JP2007254465A - REGULATOR FOR p65-MEDIATED SIGNAL TRANSDUCTION AND SCREENING METHOD THEREFOR - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To further analyze a mechanism for regulating p65-mediated signal transduction through a ubiquitin/proteasome-dependent degradation pathway in immune cells to identify a factor contributing to the pathway, and to use the findings for the development of new drugs and the like. <P>SOLUTION: The present invention provides a regulator for p65-mediated signal transduction containing a substance for regulating expression or function of SLIM, a method of screening a regulator of p65-mediated signal transduction including to judge whether a test sample can regulate the expression or function of SLIM, a SLIM-deficient non-human animal in which TLR ligand is administered, a SLIM-deficient cell treated with a TLR ligand, and a complex comprising SLIM and HSP90. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides a regulator of p65-mediated signal transduction, a screening method thereof, and a predetermined SLIM-deficient animal / cell.

  The transcription factor NF-κB / Rel family consists of five members: p65 / RelA, RelB, c-Rel, p50 and p52. In innate immune cells such as macrophages, dendritic cells and fibroblasts, p65 and p50 heterodimers can be activated by stimulation of TLR ligands and cytokines (eg, TNFα, IL-1) (Canonical NF-κB pathway) Called). When cells are stimulated, the IκB (inhibitor of κB) protein (which binds to and sequesters the NF-κB protein in the cytoplasm) is first degraded, and then the NF-κB protein translocates into the nucleus. Thus, it cooperatively induces the expression of a series of genes (including inflammatory cytokines and chemokines) essential for host defense against microbial pathogens. On the other hand, NF-κB dysregulation (resulting in constitutive activation of NF-κB signaling) in these cells can cause several human immune diseases (eg, asthma, arthritis and inflammatory bowel disease). It has been reported. This suggests the importance of NF-κB signaling terminating at the appropriate time. Several mechanisms have been proposed to explain this regulation. One is a transport pathway in which the re-synthesized IκB protein moves into the nucleus and returns NF-κB into the cytoplasm. Another important mechanism is the ubiquitin / proteasome-dependent degradation of the NF-κB · p65 protein in the nucleus, which synergizes with IκB to inactivate NF-κB signaling.

  However, the manner in which NF-κB is degraded in the nucleus and terminates its activation is not yet fully understood. Furthermore, ubiquitin ligases that mediate ubiquitination of nuclear p65 proteins have not yet been identified.

In addition, the following is mentioned as a prior art in the technical field relevant to the present research result of the present inventors.
In Non-Patent Document 1, a) p65 can associate with PML (Promyelocytic leukemia protein), b) PML overexpression is isolated in the nucleus containing PML Nuclear body (PML and some other proteins) P65 can be induced to translocate into a nucleus in a structure that is distributed in the form of dots in the nucleus), and c) overexpression of PML suppresses its transcriptional activity by inhibiting the binding of p65 to DNA. And d) PML promotes apoptosis of cells by TNFα through this inhibitory action on p65.
Non-Patent Documents 2 to 4 show that a) some proteins such as p53, Myc, and LEF-1 are transported to the PML Nuclear body, and b) PML Nuclear body has positive and negative activation of transcription factors. It has been suggested that it has a function of controlling in both directions, and c) PMLNuclear body is a special fraction responsible for proteolysis in the nucleus.
In Non-Patent Document 5, a) SLIM was identified as a nuclear ubiquitin ligase that acts on STAT4 and STAT1 in CD4 ⁺ T cells, and b) proteasome dependence of STAT4 and STAT1 due to forced expression of SLIM. C) SLIM mediates polyubiquitination and subsequent degradation of the STAT4 protein via the LIM domain, d) SLIM (− / −) mice And e) that SL1 (− / −) mouse-derived CD4 ⁺ T cells have increased Th1 cell differentiation and increased STAT4 protein levels.
Non-Patent Document 6 describes that SLIM can bind to α-actinin, which is an actin-binding protein.
Non-Patent Document 7 describes that p65 can be inactivated by being ubiquitinated and degraded in a proteasome-dependent manner.
Wu, WS et al., J. Biol. Chem. 278: 12294-12304 (2003) Zhong S. et al., Nature Cell Biol., 2: E85-90 (2000) Smith KP et al., J. Cell Biochem. 93: 1282-1296 (2004) Sachdev S. et al., Genes & Dev. 15: 3088-3013 (2001) Tanaka T. et al., Immunity 22: 729-736 (2005) Torrad M. et al., Invest Ophthalmol Vis Sci. 45: 3955-3963 (2004) Sccanti S. et al., J. Exp. Med., 200: 107-113 (2004)

  The present invention further analyzes the mechanism regulating p65-mediated signal transduction through the ubiquitin / proteasome-dependent degradation pathway in innate immune cells, identifies factors responsible for this pathway, and discovers these findings It is intended to be applied to.

  As a result of intensive studies, the present inventors have determined that SLIM (STAT-interacting LIM protein), a nuclear PDZ / LIM protein that acts as a ubiquitin E3 ligase, negatively regulates NK-κB · p65-mediated signaling And that other factors such as HSP90 are also involved in this regulatory system. Since abnormalities in NF-κB signal transduction can be associated with diseases such as inflammatory diseases, the knowledge of the present inventors is considered to enable the development of preventive and therapeutic drugs for such diseases.

