WO2001046412A2 - Interaction proteins for the stat transcription factor - Google Patents
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- WO2001046412A2 WO2001046412A2 PCT/DE2000/004557 DE0004557W WO0146412A2 WO 2001046412 A2 WO2001046412 A2 WO 2001046412A2 DE 0004557 W DE0004557 W DE 0004557W WO 0146412 A2 WO0146412 A2 WO 0146412A2
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- nucleic acid
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Classifications
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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
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions
- the invention relates to an isolated polypeptide which acts as an interaction protein of the STAT transcription factor STAT3. It also relates to an isolated nucleic acid which encodes such an interaction protein or at least parts thereof. The invention also relates to the use of this polypeptide and this nucleic acid for detecting, in particular diagnostic, therapeutic and For research purposes and it relates to reagents made using at least one of these molecules, particularly recombinant vector molecules and antibodies
- STAT transcription factors hereinafter called STAT proteins for short, are a new family of cytosolic proteins that are expressed in the non-activated state in the cytosol of different cells (Weber-Nordt et al 1998) ("STAT” stands for "SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION ”) STAT proteins are functionally involved in the processes of cell proliferation, cell maturation and apoptosis.
- STAT1 to STAT6 seven different STAT proteins are known in the prior art, namely STAT1 to STAT6, two splice variants of STAT3 being known and the Stat3 as an oncogene
- the activity of these STAT proteins and their localization in the cytoplasm or in the cell nucleus is controlled by interactions of different domains on the STAT protein, this interaction taking place by means of interacting proteins, so-called interaction proteins, or directly.
- interaction proteins or directly.
- some of these domains have been identified and described and also a few int Action proteins that control the activity of the STAT protein in question via these domains
- kinases which catalyze tyrosine and / or serine phosphorylation are known as interaction proteins.
- WO98 / 30688 describes a Stat3 interaction protein with regard to the amino acid sequence and the underlying nucleotide sequence indicated that this interaction protein reacts with the STAT proteins STAT3 and STAT6, that it tyrosine phosphorylation of Glycoproteins gpl30 (a subunit of a cytokine receptor) and the STAT protein STAT3 inhibits and that it has an SH2 region
- the activation of the STAT proteins is physiologically limited in time and dependent on the cytokine (Weber-Nordt et al 1996).
- cytokine With regard to the cytokines, it has already been described that the reaction of a cytokine with its receptor on the cell surface leads to association (intracellular) in the cell STAT proteins come to defined regions of the activated cytokine receptor chains (Weber-Nordt et al 1996).
- STAT1 The activation of the STAT proteins STAT1, STAT3 and STAT5 is described for the receptor of the cytokine interleukin-10 (Wehinger et al 1996)
- the STAT3 functions as a transcription factor that is crucial for the complexation with the IL-10 receptor as well as the transcription factors STAT1 and STAT5 (Weber-Nordt et al 1996).
- STAT3 inhibitory proteins block the constitutive activity of the STAT protein STAT3 in normal hamatopoietic cells and could thus lead to further differentiation of the immature myeloid cells
- the object of the present invention is therefore to provide interaction proteins which regulate and / or influence the activity of STAT proteins, in particular STAT3
- One solution to this problem is to provide an isolated polypeptide which acts as an interaction protein of the STAT3 transcription factor, ie an interaction protein of the transcription factor STAT3 which occurs naturally in cells, preferably sucker cells and in particular human cells, or is similar (similar in function and effect), and which has either the amino acid sequence shown in the sequence listing SEQ LD NO 2 or an amino acid sequence resulting therefrom by amino acid substitution, deletion, insertion, or inversion as an allele or derivative, the amino acid sequence being replaced by amino acid substitution, deletion, insertion, or inversion amino acid sequences formed as an allele or derivative are suitable for interaction with domains of the STAT3 transcription factor
- interaction protein according to the invention hereinafter also referred to as "interaction protein according to the invention” or “Stat3 interaction protein according to the invention” or “Stat3 interaction protein No. C 102"
- interaction protein according to the invention or “Stat3 interaction protein according to the invention” or “Stat3 interaction protein No. C 102”
- Acute Lymphocytic Leukemia from patients with CLL (Chronic Myeloid Leukemia), and from patients with CML (Chronic Lymphocytic Leukemia), and in normal mononuclear cells of human blood. It has at least two putative tyrosine phosphorylation sites (in SEQ LD NO 2 at position aa 65 and aa 171), and it interacts in the yeast two hybrid interaction system with the STAT3 transcription factor.
- the yeast two hybrid system is described in the publication by Chien C -T et al, 1991, "The two-hybrid-system a method to identify and clone genes for Proteins that interact with a protein of interest ", Proc natl Acad Sei, USA 88, 9578-9582, described in detail.
- This publication is expressly referred to here, its content belongs to present description
- the Stat3 interaction protein according to the invention can either be a native protein which is isolated or isolatable from the cells in which it is naturally expressed, using known methods, or it can be a recombinant protein
- a solution to the stated problem also consists in the provision of an isolated nucleic acid which encodes a protein according to claim 1 and the description above, and which either has the nucleotide sequence shown in the sequence listing SEQ LD NO 1 or a nucleotide sequence complementary thereto or a partial sequence of one of these nucleotide sequences or one has a nucleotide sequence that hybridizes wholly or partially with one of these nucleotide sequences, it also being possible for “U” to appear in this sequence listing instead of “T”.
- This nucleic acid according to the invention can either be obtained from a natural (in particular a cellular) or from a synthetic or a semi-synthetic source It can also be a cDNA, or a splice variant that hybridizes with the nucleotide sequence shown in the sequence listing SEQ ID NO 1, or a sense or antisense oligonucleotide that has at least 6, preferably 8 to 25 nucleotides summarizes and hybridizes with the nucleotide sequence shown in the sequence listing SEQ LD NO 1 or partial sequences thereof
- the present invention also includes those nucleotide sequences which hybridize therewith under stringent conditions.
- hybridized or “hybridization” is used in the present description of the invention according to Sambrook et al., Molecular Clonmg, A Laboratory Manual, Cold Spring Harbor, Laboratory Press, 1989, 1,101 to 1,104 used.
- hybridization occurs under stringent conditions if, after washing for 1 hour with 1 x SSC and 0, 1% SDS, preferably with a lower concentrated SSC, in particular 0.2 x SSC, at a temperature of at least 55 ° C., preferably 62 ° C. and particularly preferably 68 ° C. a positive hybridization signal can still be observed.
- any nucleotide sequence which is produced under such washing conditions with a nucleotide sequence according to SEQ LD NO 1, or hybridizing with a nucleotide sequence corresponding to this sequence in the context of the degeneration of the genetic code is part of the subject matter of the present invention
- the Stat3 interaction proteins according to the invention or the polypeptides or proteins according to the invention which act as Stat3 interaction proteins
- the nucleic acids encoding them provide the person skilled in the art with an actuator in the regulation of the STAT-mediated signal transduction and the cell cycle, with which he specifically targets regulation STAT activation and / or protein folding and / or translocation and / or protein degradation can act.
- the polypeptide according to the invention and in particular the interaction protein No. C 102 or the underlying nucleic acid can in principle be cloned into any plasmid, expression vectors and amplification vectors being preferred in particular with regard to advantageous production on an industrial scale.
- the invention consequently also relates to recombinant DNA vector molecules which comprise a nucleic acid according to the invention and which have the ability to amplify this nucleic acid or at least a part thereof and / or to express a Stat3 interaction protein or at least a part thereof in a prokaryotic or eukaryotic cell
- the invention therefore also encompasses transformed host cells which contain a nucleic acid according to the invention which is coupled to an activatable promoter which is naturally present in the respective host cell or as a result of recombination and which (as a result) has the ability to express a Stat3 interaction protein
- the invention further relates to the use of a polypeptide according to the invention for the production of an (monoclonal or polyclonal) antibody against this polypeptide and / or related proteins, said antibody itself and also its use for the production of a pharmaceutical preparation or a medicament for diagnostic and / or therapeutic treatment of leukaemias and / or other cancers and / or metabolic diseases and / or neurological diseases and for research purposes
- the invention also relates to the use of a polypeptide according to the invention and / or a nucleic acid according to the invention, which may in particular also be a sense or antisense oligonucleotide, and / or an antibody according to the invention for the production of pharmaceutical preparations / medicaments for diagnostic and / or therapeutic treatment of cancer, especially leukemia, and / or other metabolic diseases
- sense and antisense oligonucleotides are also particularly suitable as active substances for pharmacotherapies (cf. G Hartmann et al 1998 antisense oligonucleotides, Inc. Cardioeblatt 95, issue 24, Cl 1 15-C11 19) - and moreover as active substances a fundamentally new mode of action in pharmacotherapy
- a technically and economically significant possibility of using a polypeptide according to the invention and also a nucleic acid according to the invention consists in the fact that with the help of such a molecule, a very large number of available substances can be selected from a very large number of available substances in a "screening" process, specifically those specific to the Bind the relevant nucleic acid or the relevant polypeptide. These substances can then serve as the starting material (lead structure) for the development of pharmacologically usable substances and thus offer the prerequisites for the development of alternative pharmaceuticals for diagnosis and therapy, in particular the leukemia and other cancer diseases and metabolic diseases mentioned above, where the STAT protein STAT3 plays an important role
- the invention also relates to the use of a polypeptide according to the invention and / or a nucleic acid according to the invention (including sense or antisense oligonucleotides) and / or an antibody according to the invention for the identification of pharmacologically or gene-therapeutically usable substances which - preferably highly specifically - with the / react the relevant polypeptide (s) according to the invention or the relevant nucleic acid (s) according to the invention (in particular bind) and thereby influence its function and / or expression
- the invention also relates to the use of a polypeptide according to the invention and / or a nucleic acid according to the invention, including sense or antisense oligonucleotides, for the detection of a Stat3 interaction protein in a sample (material sample), in particular a serum or a cell sample, where polypeptide ( e) and / or nucleic acid (s) or oligonucleotide (s) and / or antibodies together with the serum or cells in question are subjected to an enzyme immunoassay, a polymerase chain reaction or a hybridization assay.
