WO2006048234A2

WO2006048234A2 - Diagnosis and therapy of autophagy-associated diseases

Info

Publication number: WO2006048234A2
Application number: PCT/EP2005/011680
Authority: WO
Inventors: Tassula Proikas-Cezanne; Alfred Nordheim
Original assignee: Universität Tübingen
Priority date: 2004-11-02
Filing date: 2005-11-02
Publication date: 2006-05-11
Also published as: WO2006048234A3; DE102005028117A1

Abstract

The invention relates to novel members of the WIPI family of proteins which represent valuable target structures for the modulation of cellular autophagy and also for the diagnosis and treatment of autophagy-associated diseases. The invention also relates to nucleotide sequences which encode said proteins. The invention further relates to a method for detecting and/or examining autophagy in addition to the identification of active substances which influence the autophagy, in particular in an active and/or inhibiting manner.

Description

Description Diagnosis and therapy of autophagy-associated diseases

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. In addition, 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). In the course of the process of autophagy, vesicular membrane structures are formed, so-called 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.

Autophagy can also lead to the death of individual cells in an organism ("non-apoptotic programmed cell death") .This type of 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.

It has also been shown that a misdirected autophagy may be involved in the development of a variety of diseases, particularly pathological conditions of severe severity. For example, 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 Myopathies, in particular cardiomyopathies, are based on an unregulated elimination of muscle cells In 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.Further 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.

The object is achieved by at least one protein, as described in An¬ claim 1. The corresponding nucleotide sequences can be found in claims 2 to 7. 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 hWIPMα, hWIPI-2β, hWIPI-2δ and hWIPI-2ε. Furthermore, the hWIPI-3-like protein was identified. Four members of this gene and protein family, namely hWIPM ß, WIPI-2α, 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. Furthermore, autophagy involving WIPI proteins can be triggered by cell- and / or DNA-damaging influences. In the course of the autophagy process, special forms of cellular particles, so-called autophagosomes, are formed intracellularly. Depending on the degree of maturity of the autophagosomal process, these particles take the form of "points", "sickles" or "spheres." By using so-called autophagy markers, for example an LC-3 marker, it has been shown that WIPI proteins, in particular the WIPI-1 protein, are essential components of the pre-autophagosomal structure (PAS) in the form of "dots". In addition, human WIPI-1 may play a role in late stages of autophagia. In addition, it could be demonstrated by using a WIPI-3 antiserum that the WIPI-3 protein co-localized with mitochondria. Surprisingly, it has now been found that the development of autophagy-associated diseases, for example of tumor diseases, correlates with the misprinting of WIPI proteins. In particular, WIPI proteins may have tumor-suppressing properties.

Furthermore, 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. Preferably, 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.

Furthermore, 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. In particular, 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. Depending on the intended use can but they can also be incorporated into a vector, for example an expression vector. Furthermore, these sequences can also be combined with other sequences. Furthermore, the present invention encompasses the promoters of WIPI genes that regulate gene expression, in particular induced by stress and / or DNA damage.

In a very particularly preferred embodiment of the invention, 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 Preferably, characterized in that it encodes at least one human WIPI protein from the group hWIPI-1α and hWIPI-2ß and hWIPI-2δ and hWIPI-2ε and hWIPI-3 like protein.

In the following, the term 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.

The term activation is to be understood below as meaning both the stimulation of a non-existent expression and / or function of at least one WIPI protein and the increase or enhancement of an already existing expression and / or function of at least one WIPI protein. According to the invention, at least one substance can be provided for detecting the expression and / or the function of at least one WIPI protein in eukaryotic cells. In particular, a diagnosis of diseases that are associated with a misdirected autophagy is thus possible. 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. In the ELISA test, an antibody which is specifically directed against an antigen, for example against the WIPI protein, is immobilized on a solid support, preferably cellulose or polystyrene. After formation of the immune complexes, 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. In principle, all immunoassays known from the prior art can be used.

Furthermore, 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. Likewise, 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. Thus, it may be preferred that such labeling reagents, for example GFP, and the WIPI protein to be detected are present in the cell to be investigated as a so-called fusion protein, for example GFP (Green Fluorescent Protein) WIPI.