Based on the above findings, the present inventors have completed the present invention.
That is, the present invention provides the following inventions.
[1] A regulator of p65-mediated signal transduction comprising a substance that regulates the expression or function of SLIM;
[2] The agent according to [1] above, wherein the substance that modulates SLIM expression or function is an inhibitor of p65-mediated signaling, wherein the substance promotes SLIM expression or function;
[3] The agent according to [2] above, wherein the substance that promotes the expression or function of SLIM is a SLIM expression vector or a substance that suppresses the expression or function of HSP;
[4] The agent according to [2] above, wherein the inhibitor of p65-mediated signaling is a drug for a disease selected from the group consisting of inflammatory diseases and autoimmune diseases;
[5] The agent according to [1] above, wherein the substance that modulates SLIM expression or function is an activator of p65-mediated signaling, wherein the substance suppresses SLIM expression or function;
[6] The agent according to [5] above, wherein the substance that suppresses SLIM expression or function is an HSP expression vector;
[7] The agent according to [6] above, wherein HSP is HSP90;
[8] The agent according to [5] above, wherein the activator of p65-mediated signal transduction is a drug for a disease selected from the group consisting of infectious diseases and malignant tumors;
[9] A SLIM-deficient non-human animal administered with a TLR ligand;
[10] SLIM-deficient cells treated with a TLR ligand;
[11] A screening method for a substance capable of modulating p65-mediated signal transduction, comprising evaluating whether or not a subject can modulate SLIM expression or function;
[12] 1) Measurement of SLIM expression using cells capable of measuring SLIM expression 2) Measurement of SLIM function using a reconstitution system capable of measuring SLIM function 3) Measurement of SLIM function Measurement of SLIM function using a possible cell line, 4) the screening method of [11] above, which is performed by either measuring SLIM expression or function using an animal;
[13] The evaluation is performed based on whether or not the test subject has the ability to modulate p65 ubiquitination by SLIM, or whether to have the ability to regulate p65 sequestration to PML Nuclear Body by SLIM. The method of [11] above;
[14] To evaluate whether the test modulates the ability of a complex containing SLIM and HSP90 or whether the test regulates the ability of a complex containing SLIM and actinin The screening method of [11], which is performed based on the above;
[15] The method of [11] above, which is performed under TLR ligand stimulation;
[16] A complex comprising SLIM and HSP90.

The modulator of the present invention can be used, for example, for diseases in which suppression of p65-mediated signaling is desired, for example, drugs for inflammatory diseases, autoimmune diseases, and diseases for which promotion of p65-mediated signaling is desired, for example, infectious diseases It can be useful as a medicament for malignant tumors.
The animals and cells of the present invention are useful, for example, as model animals and model cells for diseases such as inflammatory diseases and autoimmune diseases, and for analysis of p65-mediated signal transduction and development of preventive and therapeutic agents for the above diseases. obtain.
The screening method of the present invention can be useful, for example, for the development of a medicament for a disease for which modulation of p65-mediated signaling is desired.
The complex of the present invention can be useful, for example, in the screening method of the present invention.

  The present invention provides a modulator of p65-mediated signaling comprising a substance that modulates SLIM expression or function.

  In one embodiment, an agent that modulates SLIM expression or function may be an agent that promotes SLIM expression or function. As used herein, the promotion of SLIM expression includes supplementation of SLIM (protein) itself. Examples of the substance that promotes the expression or function of SLIM include SLIM or actinin (protein), an expression vector containing a nucleic acid encoding SLIM or actinin (SLIM or actinin expression vector), expression or function of HSP (eg, HSP90) (Eg, antisense nucleic acid, ribozyme, RNAi-inducible nucleic acid (eg, siRNA), HSP90 dominant negative mutant, low molecular weight compound (eg, geldanamycin, 17-AAG (17-allylamino-17-de)) (Methoxygeldanamycin), 17-AG (17-amino-geldanamycin), Radicicol, Novobiocin) These substances can be prepared by a method known per se. For human SLIM, GenBank Act Session number: See AY007729.

  The SLIM or actinin that can be contained in the agent of the present invention can be the above-mentioned animal-derived natural SLIM or actinin or a variant thereof, but preferably a human-derived natural SLIM or actinin or a variant thereof. SLIM has one or more amino acid sequences in the encoded amino acid sequence as long as it has a LIM domain responsible for ubiquitination of p65 and a PDZ domain responsible for sequestration of p65 to the PML Nuclear body, or as long as it retains its function. The amino acid mutation (eg, deletion, substitution, addition, insertion) may be present. As long as it has a domain responsible for binding to SLIM, a domain responsible for binding to actin, or retains its function, actinin has one or more amino acid mutations (eg, deletion, Substitution, addition, insertion).