- this serum or cell or other Material sample preferably obtained from suckers, in particular from humans (patients or test subjects)
- a reagent is proposed according to the invention which uses at least one polypeptide according to the invention and / or one invented nucleic acid according to the invention (including sense or antisense oligonucleotides) and / or an antibody according to the invention
- EXAMPLE 1 Obtaining an Interaction Protein of the STAT Protein mSTAT3 in the Yeast-Two-Hybrid System Yeast cells of the strain Saccharomyces cerevisiae CG 1945, which are known not to express STAT protein-activating kinases, were treated with non-phosphorylated and thus inactivated STAT protein STAT3
- the nucleotide sequence coding for the protein mSTAT3 was cloned into the yeast expression vector pAS2-1 [GAL4 DNA binding domain] and this vector was subsequently introduced into yeast cells.
- the expression of mSTAT3 was detected in the yeast cells by means of the known Western blot method using a commercially available mSTAT3-specific antibody (eg from Calbiochem)
- yeast cells containing the GAL4 DNA BD / mSTAT3 plasmid were cloned with a "mouse lymphoma CDNA bank” cloned into the yeast expression vector PACT [GAL4 Activation Domain] (ie co-transformed with an activation domain / library plasmid) and further cultivated under the conditions customary for yeast cells until colony growth.
- yeast expression vector PACT GAL4 Activation Domain
- Such colonies or clones with apparent protein-protein interaction were subjected to DNA sequencing using methods known in the art.
- the nucleotide sequences obtained were discarded in the databases provided by the NCBI.
- Frameshift mutants were discarded and the remaining clones were targeted to specific reporter genes -Activation tested, namely for the transformation of these GAL4 AD / Lymphoma cDNA Library plasmids alone, with GAL4 DNA BD / mSTAT3, with GAL4 DNA BD / LaminC or with GAL4 DNA BD / no insert.
- C 102 The clone C 102 was first propagated in yeast medium and then subjected to a plasmid isolation process using standard methods familiar to the person skilled in the art, in which the two plasmids pAS2-1 [BD / mSTAT3] and pACT [AD / C 102] were obtained using this plasmid Mixture, E coli KC8 cells were transformed and these cells were subsequently cultivated and propagated on M9 minimal medium.
- E coli KC8 cells do not inherently have a leucine gene and therefore grow on M9 minimal medium only on the condition that they have previously were transformed with a leucine gene.
- the vector pACT contains such a leucine gene so that (only) those E coli KC8 cells which contain this vector pACT grow or multiply on the M9 minimal medium.
- the pACT plasmid was isolated from minimally medium-grown E coli KC8 cells using standard methods known to those skilled in the art and then sequenced. A polypeptide integrated in the plasmid was found , which received the designation "C 102" or "Stat3 interaction protein No. C 102" and has the amino acid sequence shown in the sequence listing SEQ ID NO 2
- this Stat3 interaction protein No. C 102 was demonstrated by the following assays (1) after cotransformation of the yeast cells with the nucleotide sequences of STAT3 and the Stat3 interaction protein No C 102 in the respective vectors described above, the interaction reactions of the binding domains and activation were -Domaines of the GAL4 transcription factor in the yeast two hybrid system positive (2) The interaction reactions of the Stat3 interaction protein No. C 102 with LaminC as non-specific interaction partner were negative (3) After transformation of the yeast cells with the coding for the Stat3 interaction protein No. C 102 The nucleotide sequence alone (as an insert in the Activation Doman vector) could not trigger an auto-activation of the Yeast-Two Hybrid interaction system
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to isolated polypeptides that function as an interaction protein of the STAT transcription factor STAT3 and are provided with the amino acid sequence which is represented in the sequence protocol SEQ ID NO: 2 or an amino acid sequence that is produced by means of amino acid substitution, deletion, insertion or inversion as allelomorphic or derivative therefrom. The invention also relates to isolated nucleic acids that code such interaction proteins or at least parts thereof. The invention further relates to the use of such polypeptides and nucleic acids in therapy and research for detection and especially diagnostic purposes. The invention further relates to reagents which are produced by using at least one of said molecules, especially recombinant vector molecules and antibodies.
Description
Interaktionsprotein für STAT Transkriptionsfaktor Interaction protein for STAT transcription factor
B e s c h r e i b u n gDescription
Die Erfindung betrifft ein isoliertes Polypeptid, das als Interaktionsprotein des STAT Transkriptionsfaktors STAT3 fungiert Sie betrifft ferner eine isolierte Nukleinsaure, die ein solches Interaktionsprotein oder zumindest Teile davon kodiert Außerdem betrifft die Erfindung die Verwendung dieses Polypeptids und dieser Nukleinsaure für nachweisende, insbesondere diagnostische, therapeutische und Forschungszwecke und sie betrifft Reagenzien, die unter Verwendung wenigstens eines dieser Moleküle hergestellt sind, insbesondere rekombinante Vektormolekule und AntikörperThe invention relates to an isolated polypeptide which acts as an interaction protein of the STAT transcription factor STAT3. It also relates to an isolated nucleic acid which encodes such an interaction protein or at least parts thereof. The invention also relates to the use of this polypeptide and this nucleic acid for detecting, in particular diagnostic, therapeutic and For research purposes and it relates to reagents made using at least one of these molecules, particularly recombinant vector molecules and antibodies
STAT Transkriptionsfaktoren, im folgenden kurz STAT-Proteine genannt, sind eine neue Familie zytosolischer Proteine, die in nicht aktiviertem Zustand im Zytosol unterschiedlicher Zellen exprimiert sind (Weber-Nordt et al 1998) ("STAT" steht für "SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION") STAT- Proteine sind fünktionell involviert in die Prozesse der Zellproliferation, der Zellmaturation und der Apoptose Im Stand der Technik sind bisher sieben verschiedene STAT Proteine bekannt, nämlich STAT1 bis STAT6, wobei von STAT3 zwei Splice-Narianten bekannt sind und das Stat3 als Onkogen beschrieben wurde Die Aktivität dieser STAT- Proteine und ihre Lokalisation im Zytoplasma bzw im Zellkern wird durch Interaktionen unterschiedlicher Domänen auf dem STAT-Protein gesteuert, wobei diese Interaktion vermittels interagierender Proteine, sogenannter Interaktionsproteine, oder direkt erfolgt Bisher sind einige dieser Domänen identifiziert und beschrieben worden und auch einige wenige Interaktionsproteine, die über diese Domänen die Aktivität des betreffenden STAT-Proteins steuern Als Interaktionsproteine sind bisher vor allem Kinasen, die eine Tyrosin- und/oder Serinphosphorylierung katalysieren, bekannt In der WO98/30688 ist ein Stat3 -Interaktionsprotein hinsichtlich Aminosauresequenz und zugrundeliegender Νukleotidsequenz beschrieben und angegeben, daß dieses Interaktionsprotein das mit den STAT -Proteinen STAT3 und STAT6 reagiert, daß es die Tyrosinphosphorylierung des
Glykoproteins gpl30 (einer Untereinheit eines Zytokinrezeptors) und des STAT-Proteins STAT3 inhibiert und daß es eine SH2 Region aufweistSTAT transcription factors, hereinafter called STAT proteins for short, are a new family of cytosolic proteins that are expressed in the non-activated state in the cytosol of different cells (Weber-Nordt et al 1998) ("STAT" stands for "SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION ") STAT proteins are functionally involved in the processes of cell proliferation, cell maturation and apoptosis. So far, seven different STAT proteins are known in the prior art, namely STAT1 to STAT6, two splice variants of STAT3 being known and the Stat3 as an oncogene The activity of these STAT proteins and their localization in the cytoplasm or in the cell nucleus is controlled by interactions of different domains on the STAT protein, this interaction taking place by means of interacting proteins, so-called interaction proteins, or directly. So far, some of these domains have been identified and described and also a few int Action proteins that control the activity of the STAT protein in question via these domains So far, kinases which catalyze tyrosine and / or serine phosphorylation are known as interaction proteins. WO98 / 30688 describes a Stat3 interaction protein with regard to the amino acid sequence and the underlying nucleotide sequence indicated that this interaction protein reacts with the STAT proteins STAT3 and STAT6, that it tyrosine phosphorylation of Glycoproteins gpl30 (a subunit of a cytokine receptor) and the STAT protein STAT3 inhibits and that it has an SH2 region