Particularly preferred and of particular diagnostic value for the detection and treatment of diseases which are caused by a Fehlregu¬ lation of WIPI genes or proteins, is the detection of WIPI expression patterns of a cell. In particular, oligonucleotides, preferably cDNAs, are suitable for this purpose, on which, for example, a differential mRNA fingerprinting assay can be carried out in order to detect the expression intensity of WIPI genes at different pathological states. However, it is also possible to use all other types of 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. Also suitable as the immunological reagent is 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.

In a further preferred embodiment of the invention, 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. As an activator example, a genetically engineered mutant of the WIPI protein can be used.

According to the invention, it is particularly preferred that the active ingredient influences, preferably reacts with, evolutionarily conserved amino acids of the WIPI protein. This is particularly advantageous since the conserved amino acids in WIP1 proteins define the biological function of WIPI proteins, in particular the binding of phospholipids. Thus, for the first time, 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). By generating and, in particular, analyzing mutated variants of the WIPI-1 protein, the inventors for the first time identified evolutionarily conserved amino acids which are required for correct intracellular binding of the phospholipid. Thus, the following evolutionarily conserved amino acids could be identified for the human WIPI-iα 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. Furthermore, it can be provided according to the invention that 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. Thus, 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. In a particular embodiment of the preparation according to the invention, the active ingredient influences, in particular activates and / or inhibits, the expression and / or function of the WIPI-3 protein.

Furthermore, the application of 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.

In a further preferred embodiment of the invention, 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. Furthermore, the regulator may be the so-called TOR (Target Of Rapamycin) protein. Preferably, it may also be a protease which is responsible for the proteolytic degradation of a regulator, activator, inhibitor and / or biological precursor. However, even hitherto unknown molecules which are involved in the expression and / or function of the WIPI protein are the subject of the present invention.

In a particularly preferred embodiment of the invention, 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.

In one development of the invention, the active substance can modulate, in particular activate or inhibit, post-translational modifications of the WIPI protein. Particular mention should be made of the 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. However, any other form of post-translational modification is encompassed by the present invention. According to the invention, 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. These 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 , Thus, 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.

Advantageously, 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.

Furthermore, 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. Because of the just mentioned relationship between the number of peroxisomes and the process of autophagy, the WlPI proteins can be used as target structures for the therapy of disorders of lipid metabolism, in particular of the liver. Thus, 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).

An extreme form of disruption of cellular lipid metabolism exists in the clinical manifestation of cachexia. Cachexia is an emaciation syndrome that affects 50% of cancer patients. In the case of calcium excretion, fat and muscle tissue are broken down. In this tissue degradation, the proteolytic process is of great importance, even with the involvement of autophagy. Preferably, therefore, the present invention is also suitable for the therapy of Cache¬ xie.

Furthermore, it may be preferable, by modulation, in particular Akti¬ vation and / or inhibition, the expression and / or function of at least one WIPI protein cells either to damage, especially against cell and / or DNA damage, sensitive or desensitize. This can be advantageous for the activation of autophagic protective mechanisms or for the suppression of overshooting reactions. The invention furthermore relates to a method for detecting and / or investigating the autophagy status in eukaryotic cells, encompassing the method steps

Providing at least one WIPI protein expressing cells

Detection and / or study of expression and / or

Distribution and / or function of the at least one WIPI

Protein in the cells.

For this cellular detection system, it is advantageous to use cells which permit the detection of WIPI proteins with high efficiency. Preferred for the detection method mitotic, d. H. in the growth phase, cells provided. Preferably, the mitotic cells are G361 cells, as these express a high concentration of WIPI proteins and perform autophagy efficiently. These cells reveal detectable autophagosomal manifestations, especially in the form of "dots", even in the early stages of autophagy In a further embodiment of the method according to the invention, 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.

In a further preferred embodiment of the method according to the invention, the WIPI protein is expressed as a WIPI fusion protein, in particular as a fluorescent WIPI fusion protein. According to the invention, it is particularly preferred that 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. According to the invention, 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, in particular, include the process steps

Providing cells expressing WIPI protein

Incubation of the cells with the substance

Examination of expression and / or distribution and / or

Function of the WIPI protein in the cells.