  The SLIM and actinin that can be included in the agents of the present invention can also be proteins that can be recovered from native SLIM or actinin-expressing cells, or recombinant proteins. SLIM can be prepared by a method known per se, for example, a) SLIM may be recovered from natural SLIM-expressing cells (eg, macrophages, dendritic cells, fibroblasts, T cells, B cells), b) A transformant is produced by introducing a SLIM expression vector (described later) into a host cell (eg, Escherichia, Bacillus, yeast, insect cell, insect, animal cell), and produced by the transformant. SLIM may be recovered, or c) SLIM may be synthesized by a cell-free system using rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate or the like. Actinin can be prepared similarly. SLIM and actinin are methods using solubility such as salting out and solvent precipitation; methods using mainly differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis ; A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography and use of SLIM antibody; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as isoelectric focusing method, or the like;

  In another embodiment, an agent that modulates SLIM expression or function may be an agent that inhibits SLIM expression or function. Examples of substances that suppress the expression or function of SLIM include, for example, antisense nucleic acids, ribozymes, RNAi-inducible nucleic acids (eg, siRNA), dominant negative mutants, and HSPs (eg, HSP90) against SLIM or actinin, and these Examples thereof include an expression vector containing a nucleic acid to encode and a low molecular weight compound. These substances can be produced by a method known per se.

  When the substance that regulates the expression or function of SLIM is a nucleic acid molecule or a protein molecule, the agent of the present invention can contain an expression vector containing a nucleic acid molecule encoding the nucleic acid molecule or protein molecule as an active ingredient. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter to be used is not particularly limited as long as it can function in the administration subject. For example, viruses such as SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus-derived early promoter, etc. Examples include promoters and mammalian constituent protein gene promoters such as β-actin gene promoter, PGK gene promoter, transferrin gene promoter, and the like. Moreover, as a promoter to be used, a promoter specific to SLIM expressing cells (eg, macrophages, dendritic cells, fibroblasts, T cells, B cells) may be used. Such a promoter can be a promoter of any gene that is specifically expressed in SLIM-expressing cells.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. Furthermore, a selection marker gene for selecting transformed cells (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements an auxotrophic mutation, etc.) is further included. You can also.

  The basic skeleton vector used as the expression vector may be a plasmid or a viral vector, but suitable vectors for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia. Examples of the virus vector include viruses, poxviruses, polioviruses, Sindbis viruses, Sendai viruses, and lentiviruses.

  The agent of the present invention may contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to a substance that modulates the expression or function of SLIM. Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, sachets or tablets containing an effective amount of the substance as solids or granules. A suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified, or a powder, a granule or the like.

  Formulations suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Also, aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dosage of the agent of the present invention includes the activity and type of the active ingredient, the mode of administration (eg, oral and parenteral), the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age Usually, it is about 0.001 mg to about 2.0 g as an active ingredient amount per day for an adult, although it cannot be generally stated.

  The agent of the present invention is useful, for example, as a medicine, reagent or food.

  Specifically, the agent of the present invention can suppress p65-mediated signal transduction when a substance that regulates SLIM expression or function is included, for example, a substance that promotes SLIM expression or function. The present inventors have found that SLIM acts as an E3 ligase using p65 as a substrate and can negatively regulate, that is, suppress, p65-mediated signal transduction. In this case, the agent of the present invention can be used for the prevention / treatment of a disease for which suppression of p65-mediated signal transmission is desired, or for the preparation of a disease model (eg, cell, animal) in which the signal transmission is suppressed. . Diseases for which suppression of p65-mediated signaling is desired include, for example, inflammatory diseases (eg, Crohn's disease, ulcerative colitis, chronic bronchial asthma, atopic dermatitis, adult and pediatric respiratory distress syndrome, influenza encephalopathy ), Autoimmune diseases (eg, rheumatoid arthritis, Kawasaki disease, polyarteritis nodosa, type I diabetes, systemic lupus erythematosis). The disease in which p65-mediated signaling is suppressed in a disease model in which p65-mediated signaling is suppressed can be the same as the diseases described below in which promotion of p65-mediated signaling is desired.

  On the other hand, the agent of the present invention can promote p65-mediated signaling when it contains a substance that regulates SLIM expression or function, for example, a substance that suppresses SLIM expression or function. The present inventors have found that SLIM acts as an E3 ligase using p65 as a substrate and can negatively regulate, that is, suppress, p65-mediated signal transduction. Therefore, the agent of the present invention can promote p65-mediated signaling when it contains a substance that suppresses the expression or function of SLIM. In this case, the agent of the present invention can be used for the prevention / treatment of a disease for which promotion of p65-mediated signal transduction is desired, or the creation of a disease model (eg, cell, animal) in which p65-mediated signal transduction is promoted. Examples of the disease for which promotion of p65-mediated signal transduction is desired include infectious diseases (eg, bacterial, viral, fungal infection) and malignant tumors. The disease in which p65-mediated signal transduction is promoted in a disease model in which p65-mediated signal transduction is promoted may be similar to the above-mentioned diseases in which suppression of p65-mediated signal transduction is desired.

(Animals and cells)
The present invention provides SLIM-deficient non-human animals to which a TLR ligand is administered.

  The TLR (Toll-like receptor) ligand is not particularly limited as long as it can introduce a stimulation signal into cells via TLR. For example, LPS, CpG DNA, single-stranded or double-stranded RNA, antiviral Examples include drugs (imidazoquinolines), flagellins, peptidoglycans, bacterial lipopeptides, and mycoplasma-derived lipopeptides.