Physiologisch ist die Aktivierung der STAT Proteine zeitlich limitiert und zytokinabhangig (Weber-Nordt et al 1996) Bezuglich der Zytokine wurde bereits beschrieben, daß die Reaktion eines Zytokins mit seinem Rezeptor auf der Zelloberflache dazu führt, daß es in der Zelle (intrazellular) zur Assoziation von STAT-Proteinen an definierte Regionen der jeweiligen aktivierten Zytokin-Rezeptorketten kommt (Weber-Nordt et al 1996) Für den Rezeptor des Zytokins Interleukin-10 ist die Aktivierung der STAT-Proteine STAT1, STAT3 und STAT5 beschrieben (Wehinger et al 1996) Hierbei füngiert das STAT3 als ein Transkriptionsfaktor, der für die Komplexierung mit dem IL-10 Rezeptor als auch den Transkriptionsfaktoren STAT1 und STAT5 entscheidend ist (Weber-Nordt et al 1996) In primären Zellen akuter myeloischer Leukämien hingegen wurde die unkontrollierte Aktivierung des STAT Proteins STAT3 gezeigt (Weber-Nordt et al 1996) Da in diesen Zellen eine konstitutive Tyrosinphosphorylierung und DNA Bindungsfahigkeit des STAT-Proteins STAT3 nachgewiesen wurde, ist davon auszugehen, daß STAT3 inhibitorische Proteine die konstitutive Aktivität des STAT- Proteins STAT3 in normalen hamatopoietischen Zellen blockieren und damit zu einer Weiterdifferenzierung der unreifen myeloischen Zellen führen konnteThe activation of the STAT proteins is physiologically limited in time and dependent on the cytokine (Weber-Nordt et al 1996). With regard to the cytokines, it has already been described that the reaction of a cytokine with its receptor on the cell surface leads to association (intracellular) in the cell STAT proteins come to defined regions of the activated cytokine receptor chains (Weber-Nordt et al 1996). The activation of the STAT proteins STAT1, STAT3 and STAT5 is described for the receptor of the cytokine interleukin-10 (Wehinger et al 1996) The STAT3 functions as a transcription factor that is crucial for the complexation with the IL-10 receptor as well as the transcription factors STAT1 and STAT5 (Weber-Nordt et al 1996). In primary cells of acute myeloid leukemia, however, the uncontrolled activation of the STAT protein STAT3 was shown (Weber-Nordt et al 1996) Since in these cells constitutive tyrosine phosphorylation and DNA Bi Ability of the STAT protein STAT3 has been demonstrated, it can be assumed that STAT3 inhibitory proteins block the constitutive activity of the STAT protein STAT3 in normal hamatopoietic cells and could thus lead to further differentiation of the immature myeloid cells
Zur weiteren Aufklarung der Funktion und Wirkung von STAT-Proteinen, insbesondere der Interaktion mit ihren spezifischen Interaktionsproteinen, und damit einhergehend der ursachlichen Zusammenhange von Leukämien, mit dem Ziel neue oder besserer Ansatzpunkte für eine wirkungsvolle(re) Diagnose und Therapie zu gewinnen, besteht daher großer Bedarf nach Identifizierung, Charakterisierung und Isolierung weiterer solcher InteraktionsproteineThere is therefore a further clarification of the function and effect of STAT proteins, in particular the interaction with their specific interaction proteins, and thus the causal relationship between leukaemias, with the aim of gaining new or better starting points for effective (re) diagnosis and therapy great need for the identification, characterization and isolation of further such interaction proteins
Aufgabe der vorliegenden Erfindung ist deshalb die Bereitstellung von Interaktionsproteinen, die die Aktivität von STAT-Proteinen, insbesondere von STAT3, regulieren und/oder beeinflussen
Eine Losung dieser Aufgabe besteht in der Bereitstellung eines isolierten Polypeptids, das als Interaktionsprotein des STAT3 Transkriptionsfaktors fungiert, d h einem natürlicherweise in Zellen, vorzugsweise Saugerzellen und insbesondere Humanzellen, vorkommenden Interaktionsprotein des Transkriptionsfaktors STAT3 gleicht oder ahnlich (namlich in Funktion und Wirkung gleich) ist, und das entweder die im Sequenzprotokoll SEQ LD NO 2 dargestellte oder eine durch Aminosauresubstitution, -deletion, -insertion, -oder -inversion als Allel oder Derivat daraus entstandene Aminosauresequenz aufweist, wobei die durch Aminosauresubstitution, -deletion, -insertion, -oder -inversion als Allel oder Derivat entstandenen Aminosauresequenzen zur Interaktion mit Domänen des STAT3 Transkriptionsfaktors geeignet sindThe object of the present invention is therefore to provide interaction proteins which regulate and / or influence the activity of STAT proteins, in particular STAT3 One solution to this problem is to provide an isolated polypeptide which acts as an interaction protein of the STAT3 transcription factor, ie an interaction protein of the transcription factor STAT3 which occurs naturally in cells, preferably sucker cells and in particular human cells, or is similar (similar in function and effect), and which has either the amino acid sequence shown in the sequence listing SEQ LD NO 2 or an amino acid sequence resulting therefrom by amino acid substitution, deletion, insertion, or inversion as an allele or derivative, the amino acid sequence being replaced by amino acid substitution, deletion, insertion, or inversion amino acid sequences formed as an allele or derivative are suitable for interaction with domains of the STAT3 transcription factor
Das erfindungsgemaße Polypeptid, im folgenden auch "erfindungsgemaßes Interaktionsprotein" oder "erfindungsgemaßes Stat3 -Interaktionsprotein" oder "Stat3- Interaktionsprotein Nr C 102" genannt, weist die folgenden konstitutionellen und funktioneilen Merkmale aufThe polypeptide according to the invention, hereinafter also referred to as "interaction protein according to the invention" or "Stat3 interaction protein according to the invention" or "Stat3 interaction protein No. C 102", has the following constitutional and functional features
Es ist in Lungen-, Gehirn-, Leber-, Milz-, Herz- und Nierengewebe der Maus, in humanenIt is in the lung, brain, liver, spleen, heart and kidney tissues of the mouse, in human
Zellen von Patienten mit AML (Akute myeloische Leukämie) , von Patienten mit ALLCells from patients with AML (acute myeloid leukemia), from patients with ALL
(Akute lymphatische Leukämie) , von Patienten mit CLL (Chronische myeloische Leukämie), und von Patienten mit CML (Chronische lymphatische Leukämie), und in normalen mononuklearen Zellen von humanem Blut nachweisbar vorhanden, es weist wenigstens zwei putative Tyrosin Phosphorylierungsstellen (im SEQ LD NO 2 an Position aa 65 und aa 171), und es interagiert im Yeast-Two-Hybrid-Interaktionssystem mit dem STAT3-Transkriptionsfaktor Das Yeast-Two-Hybrid-System ist in der Publikation von Chien C -T et al , 1991, "The two-hybrid-system a method to identify and clone genes for Proteins that interact with a protein of interest", Proc natl Acad Sei , USA 88, 9578-9582, ausführlich beschrieben Auf diese Publikation wird hier ausdrucklich Bezug genommen, ihr Inhalt gehört zur vorliegenden Beschreibung(Acute Lymphocytic Leukemia), from patients with CLL (Chronic Myeloid Leukemia), and from patients with CML (Chronic Lymphocytic Leukemia), and in normal mononuclear cells of human blood. It has at least two putative tyrosine phosphorylation sites (in SEQ LD NO 2 at position aa 65 and aa 171), and it interacts in the yeast two hybrid interaction system with the STAT3 transcription factor. The yeast two hybrid system is described in the publication by Chien C -T et al, 1991, "The two-hybrid-system a method to identify and clone genes for Proteins that interact with a protein of interest ", Proc natl Acad Sei, USA 88, 9578-9582, described in detail. This publication is expressly referred to here, its content belongs to present description
Das erfindungsgemaße Stat3 -Interaktionsprotein kann entweder ein natives Protein sein, das aus den Zellen, in denen es natürlicherweise exprimiert wird, mit bekannten Nerfahren isoliert bzw isolierbar ist, oder es kann ein rekombinantes Protein sein