In a preferred embodiment of the method according to the invention, mitotic cells, preferably G361 cells, are incubated with the substance, and subsequently the influence of this substance on the formation of autophagosomes having WIPI proteins is examined. Thus, activators of autophagy can be recognized by the induction of autophageal particles. For the detection of inhibitors of autophagy, 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. Thus, 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.

Likewise, these active ingredients could influence, in particular activate and / or inhibit, the process of autophagy via other, in particular still unknown, cellular target structures.

With regard to further features of the method according to the invention, reference is made to the preceding description.

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. In this case, 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. Furthermore, the active ingredient may be a poly nucleotide, in particular a cDNA. Further, 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. Furthermore, 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.

It is further preferred that 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. Furthermore, it is possible that 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. Furthermore, it is possible that 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. For further features of the active ingredient, reference is made to the above description.

As already mentioned, life-prolonging conditions, such as hunger or reduced calorie intake, stimulate autophagy. Therefore, the use of stimulators of autophagy, preferably of the active ingredients described here for influencing, in particular for activating and / or inhibiting the expression and / or function of at least one WIPI protein, may be preferred in order to have a life-prolonging effect on an organism , in particular on plants, animals and / or humans. 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. Furthermore, with such a kit diseases can be diagnosed which are associated with an over- or under-expression or with an over- or under function of the WIPI protein. Here too, the detection of the diseases can be carried out by detecting a disturbed expression and / or function of the WIPI protein. In particular, the substance may be one which provides this detection as a poly-nucleotide and / or polypeptide. For the further characteristics of such a substance, reference is made to the corresponding previous text of the description.

Furthermore, it may be preferred to use at least one nucleotide sequence according to the above description for the detection of protein equivalents which have at least 30% homology to the nucleotide sequences encompassed by the present invention. The protein equivalents are proteins from other organisms, in particular yeast, fruit fly and / or plants.

As already mentioned, WIPI proteins are evolutionarily highly conserved and exist in all organisms, from unicellular organisms, for example yeasts, to humans, including plants. Thus, 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. For example, the 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. In particular, the yields of agricultural crops (food technology) can be improved. It is also conceivable that the biotechnological use of microorganisms, for example of yeasts in fermentation processes or microorganisms in biotechnological production processes, 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.

The described features and further features of the invention will become apparent from the following description of preferred Aus¬ embodiments in the form of examples in conjunction with the subclaims and figures. In this case, the individual features may be realized individually or in combination with each other.

Overview of the sequence protocol:

SEQ ID NO: 1: h WIP Ma cDNA

SEQ ID NO: 2: hWIPI-1a amino acid sequence

SEQ ID NO: 3: hWlPI-2β cDNA

SEQ ID NO: 4: hWIPI-2β amino acid sequence

SEQ ID NO: 5: hWIPI-2δ cDNA

SEQ ID NO: 6: hWIPI-2δ amino acid sequence SEQ ID NO: 7: hWIPI-2ε cDNA

SEQ ID NO: 8: hWIPI-2ε amino acid sequence

SEQ ID NO: 9: hWIPl-3 cDNA

SEQ ID NO: 10: hWIPI-3 amino acid sequence

SEQ ID NO: 11: hWIPI-3-like cDNA

SEQ ID NO: 12: hWIPI-3-like amino acid sequence

SEQ ID NO: 13: hWIPI-4 cDNA

SEQ ID NO: 14: hWIPI-4 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

Example I: Detection of messenger RNA expression patterns

For the preparation of specific ³² P-labeled cDNA samples for human WIPI proteins, 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 ™, 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. These PCR fragments were labeled using the random primer DNA labeling system (Fig. Invitrogen) and Chroma Spin ™ columns (BD Biosciences) according to the manufacturer's protocols cleaned. For the Northern hybridizations, 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). To control the uniform loading of the agarose gels with RNA was stained with ethidium bromide, and the arrays or blots were hybridized with ³² P labeled actin or ubiquitin (BD Biosciences) hybri¬.