  The species of the animal of the present invention is not particularly limited, and examples thereof include mammals such as rodents (eg, mice and rats) and fishes such as zebrafish. As SLIM-deficient animals, for example, SLIM-deficient mice are known (see Tanaka T. et al, Immunity 22: 729-736 (2005)).

  The animal of the present invention can be produced by administering a TRL ligand to the SLIM-deficient animal. As the TRL ligand, those described above can be used. Administration of TRL ligand to SLIM-deficient animals can be performed by a method known per se.

  The invention also provides SLIM deficient cells treated with a TLR ligand.

  The cells of the present invention can be cells derived from any animal including humans (for non-human animals, see, for example, the above-mentioned animals). The cells of the present invention can also be cells derived from any tissue, but preferably immune cells (eg, macrophages, dendritic cells, T cells, B cells), fibroblasts, bone marrow cells, spleen cells, blood vessels It can be an endothelial cell. The cell of the present invention may be either a primary cultured cell or a cell line.

  The cells of the present invention can be produced, for example, by treating cells isolated from SLIM-deficient animals with a TLR ligand. Cells derived from SLIM-deficient animals may be cells modified by a method such as genetic engineering. Isolation and modification of cells can be performed by methods known per se (see, eg, Current Protocols in Cell Biology, John Wiley & Sons, Inc. (2001); isolation and culture of functional cells, Maruzen Shoten (1987)). ). Treatment of cells with a TLR ligand can be performed by a method known per se.

  The animals of the present invention may have the characteristic that they are more sensitive to the lethal effects of treatment with TLR ligands than wild-type animals. The cells of the present invention can produce higher amounts of inflammatory cytokines by treatment with TLR ligands than wild type cells. Therefore, the animals and cells of the present invention are useful, for example, as model animals and model cells for inflammatory diseases and autoimmune diseases. The animals and cells of the present invention are also useful for analysis of p65-mediated signal transduction and development of preventive / therapeutic agents for the above diseases. For example, comprehensive analysis of gene expression in the animal of the present invention enables identification of other genes involved in pathological phenotypes including inflammatory diseases and autoimmune diseases. In this case, for example, in the animals and cells of the present invention, the gene expression profile is measured by means (for example, microarray) that enables comprehensive analysis of gene expression, and control animals such as wild-type animals or other disease model animals. Compared to gene expression profiles of (homologous or heterologous animals). It is also possible to follow the expression profile of the animals and cells of the present invention (the test subject can be administered or contacted as necessary) over time, and evaluate the link between the disease state and the change in the expression profile. .

(Screening method)
The present invention relates to a method for screening a substance capable of modulating p65-mediated signal transduction, which can be effective as a medicine, reagent or food, comprising evaluating whether a test substance can modulate SLIM expression or function, And a substance obtained by the screening method and an agent containing the substance.

  The subject to be subjected to the screening method may be any compound or composition, such as a nucleic acid (eg, nucleoside, oligonucleotide, polynucleotide), carbohydrate (eg, monosaccharide, disaccharide, oligosaccharide, Polysaccharides), lipids (eg, saturated or unsaturated linear, branched and / or cyclic fatty acids), amino acids, proteins (eg, oligopeptides, polypeptides), small organic compounds, combinatorial chemistry techniques Examples thereof include a prepared compound library, a random peptide library prepared by solid phase synthesis or a phage display method, natural components (eg, components derived from microorganisms, animals and plants, marine organisms, etc.), foods, drinking water and the like.

  The screening method of the present invention can be carried out in any form as long as it can be evaluated whether or not a subject can modulate the expression or function of SLIM. For example, the screening method of the present invention includes 1) measurement of SLIM expression using cells capable of measuring SLIM expression, 2) measurement of SLIM function using a reconstitution system capable of measuring SLIM function, 3 It can be performed based on measurement of SLIM function using a cell line capable of measuring SLIM function, 4) measurement of SLIM expression or function using an animal, and the like. The screening method of the present invention may be performed under stimulation with a TLR ligand.

In the above 1), the screening method using cells capable of measuring SLIM expression may include, for example, the following steps (a) to (c):
(A) contacting the test subject with a cell capable of measuring SLIM expression;
(B) measuring the expression level of SLIM in a cell contacted with a test substance, and comparing the expression level with the expression level of SLIM in a control cell not contacted with the test substance;
(C) A step of selecting a test substance that regulates the expression level of SLIM based on the comparison result of (b).

  In step (a) of the above method, the test subject is placed in contact with cells capable of measuring SLIM expression. Contact of the test subject with a cell capable of measuring SLIM expression can be performed in culture medium.

  A cell capable of measuring SLIM expression refers to a cell capable of directly or indirectly evaluating the expression level of a SLIM product (eg, transcription product, translation product). A cell capable of directly assessing the expression level of the SLIM product can be a SLIM-expressing cell, whereas a cell capable of indirectly assessing the expression level of the SLIM product is a reporter assay for the transcriptional regulatory region of the SLIM gene. Can be a cell. The cell capable of measuring SLIM expression may be an animal cell described above.