Eine Losung der genannten Aufgabe besteht auch in der Bereitstellung einer isolierten Nukleinsaure, die ein Protein gemäß Anspruch 1 und vorstehender Beschreibung codiert, und die entweder die im Sequenzprotokoll SEQ LD NO 1 dargestellte Nukleotidsequenz oder eine hierzu komplementäre Nukleotidsequenz oder eine Teilsequenz einer dieser Nukleotidsequenzen oder eine ganz oder teilweise mit einer dieser Nukleotidsequenzen hybridisierende Nukleotidsequenz aufweist, wobei in diesem Sequenzprotokoll anstelle von "T" auch "U" stehen kann Diese erfindungsgemaße Nukleinsaure kann entweder aus einer naturlichen (insbesondere einer zellularen) oder einer synthetischen oder einer halbsynthetischen Quelle gewonnen werden bzw worden sein Sie kann außerdem eine cDNA sein, oder auch eine Splice- Variante, die mit der im Sequenzprotokoll SEQ ID NO 1 dargestellten Nukleotidsequenz hybridisiert, oder auch ein Sense- oder Antisense-Oligonukleotid, das mindestens 6, vorzugsweise 8 bis 25 Nukleotide umfaßt und mit der im Sequenzprotokoll SEQ LD NO 1 dargestellten Nukleotidsequenz oder Teilsequenzen davon hybridisiertThe Stat3 interaction protein according to the invention can either be a native protein which is isolated or isolatable from the cells in which it is naturally expressed, using known methods, or it can be a recombinant protein A solution to the stated problem also consists in the provision of an isolated nucleic acid which encodes a protein according to claim 1 and the description above, and which either has the nucleotide sequence shown in the sequence listing SEQ LD NO 1 or a nucleotide sequence complementary thereto or a partial sequence of one of these nucleotide sequences or one has a nucleotide sequence that hybridizes wholly or partially with one of these nucleotide sequences, it also being possible for “U” to appear in this sequence listing instead of “T”. This nucleic acid according to the invention can either be obtained from a natural (in particular a cellular) or from a synthetic or a semi-synthetic source It can also be a cDNA, or a splice variant that hybridizes with the nucleotide sequence shown in the sequence listing SEQ ID NO 1, or a sense or antisense oligonucleotide that has at least 6, preferably 8 to 25 nucleotides summarizes and hybridizes with the nucleotide sequence shown in the sequence listing SEQ LD NO 1 or partial sequences thereof
Neben der in dem Sequenzprotokoll SEQ ID NO 1 gezeigten Nukleotidsequenzen und den dieser Sequenz im Rahmen der Degeneration des genetischen Codes entsprechenden Nukleotidsequenzen umfaßt die vorliegende Erfindung auch solche Nukleotidsequenzen, die damit unter stringenten Bedingungen hybridisieren Der Begriff "hybridisiert" bzw "Hybridisierung" ist in der vorliegenden Erfindungsbeschreibung gemäß Sambrook et al., Molecular Clonmg, A Laboratory Manual, Cold Spring Harbor, Laboratory Press, 1989, 1.101 bis 1.104 verwendet Demnach liegt eine Hybridisierung unter stringenten Bedingungen vor, wenn nach Waschen für eine Stunde mit 1 x SSC und 0,1% SDS, vorzugsweise mit niedriger konzentriertem SSC, insbesondere 0,2 x SSC, bei einer Temperatur von wenigstens 55°C, vorzugsweise 62°C und besonders bevorzugt 68°C noch ein positives Hybridisierungssignal beobachtet werden kann Jede Nukleotidsequenz, die unter derartigen Waschbedingungen mit einer Nukleotidsequenz gemäß SEQ LD NO 1, oder mit einer dieser Sequenz im Rahmen der Degeneration des genetischen Codes entsprechenden Nukleotidsequenz hybridisiert, gehört zum Gegenstand der vorliegenden Erfindung
Die erfindungsgemaßen Stat3 -Interaktionsproteine (bzw die erfindungsgemaßen, als Stat3 -Interaktionsprotein fungierenden Polypeptide bzw Proteine) und ebenso die diese kodierenden Nukleinsäuren geben dem Fachmann ein Stellglied in der Regulation der STAT vermittelten Signaltransduktion und des Zellzykluses an die Hand, mit dem er gezielt regulatorisch auf die STAT Aktivierung und/oder Proteinfaltung und/oder Translokation und/oder den Proteinabbau einwirken kann. Sie eröffnen ihm insbesondere die Möglichkeiten, durch Einsatz dieser erfindungsgemaßen Polypeptide (Proteine) oder Nukleinsäuren selbst (direkt) oder dafür spezifischer Antikörper, die Aktivierung des STAT-Proteins STAT3 zu beeinflussen, die STAT3 -Interaktionspartner im Zytoplasma zu lokalisieren und eine nukleare Translokation des STAT3 zu beeinflusse, den Rucktransports des STAT3 in das Zytoplasma zu beeinflussen und dadurch naher aufzuklaren, sowie die intrazytoplasmatischen Proteinfaltung zu beeinflussen und somit naher aufzuklaren Mit der Bereitstellung der erfindungsgemaßen Interaktionsproteine und der diese kodierenden Nukleinsäuren ist außerdem die Voraussetzung geschaffen worden für eine Analyse verschiedener Gewebs- und Zelltypen (Normalzellen und maligne entartete Zellen) auf Exprimierung (Expression) des betreffenden Proteins, für eineIn addition to the nucleotide sequences shown in the sequence listing SEQ ID NO 1 and the nucleotide sequences corresponding to this sequence in the context of the degeneration of the genetic code, the present invention also includes those nucleotide sequences which hybridize therewith under stringent conditions. The term "hybridized" or "hybridization" is used in the present description of the invention according to Sambrook et al., Molecular Clonmg, A Laboratory Manual, Cold Spring Harbor, Laboratory Press, 1989, 1,101 to 1,104 used. Accordingly, hybridization occurs under stringent conditions if, after washing for 1 hour with 1 x SSC and 0, 1% SDS, preferably with a lower concentrated SSC, in particular 0.2 x SSC, at a temperature of at least 55 ° C., preferably 62 ° C. and particularly preferably 68 ° C. a positive hybridization signal can still be observed. Any nucleotide sequence which is produced under such washing conditions with a nucleotide sequence according to SEQ LD NO 1, or hybridizing with a nucleotide sequence corresponding to this sequence in the context of the degeneration of the genetic code is part of the subject matter of the present invention The Stat3 interaction proteins according to the invention (or the polypeptides or proteins according to the invention which act as Stat3 interaction proteins) and also the nucleic acids encoding them provide the person skilled in the art with an actuator in the regulation of the STAT-mediated signal transduction and the cell cycle, with which he specifically targets regulation STAT activation and / or protein folding and / or translocation and / or protein degradation can act. They open it in particular ways by using this inventive polypeptides (P r oteine) or nucleic acids themselves (direct) or for specific antibodies to influence the activation of STAT protein STAT3 to locate the STAT3 -Interaktionspartner in the cytoplasm and nuclear translocation to influence the STAT3, to influence the return transport of the STAT3 into the cytoplasm and thereby to elucidate it more closely, and to influence the intracytoplasmic protein folding and thus to elucidate it more closely. With the provision of the interaction proteins according to the invention and the nucleic acids coding for them, the prerequisites for an analysis of various others have also been created Tissue and cell types (normal cells and malignant degenerate cells) on expression of the protein in question, for one
Koimmunoprazipitation des STAT-Proteines STAT3 und des erfindungsgemaßen Interaktionsproteins Nr C 102 zwecks weiterer Aufklärung deren Interaktion, für eine Kotransfektion von murinen und humanen Zellen in Kultur mit Stat3 bzw Interaktionsprotein Nr C 102 enthaltenden Sauger-Expressionsvektoren, um die Lokalisation des STAT-Proteins STAT3 und der erfindungsgemaßen Interaktionsproteine, insbesondere des Interaktionsproteins Nr C 102, in der Zelle und damit einhergehend auch die Aktivierung des STAT-Proteins STAT3 weiter zu untersuchen, für Transformationsversuche mit Mutanten des STAT-Proteins STAT3, denen einzelne Domänen fehlen, um die interagierende(n) Domane(n) des STAT-Proteins STAT3 bestimmen zu können, für Untersuchungen zur chromosomale Lokalisation des Gens für das erfindungsgemaße Interaktionsproteins Nr C 102 im Vergleich zur Lokalisation des STAT-Proteins STAT3, für eine Generierung und Analyse von -/- Knock-Out -Mausen, für funktionelle Untersuchungen zur Uberexpression des erfindungsgemaßen Interaktionsproteins Nr C 102 im Hinblick auf das Proliferationsverhalten, die
