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy" ONCOGENE, Bd. 23, Nr. 58, 16. Dezember 2004 (2004-12-16), Seiten 9314-9325, XP002377035 ISSN: 0950-9232 *

Also Published As

Publication number Publication date
DE102005028117A1 (de) 2006-05-24
WO2006048234A3 (fr) 2007-01-18

Similar Documents

Publication Publication Date Title
Smeyne et al. Fos-IacZ transgenic mice: mapping sites of gene induction in the central nervous system
DE69434670T2 (de) Verfahren und produkt für die regulation des reaktionsvermögens von zellen auf äussere signale
Shamloo et al. Npas4, a novel helix–loop–helix PAS domain protein, is regulated in response to cerebral ischemia
DE60126115T2 (de) Oberflächentransfektion und expressionsverfahren
EP1469316B1 (fr) Procédé de criblage
DE10123055A1 (de) Screeningverfahren mit PIM1-Kinase oder PIM3-Kinase
DE19957065A1 (de) Screening-Verfahren für Arzneistoffe
DE69935414T2 (de) Humanes h-TRCP Protein
AU2001295457B2 (en) Use of CARP inhibitors for the treatment of heart diseases
WO2006048234A2 (fr) Diagnostic et traitement de maladies associees a l'autophagie
AU2001295457A1 (en) Use of CARP inhibitors for the treatment of heart diseases
DE69231710T2 (de) Tyrosinkinase
KR100940092B1 (ko) 아토피 피부염 치료 후보 물질의 스크리닝 방법
DE602004013319T2 (de) Hefemodell für die toxizität amyloidogener proteine
DE10018464A1 (de) Testsystem zur Charakterisierung von Modulatoren des Spleißprozesses von mRNA in lebenden Zellen (in vivo), dessen Herstellung und Verwendung
DE19838837A1 (de) Zellspezifische Promoter des Entkopplungsproteins 3
DE10011926A1 (de) Genetisch veränderte Fibroblastenzellen
DE69918260T2 (de) Methoden zur Identifizierung und Validierung von funktionellen Zielen mittels Intrameren oder in vivo Selektion
EP1395834A2 (fr) Procede de criblage a l'aide des proteines bnpi et dnpi
EP4141102A1 (fr) Acide nucléique capable d'interagir avec un récepteur de perturbateurs endocriniens, et son utilisation
DE19964386B4 (de) Verfahren zur Herstellung von Makrophagenmigrations-Inhibitionsfaktor (MIF)
Hong et al. Piezo1 transduces inflammatory pain signals in nociceptors
EP1098985A1 (fr) Genes de la famille des proteines de la boite dead, leurs produits d'expression et leur utilisation
US20080115235A1 (en) Drugs For Diseases Accompanying Changes In Total Bile Acid Pool Or Lipid Metabolism Disorders And Method Of Screening These Drugs
EP0797669A1 (fr) Modeles de criblage

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05803912

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载