  The SLIM-expressing cell is not particularly limited as long as it potentially expresses SLIM. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. It is also preferable to use immune cells such as macrophages as SLIM expressing cells. Moreover, you may use the cell derived from the model animal of the above-mentioned diseases, such as an inflammatory disease.

  A cell that enables a reporter assay for the transcriptional regulatory region of the SLIM gene is a cell that includes a transcriptional regulatory region of the SLIM gene and a reporter gene operably linked to the region. The transcriptional regulatory region of SLIM gene, the reporter gene can be inserted into an expression vector. The transcriptional regulatory region of the SLIM gene is not particularly limited as long as it is a region that can control SLIM expression. For example, the region from the transcription start point of each SLIM gene to about 2 kbp upstream, or one or more in the nucleotide sequence of the region And a region having the ability to control the transcription of these SLIMs. The reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance. For example, a GFP (green fluorescent protein) gene, GUS (β-glucuronidase) gene, LUC (luciferase) gene, CAT (Chloramphenicol acetyltransferase) gene and the like.

  The transcriptional regulatory region of the SLIM gene and a cell into which the reporter gene operably linked to the region is introduced, as long as the transcriptional regulatory function of the SLIM gene can be evaluated, that is, the expression level of the reporter gene can be quantitatively analyzed. As long as it is, it is not particularly limited. However, SLIM-expressing cells are preferred as the cells to be introduced because they express a physiological transcriptional regulatory factor for SLIM and are considered to be more suitable for evaluation of SLIM expression regulation.

  The medium in which the test substance is contacted with the cells capable of measuring SLIM expression is appropriately selected according to the type of cells used. For example, a minimal essential medium containing about 5 to 20% fetal bovine serum is used. (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like. The culture conditions are also appropriately determined according to the type of cells used, etc. For example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C., and the culture time is About 12 to about 72 hours.

  In step (b) of the above method, first, the expression level of SLIM in the cell contacted with the test substance is measured. The measurement of the expression level can be performed by a method known per se described above in consideration of the type of cells used. When a cell capable of measuring a SLIM transcription regulatory region is used as a cell capable of measuring SLIM expression, the expression level can be measured based on the signal intensity of the reporter.

  Next, the expression level of SLIM in the cells contacted with the test object is compared with the expression level of SLIM in the control cells not contacted with the test object. The comparison of expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of SLIM in the control cells not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance compared to the measurement of SLIM expression level in the cells contacted with the test substance. However, the expression level is preferably measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In step (c) of the above method, a test substance that regulates the expression level of SLIM is selected. For example, a test subject that increases (promotes the expression of) SLIM expression is useful for the prevention or treatment of diseases in which suppression of p65-mediated signaling is desired, and reduces the expression level of SLIM (reducing expression). The (suppressed) test substance is useful for the prevention / treatment of diseases in which promotion of p65-mediated signaling is desired.

  In the above 2), the reconstitution system capable of measuring the SLIM function is a non-cultured cell system capable of evaluating the ability of SLIM to regulate SLIM function, including SLIM (protein) and other factors (eg, protein) Say.

In one embodiment, the screening method of the present invention using a reconstitution system capable of measuring the function of SLIM has the ability to form a complex in which the test subject contains SLIM and its binding pair (eg, HSP such as HSP90, actinin). This can be done by evaluating whether to adjust. Such screening methods can include, for example, the following steps (a1) to (c1):
(A1) contacting the test object and SLIM and its binding pair;
(B1) a step of measuring the amount of a complex containing SLIM and its binding pair when the test substance is contacted, and comparing the amount of the complex with the amount of the complex when the test object is not contacted;
(C1) A step of selecting a test substance that regulates the amount of the complex containing SLIM and its binding pair, based on the comparison result of (b1).

In step (a1) of the above method, the test subject, SLIM and its binding pair are contacted in an assay system capable of forming a complex comprising SLIM and its binding pair. Examples of the binding pair include HSP such as HSP90 and actinin. It should be noted that one or both of SLIM and its binding pair may be labeled to facilitate detection of their complexes. Examples of the label include a labeling substance (eg, a fluorescent substance such as FITC and FAM, a luminescent substance such as luminol, luciferin, and lucigenin, a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, In addition to labeling with an affinity substance such as biotin and streptavidin), fusion with a protein that can be encoded by a reporter gene can be mentioned. In this assay system, a cell homogenate containing SLIM and / or a binding pair thereof (eg, a homogenate of a cell transfected with an SLIM expression vector and / or an expression vector of a SLIM binding pair) can also be used.

In step (b1) of the above method, first, the amount of the complex when the test substance is contacted is measured. The amount of complex can be measured by a method known per se, for example, immunological techniques (eg, immunoprecipitation, ELISA), interaction analysis methods using surface plasmon resonance (eg, use of Biacore ^™ ) ).