Klonogenitat und die Apoptoserate der transfizierten Zellen, für eine Proteinexpression z B im Baculo- Virus-System, und Kristallographie des erfindungsgemaßen Interaktionsproteins Nr C 102 und des STAT-Proteins STAT3 als Interaktionspartner, und nicht zuletzt für Untersuchungen zur Uberexpression des erfindungsgemaßen Interaktionsproteins Nr C 102 in AML Zellen mit dem Ziel der Entwicklung eines kausal wirkenden gentherapeutischen Ansatzes in humanen Zellen in vitro und in vivoCoimmunoprecipitation of the STAT protein STAT3 and the interaction protein No. C 102 according to the invention for the purpose of further elucidating their interaction, for a co-transfection of murine and human cells in culture with Stat3 or interaction protein No. C 102 containing sucker expression vectors in order to localize the STAT protein STAT3 and to further investigate the interaction proteins according to the invention, in particular interaction protein No. C 102, in the cell and, along with this, also the activation of the STAT protein STAT3, for transformation experiments with mutants of the STAT protein STAT3, which lack individual domains, in order to understand the interacting (n) To be able to determine the domain (s) of the STAT protein STAT3, for studies on the chromosomal localization of the gene for the interaction protein No. C 102 according to the invention in comparison to the localization of the STAT protein STAT3, for generation and analysis of - / - knock-out - Mausen, for functional examinations Expression of the interaction protein No. C 102 according to the invention with regard to the proliferation behavior, the Clonogenicity and the apoptosis rate of the transfected cells, for protein expression, for example in the baculo virus system, and crystallography of the interaction protein No. C 102 according to the invention and the STAT protein STAT3 as interaction partner, and last but not least for studies on overexpression of the interaction protein No. C 102 according to the invention in AML cells with the aim of developing a causal gene therapy approach in human cells in vitro and in vivo
Das erfindungsgemaße Polypeptid und insbesondere das Interaktionsproteins Nr C 102 bzw die jeweils zurundeliegende Nukleinsaure kann prinzipiell in jedes beliebige Plasmid kloniert werden, wobei Expressionsvektoren und Amplifikationsvektoren insbesondere auch im Hinblick auf eine vorteilhafte Herstellung im großtechnischen Maßstab bevorzugt sind Die Erfindung betrifft infolgedessen auch rekombinante DNS-Vektormolekule, die eine erfindungsgemaße Nukleinsaure umfassen, und die die Fähigkeit zur Amplifikation dieser Nukleinsaure oder zumindest eines Teils davon und/oder zur Expression eines Stat3 -Interaktionsproteins oder zumindest eines Teils davon in einer prokaryontischen oder eukaryontischen Zelle aufweisenThe polypeptide according to the invention and in particular the interaction protein No. C 102 or the underlying nucleic acid can in principle be cloned into any plasmid, expression vectors and amplification vectors being preferred in particular with regard to advantageous production on an industrial scale. The invention consequently also relates to recombinant DNA vector molecules which comprise a nucleic acid according to the invention and which have the ability to amplify this nucleic acid or at least a part thereof and / or to express a Stat3 interaction protein or at least a part thereof in a prokaryotic or eukaryotic cell
Die Erfindung umfaßt deshalb auch transformierte Wirtszellen, die eine erfindungsgemaße Nukleinsaure enthalten, welche mit einem aktivierbaren Promotor gekoppelt ist, der in der jeweiligen Wirtszelle natürlicherweise oder als Folgen einer Rekombination enthalten ist, und die (infolgedessen) die Fähigkeit zur Expression eines Stat3 -Interaktionsproteins aufweisenThe invention therefore also encompasses transformed host cells which contain a nucleic acid according to the invention which is coupled to an activatable promoter which is naturally present in the respective host cell or as a result of recombination and which (as a result) has the ability to express a Stat3 interaction protein
Die Erfindung betrifft ferner die Verwendung eines erfindungsgemaßen Polypeptids zur Herstellung eines (monoklonalen oder polyklonalen) Antikörpers gegen dieses Polypeptid und/oder damit verwandter Proteine, den besagten Antikörper selbst und ebenso seine Verwendung zur Herstellung einer pharmazeutischen Zubereitung bzw eines Arzneimittels für die diagnostische und/oder therapeutische Behandlung von Leukämien und/oder anderen Krebserkrankungen und/oder metabolischen Erkrankungen und/oder neurologischer Erkrankungen sowie für Forschungszwecke
Die Erfindung betrifft auch die Verwendung eines erfindungsgemaßen Polypeptids und/oder einer erfindungsgemaßen Nukleinsaure, wobei es sich insbesondere auch um ein Sense- oder Antisense-Oligonukleotids handeln kann, und/oder eines erfindungsgemaßen Antikörpers zur Herstellung von pharmazeutischen Zubereitungen/ Arzneimitteln für die diagnostische und/oder therapeutische Behandlung von Krebserkrankungen, vor allem Leukämien, und/oder von anderen metabolischen ErkrankungenThe invention further relates to the use of a polypeptide according to the invention for the production of an (monoclonal or polyclonal) antibody against this polypeptide and / or related proteins, said antibody itself and also its use for the production of a pharmaceutical preparation or a medicament for diagnostic and / or therapeutic treatment of leukaemias and / or other cancers and / or metabolic diseases and / or neurological diseases and for research purposes The invention also relates to the use of a polypeptide according to the invention and / or a nucleic acid according to the invention, which may in particular also be a sense or antisense oligonucleotide, and / or an antibody according to the invention for the production of pharmaceutical preparations / medicaments for diagnostic and / or therapeutic treatment of cancer, especially leukemia, and / or other metabolic diseases
Nach neueren wissenschaftlichen Erkenntnissen kommen gerade auch Sense- und Antisense-Oligonukleotide als Wirkstoffe für Pharmakotherapien in Betracht (vgl G Hartmann et al 1998 Antisense-Oligonukleotide, Deutsches Arzteblatt 95, Heft 24, Cl l 15-C11 19) - und überdies als Wirkstoffe mit einem in der Pharmakotherapie grundsatzlich neuen WirkprinzipAccording to recent scientific findings, sense and antisense oligonucleotides are also particularly suitable as active substances for pharmacotherapies (cf. G Hartmann et al 1998 antisense oligonucleotides, Deutsches Arzteblatt 95, issue 24, Cl 1 15-C11 19) - and moreover as active substances a fundamentally new mode of action in pharmacotherapy
Eine technisch und wirtschaftlich bedeutende Einsatzmoglichkeit eines erfindungsgemaßen Polypeptids und ebenso einer erfindungsgemaßen Nukleinsaure besteht nicht zuletzt auch darin, daß mit Hilfe eines solchen Moleküls in einem "Screening"-Verfahren aus einer sehr großen Anzahl bereitstehender Stoffe solche heraus selektiert werden können, die spezifisch an die betreffende Nukleinsaure oder das betreffende Polypeptid binden Diese Stoffe können dann als Ausgangsmaterial (Leitstruktur) für die Entwicklung pharmakologisch einsetzbarer Substanzen dienen und bieten damit die Voraussetzungen für die Entwicklung alternativer Pharmazeutika zur Diagnose und Therapie, insbesondere der bereits erwähnten Leukämien und anderen Krebserkrankungen sowie metabolischen Erkrankungen, bei denen das STAT Protein STAT3 eine wichtige Rolle spieltLast but not least, a technically and economically significant possibility of using a polypeptide according to the invention and also a nucleic acid according to the invention consists in the fact that with the help of such a molecule, a very large number of available substances can be selected from a very large number of available substances in a "screening" process, specifically those specific to the Bind the relevant nucleic acid or the relevant polypeptide. These substances can then serve as the starting material (lead structure) for the development of pharmacologically usable substances and thus offer the prerequisites for the development of alternative pharmaceuticals for diagnosis and therapy, in particular the leukemia and other cancer diseases and metabolic diseases mentioned above, where the STAT protein STAT3 plays an important role