Example II: Isolation of WIPI genes

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. In order to produce a complete hWIPI-1α cDNA clone, 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-1α. In order to obtain a complete GFP hWIPI-1α, the full hWIPMα 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-1α 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). The cloning of members of the human WIPI family was carried out by RT-PCR (TITANUM one-step RT-PCR, BD Biosciences), in which the mRNA of human testes (BD Biosciences) and the oligonucleotides according to the sequences 27- 34, which were prepared according to the BLAST search results at NCBI, were used: hWIPI-2α SEQ ID NO: 27-28, hWIPI-2β 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. All constructs were verified by PCR and overlapping automatic DNA sequencing (GENterprise Genomics, Germany). The following Genbank accession numbers were assigned to our WIPI isolates: AY691424 (hWIPMα), AY691425 (hWIPI-2β), AY691426 (hWIPI-2δ), AY691427 (hWIPI-3), AY691428 (hWIPI-4).

Claims

claims

1. WIPI protein (WD-repeat proteins interfering with phospholipid inosides), characterized in that it is at least partially encoded by a nucleotide sequence or parts thereof, which zu¬ at least 70%, in particular at least 80%, identical to a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 11.

2. Nucleic acid or parts thereof, characterized in that it is at least 70%, in particular at least 80%, identical to a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 11.

3. Nucleic acid encoding the human WIPI-iα with the nucleotide sequence according to SEQ ID NO: 1, or parts thereof.

4. nucleic acid encoding the human WIPI-2ß with the nucleotide tidsequenz according to SEQ ID NO: 3, or parts thereof.

5. Nucleic acid encoding human WIPI-2δ with the nucleotide sequence according to SEQ ID NO: 5, or parts thereof.

6. Nucleic acid encoding human WIPI-2ε with the nucleotide sequence according to SEQ ID NO: 7, or parts thereof.

7. nucleic acid encoding the human WIPI-3 similar protein with the nucleotide sequence according to SEQ ID NO: 11, or parts da¬ of.

8. Provision of at least one substance for detecting the expression and / or function of at least one WIPI protein in eukaryotic cells for the diagnosis of pathological conditions, in particular of autophagy-associated diseases.

9. Provision of at least one active substance 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 of autophagy-associated diseases.

10. Provision of at least one active substance 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 production of a medicament or a pharmaceutical composition for the treatment of pathological States, especially of autophagy-associated diseases.

11. Provision according to one of claims 8 to 10, characterized in that it is the WIPI protein from a triester from the family of WIPI-1 protein, WIPI-2 protein, WIPI-3 protein, WIPI-3-like protein and / or WIPI-4 protein and / or a splice variant thereof.

12. Provision according to one of claims 8 to 11, characterized in that the WIPI protein is encoded by a nucleotide sequence or parts thereof which are at least 70%, in particular at least 80%, identical to a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 11.

13. Provision according to one of claims 8 to 12, characterized in that the WIPI protein is encoded by a nucleotide sequence, or parts thereof, identical to a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 11.

14. Provision of an active substance according to any one of claims 9 to 13, characterized in that the active ingredient is a polypeptide or protein, in particular an enzyme or antibody.

15. Provision of an active ingredient according to any one of claims 9 to 14, characterized in that the active ingredient is a poly nucleotide, in particular a cDNA and / or an antisense molecule.

16. Provision of an active ingredient according to one of claims 9 to 15, characterized in that the active ingredient is directed against the WIPI protein.

17. Provision of an active substance according to any one of claims 9 to 16, characterized in that the active ingredient is a poly nucleotide which encodes a polypeptide which influences the expression and / or function of the WIPI protein, in particular activated or inhibited.

18. Provision of an active substance according to one of claims 9 to 17, characterized in that the active ingredient against at least one regulator, in particular TOR (Target Of Rapamycin), activator, inhibitor and / or against a biological Vor¬ runner of WIPI Protein is directed.

19. Provision of an active ingredient according to claim 18, characterized in that the regulator sulfatase.3 and / or an ILF-1 (interleukin enhancer-binding factor) is similar protein.