  Next, the amount of the complex when the test object is contacted is compared with the amount of the complex when the test object is not contacted. The comparison of the amount of complex is preferably performed based on the presence or absence of a significant difference. The amount of the complex when the test object is not contacted may be the amount of the complex measured in advance or the amount of the complex measured simultaneously with respect to the measurement of the amount of the complex when the test object is contacted. Although it is good, it is preferable that it is the amount of the complex measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

  In step (c1) of the above method, a test substance for adjusting the amount of the complex is selected. For example, a subject that increases the amount of a complex comprising SLIM and HSP (facilitates the formation of a complex) or decreases the amount of a complex comprising SLIM and actinin (suppresses the formation of a complex) is p65-mediated. A subject that is useful for the prevention and treatment of diseases in which promotion of signal transduction is desired, and that reduces the amount of the complex containing SLIM and HSP or increases the amount of the complex containing SLIM and actinin, is a p65-mediated signal. This is useful for the prevention and treatment of diseases for which suppression of transmission is desired.

In another embodiment, the screening method of the invention using a reconstitution system capable of measuring SLIM function is performed, for example, by assessing whether the subject has the ability to modulate p65 ubiquitination by SLIM. Can be broken. Such screening methods can include, for example, the following steps (a2) to (c2):
(A2) contacting a test substance and a factor necessary for SLIM and ubiquitination reaction;
(B2) measuring the amount of ubiquitinated SLIM when the test substance is contacted, and comparing the amount of the complex with the amount of ubiquitinated SLIM when the test substance is not contacted;
(C2) A step of selecting a test substance that regulates the amount of the complex including the amount of ubiquitinated SLIM based on the comparison result of (b2) above.

  In step (a2) of the above method, the test substance, SLIM and factors necessary for the ubiquitination reaction are contacted in an assay system capable of self-ubiquitination of SLIM. Examples of factors necessary for the ubiquitination reaction include ubiquitin, E1, and E2. In this assay system, p65 can be further contacted.

  In step (b2) of the above method, first, the amount of ubiquitinated SLIM when the test substance is contacted is measured. The amount of ubiquitinated SLIM can be measured by a method known per se, such as an immunological technique such as Western blotting.

  Next, the amount of ubiquitinated SLIM when the test substance is contacted (or the amount of ubiquitinated p65 when p65 is further contacted) is compared with the amount of ubiquitinated SLIM when the test substance is not contacted. The The comparison of the amount of ubiquitinated SLIM is preferably performed based on the presence or absence of a significant difference. The amount of ubiquitinated SLIM when not contacting the test object is the amount of ubiquitinated SLIM measured at the same time even if the amount of ubiquitinated SLIM when the test object is contacted is the amount of ubiquitinated SLIM measured in advance. However, the amount of ubiquitinated SLIM measured at the same time from the viewpoint of the accuracy and reproducibility of the experiment is preferable.

  In step (c2) of the above method, a test substance that regulates the amount of ubiquitinated SLIM is selected. For example, a subject that decreases the amount of ubiquitinated SLIM (suppresses ubiquitination) is useful for the prevention and treatment of diseases in which promotion of p65-mediated signaling is desired, and increases the amount of ubiquitinated SLIM (ubiquitin) The test subject (which promotes conversion) is useful for the prevention / treatment of diseases in which suppression of p65-mediated signal transduction is desired. For details of this self-ubiquitination assay, see, for example, Tanaka T. et al, Immunity 22: 729-736 (2005). Note that the ability of SLIM to self-ubiquitinate in the reconstituted system is considered to reflect the activity of the LIM domain of SLIM to ubiquitinate an actual substrate in the cell.

In the above 3), the screening method for measuring SLIM function using a cell line capable of measuring SLIM function is, for example, i) whether or not the test substance has the ability to modulate p65 ubiquitination by SLIM, ii This is done by evaluating whether SLIM has the ability to regulate the sequestration of p65 to PML Nuclear Body, or iii) whether to regulate the amount of the complex containing SLIM and its binding pair (eg, HSP, actinin). Can be broken. Such screening methods can include, for example, the following steps (a) to (c):
(A) a step of contacting a test subject with SLIM-expressing cells;
(B) measuring the functional level of SLIM in a cell contacted with the test and comparing the functional level with a functional level in a control cell not contacted with the test;
(C) A step of selecting a test substance that adjusts the functional level of SLIM based on the comparison result of (b).

  In step (a) of the above method, the test subject is placed in contact with SLIM-expressing cells. Contact of the test subject with the SLIM-expressing cells can be performed in culture medium. As used herein, SLIM expressing cells can be cells that can express SLIM to the extent that SLIM assay at the protein level is possible. Preferred examples of such SLIM-expressing cells include cells transfected with SLIM expression vectors and / or HSP expression vectors (eg, mast cells, T cells), SLIM and HSP naturally expressing cells (eg, macrophages, etc.). Immune cells). Contact of the test subject with the SLIM-expressing cells can be performed in culture medium. This step can also be performed under TLR ligand treatment (stimulation).

  In step (b) of the above method, first, the functional level of SLIM in the cell contacted with the test substance is measured. For example, i) can be performed by measuring the amount of ubiquitinated p65 (see, eg, Examples and Tanaka T. et al, Immunity 22: 729-736 (2005)). For ii), by measuring the amount of p65 in the insoluble fraction of the nucleus (corresponding to PML Nuclear Body) (eg, see Examples), or by marker proteins (eg, fluorescent proteins such as GFP, tags such as Flag, His, etc.) This can be done by measuring the nuclear localization of p65 fused to the additional protein). iii) can be measured by a two-hybrid system in addition to the method (b1) of 2) above. In addition, the comparison of the function level in this process (b) can be performed similarly to the said method 2).