Im Hinblick darauf betrifft die Erfindung auch die Verwendung eines erfindungsgemaßen Polypeptids und/oder einer erfindungsgemaßen Nukleinsaure (Sense- oder Antisense- Oligonukleotide eingeschlossen) und/oder eines erfindungsgemaßen Antikörpers zur Identifizierung von pharmakologisch oder gentherapeutisch einsetzbaren Substanzen, die - vorzugsweise hoch spezifisch - mit dem/den betreffenden erfindungsgemaßen Polypeptid(en) bzw der/den betreffenden erfindungsgemaßen Nukleinsaure(n) reagieren
(insbesondere binden) und dadurch dessen/deren Funktion und/oder Expression beeinflussenIn view of this, the invention also relates to the use of a polypeptide according to the invention and / or a nucleic acid according to the invention (including sense or antisense oligonucleotides) and / or an antibody according to the invention for the identification of pharmacologically or gene-therapeutically usable substances which - preferably highly specifically - with the / react the relevant polypeptide (s) according to the invention or the relevant nucleic acid (s) according to the invention (in particular bind) and thereby influence its function and / or expression
Die Erfindung betrifft darüber hinaus auch die Verwendung eines erfindungsgemaßen Polypeptids und/oder einer erfindungsgemaßen Nukleinsaure, einschließlich Sense- oder Antisense-Oligonukleotide, zum Nachweis eines Stat3 -Interaktionsproteins in einer Probe (Materialprobe), insbesondere einer Serum- oder einer Zellprobe, wobei Polypeptid(e) und/oder Nukleinsaure(n) bzw Oligonukleotid(e) und/oder Antikörper zusammen mit dem betreffenden Serum oder den betreffenden Zellen einem Enzymimmunoassay, einer Polymerasekettenreaktion oder einem Hybridisierungsassay unterworfen werden In der praktischen Anwendung ist diese Serum- oder Zeil- oder sonstige Materialprobe vorzugsweise von Saugern, insbesondere von Menschen (Patienten oder Probanden) gewonnen Zur Durchführung eines solchen Stat3 -Interaktionsprotein-Nachweises, d h einer solchen Nachweisreaktion, wird erfindungsgemaß ein Reagenz vorgeschlagen, das unter Einsatz wenigstens eines erfindungsgemaßen Polypeptids und/oder einer erfindungsgemaßen Nukleinsaure (einschließlich Sense- oder Antisense-Oligonukleotide) und/oder eines erfindungsgemaßen Antikörpers hergestellt istThe invention also relates to the use of a polypeptide according to the invention and / or a nucleic acid according to the invention, including sense or antisense oligonucleotides, for the detection of a Stat3 interaction protein in a sample (material sample), in particular a serum or a cell sample, where polypeptide ( e) and / or nucleic acid (s) or oligonucleotide (s) and / or antibodies together with the serum or cells in question are subjected to an enzyme immunoassay, a polymerase chain reaction or a hybridization assay. In practical use, this serum or cell or other Material sample preferably obtained from suckers, in particular from humans (patients or test subjects) To carry out such Stat3 interaction protein detection, ie such a detection reaction, a reagent is proposed according to the invention which uses at least one polypeptide according to the invention and / or one invented nucleic acid according to the invention (including sense or antisense oligonucleotides) and / or an antibody according to the invention
Die Erfindung wird im folgenden anhand von Herstellungs- und Anwendungsbeispielen naher erläutertThe invention is explained in more detail below with reference to manufacturing and application examples
Beispiel 1: Gewinnung eines Interaktionsproteins des STAT-Proteins mSTAT3 im Yeast-Two-Hybrid System Hefezellen des Stamms Saccharomyces cerevisiae CG 1945, die bekanntermaßen keine STAT-Protein aktivierenden Kinasen exprimieren, wurden mit nicht-phosphoryliertem und somit nicht-aktiviertem STAT-Protein STAT3 transformiert Hierfür wurde die für das Protein mSTAT3 kodierende Nukleotidsequenz in den Hefe-Expressions- Vektor pAS2-l [GAL4 DNA Binding Domain] kloniert und dieser Vektor anschließend in Hefezellen eingeschleust Der Nachweis der Expression von mSTAT3 in den Hefezellen erfolgte
mittels des bekannten Western-Blot- Verfahrens unter Verwendung eines handelsüblichen mSTAT3 spezifischen Antikörpers (z B der Firma Calbiochem)EXAMPLE 1 Obtaining an Interaction Protein of the STAT Protein mSTAT3 in the Yeast-Two-Hybrid System Yeast cells of the strain Saccharomyces cerevisiae CG 1945, which are known not to express STAT protein-activating kinases, were treated with non-phosphorylated and thus inactivated STAT protein STAT3 For this purpose, the nucleotide sequence coding for the protein mSTAT3 was cloned into the yeast expression vector pAS2-1 [GAL4 DNA binding domain] and this vector was subsequently introduced into yeast cells. The expression of mSTAT3 was detected in the yeast cells by means of the known Western blot method using a commercially available mSTAT3-specific antibody (eg from Calbiochem)
Zum Nachweis dafür, daß bei den Saccharomyces cerevisiae CG 1945 enthaltenen Reportergenen, namlich (1 ) Histidin, dessen Promotor eine GAL4-responsive Upstream Activating Sequence enthalt, und (2 ) lacz, dessen Promotor ebenfalls eine GAL4- responsive Upstream Activating Sequence UAS enthalt), keine Autoaktivierung stattfand bzw stattfindet, wurde in Kontrollversuchen untersucht, ob die betreffenden Reportergene allein durch das GAL4 DNA BD/mSTAT3-Plasmid zu aktivieren sind Diese Kontrollversuche lieferten ein negatives Ergebnis und zeigten damit, daß keine Autoaktivierung möglich warTo prove that the reporter genes contained in the Saccharomyces cerevisiae CG 1945, namely (1) histidine, whose promoter contains a GAL4-responsive upstream activating sequence, and (2) lacz, whose promoter also contains a GAL4-responsive upstream activating sequence UAS) , no auto-activation took place or takes place, control tests were carried out to determine whether the reporter genes in question can be activated solely by the GAL4 DNA BD / mSTAT3 plasmid. These control tests gave a negative result and therefore showed that no auto-activation was possible
Die das GAL4 DNA BD/mSTAT3-Plasmid (DNA Binding Domain/mSTAT3-Plasmid) enthaltenden Hefezellen wurden mit einer "Maus Lymphoma CDNA-Bank", die in den Hefe-Expressions- Vektor PACT [GAL4 Activation Domain] kloniert war, (d h mit einem Aktivation Domain/Library-Plasmid) kotransformiert und unter den für Hefezellen üblichen Bedingungen bis zum Koloniewachstum weiter kultiviert Zum Nachweis einer Protein-Protein-Interaktion wurden Kolonien dieser kotransformierten Hefezellen zunächst auf Histidin-Selektionsplatten gepickt und anschließend auf die Expression vonThe yeast cells containing the GAL4 DNA BD / mSTAT3 plasmid (DNA binding domain / mSTAT3 plasmid) were cloned with a "mouse lymphoma CDNA bank" cloned into the yeast expression vector PACT [GAL4 Activation Domain] (ie co-transformed with an activation domain / library plasmid) and further cultivated under the conditions customary for yeast cells until colony growth. To detect a protein-protein interaction, colonies of these co-transformed yeast cells were first picked on histidine selection plates and then on the expression of
Beta-Galaktosidase getestet Kolonien, die sowohl auf Histidin-Selektionsplatten wuchsen als auch zur Beta-Galaktosidase-Expression befähigt waren, wurden als Kolonien mit erfolgter Protein-Protein-Interaktion bewertetBeta-galactosidase tested Colonies that both grew on histidine selection plates and were capable of beta-galactosidase expression were evaluated as colonies with protein-protein interaction
Solche Kolonien bzw Klone mit augenscheinlich erfolgter Protein-Protein-Interaktion wurden einer DNA-Sequenzierung mit im Stand der Technik gelaufigen Verfahren unterworfen Die dabei erhaltenen Nukleotidsequenzen wurden in die vom NCBI bereitgestellten Datenbanken eingegeben Frameshift-Mutanten wurden verworfen und die verbliebenen Klone wurden auf spezifische Reportergen-Aktivierung getestet, namlich auf die Transformation dieser GAL4 AD/Lymphoma cDNA Library-Plasmide allein, mit GAL4 DNA BD/mSTAT3, mit GAL4 DNA BD/LaminC oder mit GAL4 DNA BD/no insert Nur ein einziger Klon zeigte die Aktivierung beider Reportergene ausschließlich bei Kotransformation mit GAL4 DNA BD/mSTAT3 Dieser Klon erhielt die Bezeichnung C 102