20. Provision of an active ingredient according to any one of claims 9 to 19, characterized in that the active ingredient is a poly nucleotide which encodes a polypeptide which comprises the expression and / or function of at least one regulator, activator, inhibitor and / or one biological precursor of the WIPI protein, in particular activated or inhibited.

21. Provision of an active substance according to any one of claims 9 to 20, characterized in that the active substance influences the post-translational modification of the WIPI protein, in particular activates or inhibits.

22. Provision of an active ingredient according to any one of claims 9 to 21, characterized in that the active ingredient affects the Phospho¬ rylation and / or sumoylation of the WIPI protein, in particular activated or inhibited.

23. Provision of an active ingredient according to any one of claims 9 to 22, characterized in that the active substance an enzyme, in particular a kinase and / or ligase, preferably a sumo ligase, influenced, in particular activated or inhibited.

24. Provision of an active ingredient according to any one of claims 9 to 23, characterized in that the active ingredient affects the Assemb¬ lation of WIPI protein with at least one binding partner, in particular activated or inhibited.

25. Provision of an active compound according to claim 24, characterized in that the binding partner is a protein, in particular a tumor suppressor protein or a regulator protein or a steroid hormone.

26. Provision of an active ingredient according to any one of claims 9 to 25, characterized in that the active ingredient is a nieder¬ molecular molecule having a molecular weight <1000.

27. Provision according to one of claims 8 to 26, characterized in that the diseases are tumors, neurodegenerative diseases, myopathies, infectious diseases, inflammatory diseases, aging diseases and / or diseases of the lipid metabolism.

28. A method for detecting and / or investigating autophagy status in eukaryotic cells comprising the steps of Ver¬

Providing at least one WIPI protein expressing cells

Detection and / or investigation of the expression and / or distribution and / or function of the at least one WIPI protein in the cells.

29. The method according to claim 28, characterized in that mito¬ tables cells, preferably G361 cells are provided.

30. The method according to claim 28 or 29, characterized in that the WIPI protein with a labeling reagent, in particular a fluorescent labeling reagent, preferably a fluorescent antibody and / or fluorescent peptide aptamer coupled.

31. The method according to any one of claims 28 to 29, characterized gekenn¬ characterized in that the WIPI protein is expressed as WIPI fusion protein, in particular as a fluorescent WIPI fusion protein, preferably as GFP-WIPI.

32. The method according to any one of claims 28 to 31, characterized in that the expression and / or distribution and / or function of the WIPI protein nachge¬ by visualization methods, in particular fluorescence microscopy, fluorimetric, fluorescence cytometric and / or immunological methods and / or examined.

33. Method for the investigation of the activity of at least one substance for influencing the autophagy process, in particular by activation and / or inhibition, of the expression and / or function of at least one WIPI protein in eukaryotic cells for the treatment of pathological conditions, in particular of autophagy associated diseases, comprising the procedural steps

Providing cells expressing WIPI protein

Incubation of the cells with the substance

Examination of expression and / or distribution and / or

Function of the WIPI protein in the cells.

34. The method according to claim 33, further characterized by at least one of the features of the characterizing part of claims 29 to 32.

35. A pharmaceutical composition comprising an effective amount of at least one active substance which influences the expression and / or function of at least one WIPI protein, especially activated or inhibited, and optionally a pharmaceutical carrier material.

36. Pharmaceutical composition according to claim 35, characterized in that the active ingredient influences the expression and / or function of at least one regulator, activator, inhibitor and / or biological precursor of the WIPI protein, in particular activates or inhibits.

37. Pharmaceutical composition according to claim 35 or 36, characterized in that the active substance is a low molecular weight molecule with a molecular weight of <1000.

38. A diagnostic kit comprising at least one substance 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 auto-phagia-associated diseases.

39. Diagnosis kit according to claim 38, characterized in that the pathological states are associated with an over- and / or under expression or function of the WIPI protein.

40. Use of an active substance with at least one feature from claims 11 to 27 in biotechnology, in particular for improving the properties of crops and / or microorganisms, and in biosensing.