  In step (c) of the above method, a test substance that modulates the functional level of SLIM is selected. For example, a subject that increases the SLIM function level (promotes the function) is useful for prevention or treatment of a disease in which suppression of p65-mediated signaling is desired, and decreases the SLIM function level (function The (suppressed) test substance is useful for the prevention / treatment of diseases in which promotion of p65-mediated signaling is desired.

In the above 4), the screening method of the present invention using an animal can include, for example, the following steps (a) to (c):
(A) administering a test article to an animal;
(B) measuring the expression level or functional level of SLIM in an animal administered with the test, and comparing the expression level with the expression level or functional level of SLIM in a control animal not administered with the test;
(C) A step of selecting a test substance that regulates the expression level or functional level of SLIM based on the comparison result of (b).
In addition, this methodology can also make only the process of (b) and (c) essential.

  In step (a) of the above method, any animal, for example, an animal of the present invention (an animal to which a TLR ligand has been administered) or a disease model animal can be used as the animal. The test substance can be administered to the animal by a method known per se.

  In step (b) of the above method, the expression level or functional level of SLIM can be measured by a method known per se. For example, the expression level or functional level of SLIM in immune cells isolated or collected from animals can be measured by the same methodology as in step (b) of the methods 1) to 3) above. The comparison of the expression level in this step (b) and the step (c) of the above method can also be performed in the same manner as the methodologies of 1) to 3) above.

(Other)
The present invention provides a complex comprising SLIM and HSP (eg, HSP90), and a method for producing and detecting the same. Such a complex can be produced by a method known per se using a purified protein or a recombinant protein. Such a complex can be detected by an antibody immunological technique using an anti-SLIM antibody and / or an anti-HSP antibody. The complex of the present invention is useful for the screening method of the present invention.

  The present invention also includes HSP natural expression cells into which SLIM expression vectors have been introduced, SLIM natural expression cells into which HSP expression vectors have been introduced, one or two vectors that express SLIM and HSP (eg, combinations of expression vectors, A cell into which an expression vector is introduced is provided. Such cells of the present invention are useful for the screening of the present invention.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Materials and methods
1. Plasmids, reagents and antibody p65 expression plasmid (Flag-tag and p65 coding region) were inserted into pCDNA3 (Invtorgen). Flag-p65-IRES-GFP was made by inserting Flag-p65 into pCII-CMV-MCS-IRES-Venus (kindly provided by H. Miyoshi and A. Miyawaki). C-Myc-SLIM is an M.I. I got it from Grusby. For SLIM mutants ΔLIM and ΔPDZ, amino acids 80-350 or 1-280 of mouse SLIM were subcloned into pCMV-Myc (Clontech), respectively (kindly provided by M. Grusby). The luciferase reporter construct containing the ELAM-1 promoter region is I got it from Golenbook. LPS (Salmonella enterica) was purchased from Sigma. CpG oligonucleotide (ODN1668) was purchased from Hokaido System Science. Poly (I: C) was purchased from Amersham Biosciences. Human TNFα was purchased from R & D systems. MG132 was purchased from Calbiochem. D-galactosamine, latrunculin A, cytochalasin D and nocodazole were purchased from Sigma. Geldanamycin was purchased from WAKO Chemicals. Anti-p65, p50, Sp1, HSP90, PKC, LaminB, I-κBα and PML antibodies were purchased from Santa Cruz Biotechnology. Anti-Flag, SC-35, and α-actinin antibodies were purchased from Sigma. Anti-ubiquitin antibody was purchased from Zymed. Anti-20S proteasome antibody was purchased from Affiniti Research Products. Anti-phospho p65 (Ser536) and phospho I-κBα (Ser32) antibodies were purchased from Cell Signaling.

2. Cells, transfection, reporter assay 293T, RAW267.4, COS7, 3T3 cells were maintained in DMEM supplemented with 10% FCS. Resident macrophages used for experiments in SLIM-deficient mice were collected from the mouse abdominal cavity with PBS. Peritoneal macrophages (PM) were collected from the abdominal cavity 4 days after the intraperitoneal injection of 4% thioglycolate. Mouse embryonic fibroblasts (MEF) were prepared from 13.5 dpc embryos as described previously. For transfection, the indicated cells were transiently transfected with Effectene (QIAGEN). For the reporter assay, RAW267.4 cells or MEFs were treated with ELAM-1 luciferase construct (0.1 μg) with or without SLIM or its variant (0.6 μg) and p65 (0.1 μg). Transfected. Cells were lysed 24 hours after transfection and luciferase activity was measured as per manufacturer's protocol (Promega).

3. Subcellular fractional cytoplasm, nuclear soluble, and nuclear insoluble extracts were prepared as follows. First, cells hypotonic buffer _{(20mM HEPES, 10mM KCl, 1mM} MgCl 2, 0.1% Triton X-100,20% glycerol) was dissolved on ice for 10 minutes with. After centrifugation (5000 rpm, 1 minute), the supernatant was collected and used as the cytoplasmic fraction. Next, hypertonic buffer (20 mM HEPES, 1 mM EDTA, 20% glycerol, 0.1% Triton X-100, 400 mM NaCl) was added to the pellet, vortexed briefly, and then dissolved on ice for 20 minutes. After centrifugation (15000 rpm, 5 minutes), the supernatant was collected and used as a nuclear soluble fraction. Next, an insoluble buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% SDS, 1% NP-40, 10 mM iodoacetamide) was added to the pellet, and extraction was performed by vortexing for 50 minutes. The supernatant after centrifugation (15000 rpm, 5 minutes) was used as a nuclear insoluble fraction.