Der Klon C 102 wurde zunächst in Hefemedium vermehrt und anschließend einem Plasmid-Isolationsverfahren mit dem Fachmann gelaufigen Standard-Methoden unterworfen, bei dem die beiden Plasmide pAS2-l[BD/mSTAT3] und pACT[ AD/C 102] gewonnen wurden Mit diesem Plasmid-Gemisch wurden E coli KC8 Zellen transformiert und diese Zellen anschließend auf M9-Minimal-Medium kultiviert und vermehrt E coli KC8 Zellen besitzen von Haus aus kein Leucin-Gen und wachsen deshalb auf M9- Minimal-Medium nur unter der Voraussetzung, daß sie zuvor mit einem Leucin-Gen transformiert wurden Der Vektor pACT enthalt ein solches Leucin-Gen, so daß (nur) solche E coli KC8 Zellen, die diesen Vektor pACT enthalten, auf dem M9-Minimal- Medium wachsen bzw sich vermehren Aus diesen auf M9-Minimal-Medium gezüchteten E coli KC8 Zellen wurde das pACT-Plasmid mit dem Fachmann bekannten und gelaufigen Standard-Methoden isoliert und anschließend sequenziert Dabei wurde ein in dem Plasmid integriertes Polypeptid gefunden, das die Bezeichnung "C 102" bzw "Stat3- Interaktionsprotein Nr C 102" erhielt und die im Sequenzprotokoll SEQ ID NO 2 dargestellte Aminosauresequenz aufweistSuch colonies or clones with apparent protein-protein interaction were subjected to DNA sequencing using methods known in the art. The nucleotide sequences obtained were discarded in the databases provided by the NCBI. Frameshift mutants were discarded and the remaining clones were targeted to specific reporter genes -Activation tested, namely for the transformation of these GAL4 AD / Lymphoma cDNA Library plasmids alone, with GAL4 DNA BD / mSTAT3, with GAL4 DNA BD / LaminC or with GAL4 DNA BD / no insert. Only one clone showed the activation of both reporter genes exclusively in co-transformation with GAL4 DNA BD / mSTAT3 This clone was given the designation C 102 The clone C 102 was first propagated in yeast medium and then subjected to a plasmid isolation process using standard methods familiar to the person skilled in the art, in which the two plasmids pAS2-1 [BD / mSTAT3] and pACT [AD / C 102] were obtained using this plasmid Mixture, E coli KC8 cells were transformed and these cells were subsequently cultivated and propagated on M9 minimal medium. E coli KC8 cells do not inherently have a leucine gene and therefore grow on M9 minimal medium only on the condition that they have previously were transformed with a leucine gene. The vector pACT contains such a leucine gene so that (only) those E coli KC8 cells which contain this vector pACT grow or multiply on the M9 minimal medium. The pACT plasmid was isolated from minimally medium-grown E coli KC8 cells using standard methods known to those skilled in the art and then sequenced. A polypeptide integrated in the plasmid was found , which received the designation "C 102" or "Stat3 interaction protein No. C 102" and has the amino acid sequence shown in the sequence listing SEQ ID NO 2
Die Spezifitat dieses Stat3 -Interaktionsproteins Nr C 102 ist durch folgende Assays nachgewiesen (1) nach Kotransformation der Hefezellen mit den Nukleotidsequenzen von STAT3 und dem Stat3 -Interaktionsproteins No C 102 in den jeweiligen, oben beschriebenen Vektoren waren die Interaktionsreaktionen der Bindungs-Domanen und Activation-Domanen des GAL4 Transkriptionsfaktors im Yeast-Two-Hybrid System positiv (2) Die Interaktionsreaktionen des Stat3 -Interaktionsproteins Nr C 102 mit LaminC als unspezifischem Interaktionspartner waren negativ (3) Nach Transformation der Hefezellen mit der für das Stat3 -Interaktionsprotein Nr C 102 kodierenden Nukleotidsequenz allein (als Insert im Activation Doman- Vektor) war keine Autoaktivierung des Yeast-Two Hybrid Interaktionssystems auslosbar
The specificity of this Stat3 interaction protein No. C 102 was demonstrated by the following assays (1) after cotransformation of the yeast cells with the nucleotide sequences of STAT3 and the Stat3 interaction protein No C 102 in the respective vectors described above, the interaction reactions of the binding domains and activation were -Domaines of the GAL4 transcription factor in the yeast two hybrid system positive (2) The interaction reactions of the Stat3 interaction protein No. C 102 with LaminC as non-specific interaction partner were negative (3) After transformation of the yeast cells with the coding for the Stat3 interaction protein No. C 102 The nucleotide sequence alone (as an insert in the Activation Doman vector) could not trigger an auto-activation of the Yeast-Two Hybrid interaction system
Claims
A n s p r ü c h e Expectations
Isoliertes Polypeptid, das als Interaktionsprotein des Stat3 Transkriptionsfaktors fungiert, und das entweder die im Sequenzprotokoll SEQ ID NO 2 dargestellte oder eine durch Aminosauresubstitution, -deletion, -insertion, -oder -inversion als Allel oder Derivat daraus entstandene Aminosauresequenz aufweist, wobei SEQ D NO 2, Bestandteil dieses Anspruchs ist, und wobei die durch Aminosauresubstitution, -deletion, -insertion, - oder -inversion als Allel oder Derivat entstandenen A inosauresequenzen zur Interaktion mit Domänen des Stat3 Transkriptionsfaktors geeignet sindIsolated polypeptide which functions as an interaction protein of the Stat3 transcription factor and which has either the amino acid sequence shown in the sequence listing SEQ ID NO 2 or an amino acid sequence resulting therefrom as an allele or derivative by amino acid substitution, deletion, insertion, or inversion, where SEQ D NO 2, is part of this claim, and wherein the amino acid sequences created as an allele or derivative by amino acid substitution, deletion, insertion, or inversion are suitable for interaction with domains of the Stat3 transcription factor
Isoliertes Polypeptid nach Anspruch 1, dadurch gekennzeichnet, daß es ein Saugerprotein istIsolated polypeptide according to claim 1, characterized in that it is a sucker protein
Isoliertes Polypeptid nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß es ein Mausprotein oder ein Humanprotein istIsolated polypeptide according to claim 1 or 2, characterized in that it is a mouse protein or a human protein
Isoliertes Polypeptid nach einem der Ansprüche 1 bis 3, dadurch gekennzeichnet, daß es ein natives Protein istIsolated polypeptide according to any one of claims 1 to 3, characterized in that it is a native protein
Isoliertes Polypeptid nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, daß es ein rekombinantes Protein istIsolated polypeptide according to any one of claims 1 to 4, characterized in that it is a recombinant protein
Isolierte Nukleinsaure, die ein Polypeptid gemäß Anspruch 1 codiert, und die entweder die im Sequenzprotokoll SEQ LD NO 1 dargestellte Nukleotidsequenz oder eine hierzu komplementäre Nukleotidsequenz oder eine Teilsequenz einer dieser Nukleotidsequenzen oder eine ganz oder teilweise mit einer dieser Nukleotidsequenzen hybridisierende Nukleotidsequenz aufweist, wobei SEQ ID NO 1 Bestandteil dieses Anspruchs ist, und wobei in diesem Sequenzprotokoll anstelle von "T" auch "U" stehen kann
Isolierte Nukleinsaure nach Anspruch 6, dadurch gekennzeichnet, daß diese Nukleinsaure aus einer naturlichen oder einer synthetischen oder einer halbsynthetischen Quelle gewonnen istIsolated nucleic acid which encodes a polypeptide according to claim 1 and which has either the nucleotide sequence shown in the sequence listing SEQ LD NO 1 or a nucleotide sequence complementary thereto or a partial sequence of one of these nucleotide sequences or a nucleotide sequence which hybridizes in whole or in part with one of these nucleotide sequences, where SEQ ID NO 1 is part of this claim, and “U” can also be used instead of “T” in this sequence listing Isolated nucleic acid according to claim 6, characterized in that this nucleic acid is obtained from a natural or a synthetic or a semi-synthetic source
Isolierte Nukleinsaure nach Anspruch 6 oder 7 dadurch gekennzeichnet, daß diese Nukleinsaure eine cDNA istIsolated nucleic acid according to claim 6 or 7, characterized in that this nucleic acid is a cDNA
Isolierte Nukleinsaure nach einem der Ansprüche 6 oder 7, dadurch gekennzeichnet, daß diese Nukleinsaure ein Sense- oder Antisense-Oligonukleotid ist, das mindestens 6, vorzugsweise 8 bis 25 Nukleotide umfaßt und mit der im Sequenzprotokoll SEQ ID NO 1 dargestellten Nukleotidsequenz oder Teilsequenzen davon hybridisiertIsolated nucleic acid according to one of claims 6 or 7, characterized in that this nucleic acid is a sense or antisense oligonucleotide which comprises at least 6, preferably 8 to 25 nucleotides and hybridizes with the nucleotide sequence or partial sequences thereof shown in the sequence listing SEQ ID NO 1
Isolierte Nukleinsaure nach einem der Ansprüche 6 oder 7, dadurch gekennzeichnet, daß diese Nukleinsaure eine Splice- Variante ist, die mit der im Sequenzprotokoll SEQ LD NO 1 dargestellten Nukleotidesquenz hybridisiertIsolated nucleic acid according to one of claims 6 or 7, characterized in that this nucleic acid is a splice variant which hybridizes with the nucleotide sequence shown in the sequence listing SEQ LD NO 1
Rekombinantes DNS-Vektormolekul, das eine Nukleinsaure nach einem derRecombinant DNA vector molecule containing a nucleic acid according to one of the
Ansprüche 6 bis 10 umfaßt, und das die Fähigkeit zur Expression eines Stat3- Interaktionsproteins in einer prokaryontischen oder eukaryontischen Zelle aufweistClaims 6 to 10, and which has the ability to express a Stat3 interaction protein in a prokaryotic or eukaryotic cell
Rekombinantes DNS-Vektormolekul das eine Nukleinsaure nach einem der Ansprüche 6 bis 10 umfaßt, und das die Fähigkeit zur A plifikation eines Stat3- Interaktionsproteins in einer prokaryontischen oder eukaryontischen Zelle aufweistRecombinant DNA vector molecule which comprises a nucleic acid according to any one of claims 6 to 10 and which has the ability to amplify a Stat3 interaction protein in a prokaryotic or eukaryotic cell
Transformierte Wirtszelle, die eine Nukleinsaure nach einem der Ansprüche 6 bis 1 1 enthalt, welche mit einem aktivierbaren Promotor gekoppelt ist, der in der Wirtszelle natürlicherweise oder als Folgen einer Rekombination enthalten ist, und die die Fähigkeit zur Expression eines Stat3 -Interaktionsproteins aufweist
Verwendung eines Polypeptids nach einem der Ansprüche 1 bis 5 zur Herstellung eines Antikörpers gegen dieses Polypeptid und/oder damit verwandter ProteineTransformed host cell containing a nucleic acid according to any one of claims 6 to 11, which is coupled to an activatable promoter which is contained in the host cell naturally or as a result of recombination, and which has the ability to express a Stat3 interaction protein Use of a polypeptide according to any one of claims 1 to 5 for producing an antibody against this polypeptide and/or proteins related thereto
Antikörper, der spezifisch mit einem Polypeptid gemäß einem der Ansprüche 1 bis 5 reagiertAntibody which reacts specifically with a polypeptide according to any one of claims 1 to 5
Verwendung eines Antikörpers nach Anspruch 14 oder 15 , dadurch gekennzeichnet, daß der Antikörper zur Herstellung einer pharmazeutischen Zubereitung/eines Arzneimittels für die diagnostische und/oder therapeutische Behandlung von Leukämien und/oder anderen Krebserkrankungen bzw metabolischen Erkrankungen eingesetzt wirdUse of an antibody according to claim 14 or 15, characterized in that the antibody is used to produce a pharmaceutical preparation/medicine for the diagnostic and/or therapeutic treatment of leukemias and/or other cancers or metabolic diseases
Verwendung eines Polypeptids nach einem der Ansprüche 1 bis 5 und/oder einer Nukleinsaure nach einem der Ansprüche 6 bis 11 (insbesondere auch eines Sense- oder Antisense-Oligonukleotids) und/oder eines Antikörpers nach Anspruch 14 oder 15 zur Herstellung von pharmazeutischen Zubereitungen/ Arzneimitteln für die diagnostische und/oder therapeutische Behandlung von Leukämien und/oder anderen Krebserkrankungen bzw metabolischen ErkrankungenUse of a polypeptide according to one of claims 1 to 5 and/or a nucleic acid according to one of claims 6 to 11 (in particular also a sense or antisense oligonucleotide) and/or an antibody according to claim 14 or 15 for the production of pharmaceutical preparations/medicines for the diagnostic and/or therapeutic treatment of leukemias and/or other cancers or metabolic diseases
Verwendung eines Polypeptids nach einem der Ansprüche 1 bis 5 oder einer Nukleinsaure nach einem der Ansprüche 6 bis 11 oder eines Antikörpers nach Anspruch 14 oder 15 zur Identifizierung von pharmakologisch oder gentherapeutisch einsetzbaren Substanzen, die mit dem Polypeptid gemäß Anspruch 1 bzw der Nukleinsaure gemäß Anspruch 6 reagieren und dadurch dessen/deren Funktion und/oder Expression beeinflussen
Use of a polypeptide according to one of claims 1 to 5 or a nucleic acid according to one of claims 6 to 11 or an antibody according to claim 14 or 15 for identifying substances which can be used pharmacologically or in gene therapy and which are compatible with the polypeptide according to claim 1 or the nucleic acid according to claim 6 react and thereby influence its function and/or expression
19. Verwendung eines Polypeptids nach einem der Ansprüche 1 bis 5 und/oder einer Nukleinsaure nach einem der Ansprüche 6 bis 11 (insbesondere auch eines Sense- oder Antisense-Oligonukleotids) und oder eines Antikörpers nach Anspruch 14 oder 15 zum Nachweis eines Stat3-Interaktionsproteins dadurch gekennzeichnet, daß Polypeptid(e) und/oder Nukleinsäure(n) bzw. Olygonukleotid(e) und/oder Antikörper zusammen mit einer Materialprobe eines Säugers, einem Enzymimmunoassay, einer Polymerasekettenreaktion oder einem Hybridisierungsassay unterworfen werden.19. Use of a polypeptide according to one of claims 1 to 5 and / or a nucleic acid according to one of claims 6 to 11 (in particular also a sense or antisense oligonucleotide) and or an antibody according to claim 14 or 15 for detecting a Stat3 interaction protein characterized in that polypeptide(s) and/or nucleic acid(s) or olygonucleotide(s) and/or antibodies are subjected together with a material sample from a mammal to an enzyme immunoassay, a polymerase chain reaction or a hybridization assay.
20. Reagenz zum Nachweis eines Stat3 -Interaktionsproteins, dadurch gekennzeichnet, daß das Reagenz unter Verwendung wenigstens eines Polypeptids gemäß einem der Ansprüche 1 bis 5 und/oder wenigstens einer Nukleinsaure nach einem der Ansprüche 6 bis 1 1 und/oder wenigstens eines Antikörpers nach Anspruch 14 oder 15 hergestellt ist.
20. Reagent for detecting a Stat3 interaction protein, characterized in that the reagent using at least one polypeptide according to one of claims 1 to 5 and / or at least one nucleic acid according to one of claims 6 to 1 1 and / or at least one antibody according to claim 14 or 15 is made.
Priority Applications (1)
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|---|---|---|---|
| AU33593/01A AU3359301A (en) | 1999-12-21 | 2000-12-19 | Interaction proteins for the stat transcription factor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1999161899 DE19961899A1 (en) | 1999-12-21 | 1999-12-21 | Interactone proteins for STAT transcription factor |
| DE19961899.2 | 1999-12-21 |
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| WO2001046412A2 true WO2001046412A2 (en) | 2001-06-28 |
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| AU (1) | AU3359301A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1998030688A1 (en) * | 1997-01-10 | 1998-07-16 | Tadamitsu Kishimoto | Novel stat function-regulatory protein |
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-
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2000
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|---|---|---|---|---|
| WO1998030688A1 (en) * | 1997-01-10 | 1998-07-16 | Tadamitsu Kishimoto | Novel stat function-regulatory protein |
Non-Patent Citations (5)
| Title |
|---|
| CHIEN C-T ET AL: "THE TWO-HYBRID SYSTEM: A METHOD TO IDENTIFY AND CLONE GENES FOR PROTEINS THAT INTERACT WITH A PROTEIN OF INTEREST" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, Bd. 88, 1. November 1991 (1991-11-01), Seiten 9578-9582, XP000647704 ISSN: 0027-8424 in der Anmeldung erw{hnt * |
| FRANK DAVID A: "STAT signaling in the pathogenesis and treatment of cancer." MOLECULAR MEDICINE (NEW YORK), Bd. 5, Nr. 7, Juli 1999 (1999-07), Seiten 432-456, XP001021603 ISSN: 1076-1551 * |
| GABLER ANGELIKA ET AL: "C102: A novel STAT3 interacting protein." BLOOD, Bd. 96, Nr. 11 Part 1, 16. November 2000 (2000-11-16), Seite 94a XP001025916 42nd Annual Meeting of the American Society of Hematology;San Francisco, California, USA; December 01-05, 2000 ISSN: 0006-4971 * |
| NAKA T ET AL: "NEGATIVE REGULATION OF CYTOKINE SIGNALING: STAT-INDUCED STAT INHIBITOR" TIBS TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER PUBLICATION, CAMBRIDGE, EN, Bd. 24, Nr. 10, Oktober 1999 (1999-10), Seiten 394-398, XP001019044 ISSN: 0968-0004 * |
| NAKA T ET AL: "Structure and function of a new STAT-induced STAT inhibitor" NATURE, MACMILLAN JOURNALS LTD. LONDON, GB, Bd. 387, Nr. 6636, 26. Juni 1997 (1997-06-26), Seiten 924-929, XP002088455 ISSN: 0028-0836 * |
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| WO2001046412A3 (en) | 2002-03-14 |
| AU3359301A (en) | 2001-07-03 |
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