4). Immunoprecipitation whole cell extract was prepared by lysing cells in 250 mM NaCl, 50 mM Tris (pH 8.0), 0.5% NP-40, and protease inhibitors. The extract was incubated with the indicated antibody and protein G-Sepharose (Amersham Bioscience), washed 4 times, and subjected to immunoblot analysis with the indicated antibody.

5. Ubiquitination assay For endogenous p65 ubiquitination assay, whole cell extract prepared using RIPA buffer containing 10 mM N-ethylamide, or cytoplasm / nuclear extract was subjected to immunoprecipitation with anti-p65 antibody and anti-ubiquitin. The antibody was subjected to immunoblotting. For the overexpressed p65 ubiquitination assay, 293T cells were transfected with p65, histidine-tagged ubiquitin and SLIM or variants thereof, and the histidine-tagged protein was purified as previously described to obtain anti-p65 antibody And subjected to immunoblotting.

6). Immunofluorescence and confocal microscope 293T cells or MEFs were seeded on slides coated with poly-L-lysine. For 293T cells, transiently transfected cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. For MEF, cells were sequentially extracted with 3 different buffers on slides as described previously, followed by fixation with 4% paraformaldehyde for 15 minutes. Cells were blocked with 10% FCS and incubated with primary and secondary antibodies (diluted 1: 100 and 1: 500 with TBST, respectively). AlexaFluor series conjugated secondary antibody was purchased from Molecular Probe. Nuclei were stained with DAPI (4 ′, 6′-diamidino-2-phenylindole hydrochloride, Vector Laboratories, Inc.). Images were obtained with a Leica confocal microscope.

7). RT-PCR
Total RNA was prepared using Sepasol (nacalai quest), and cDNA was amplified with SuperScript II reverse transcriptase (Invitrogen). The primer pairs used for PCR are as follows; Flag-p65,5′-GACTACAAAGACGATGGAGAC-3 ′ (SEQ ID NO: 1) and 5′-GTCAGCCTCATAGTAGCCATC-3 ′ (SEQ ID NO: 2); human actin, 5′- CTCACCATGGATGATGATATCGCC-3 ′ (SEQ ID NO: 3) and 5′-TTCATGAGGTAGCAGTCAGGTCC-3 ′ (SEQ ID NO: 4).

8). Experiments with SLIM (− / −) mice The generation of SLIM (− / −) mice has been described previously (Tanaka T. et al, Immunity 22: 729-736 (2005)). All mice were used after maintaining certain pathogen-free conditions and backcrossing with Balb / c seven times. Resident macrophages were collected from the abdominal cavity by washing with PBS and stimulated with LPS for 24 hours. Production of IL-12p40 and IL-6 into the supernatant was assayed by ELISA. For endotoxin shock experiments, mice were injected intraperitoneally with the doses of LPS alone or LPS plus D-galactosamine as shown in the figure, and survival was monitored.

Example 1: Nuclear p65 ubiquitination / degradation following TLR ligand stimulation, and translocation to actin-dependent nuclear insoluble compartment . We first started with endogenous NF-κB · p65 protein stimulated by TLR ligand. It was investigated whether it can be ubiquitinated by the response to. RAW264.7 cells were stimulated with LPS in the absence or presence of the proteasome inhibitor MG132. Whole cell extracts were immunoprecipitated with anti-p65 and blotted with anti-ubiquitin. Polyubiquitination of the p65 protein was detected in the presence of MG132 and was further enhanced by LPS stimulation (FIG. 1a). Similar results were obtained using primary cultured peritoneal macrophages and primary cultured fetal fibroblasts (MEF) stimulated with LPS. Furthermore, p65 was also ubiquitinated upon stimulation with other TLR ligands, CpG and poly I: C (FIG. 1b). In order to determine the intracellular site where ubiquitination of the p65 protein occurs, the present inventors further prepared a cytoplasm / nuclear extract from cells stimulated with LPS and / or MG132. FIG. 1c clearly shows that nuclear p65 but not cytoplasm was ubiquitinated. Since ubiquitinated proteins tend to be degraded via a proteasome-dependent pathway, we next examined the steady state level of p65 protein in the nucleus (FIG. 1d). When cells were stimulated with LPS, p65 was readily detected in the soluble nuclear fraction. In addition, p65 was also detected in the insoluble nuclear fraction, albeit in a smaller amount. The cells were then washed to remove LPS from the medium and the nuclear p65 in the soluble fraction was reduced to the initial level in 2 hours. Interestingly, treatment of these cells with MG132 restored p65 protein levels only in the insoluble fraction and not in the soluble fraction. These data indicate that part of the p65 protein that enters the nucleus upon LPS stimulation is translocated from the soluble compartment to the insoluble compartment of the nucleus, and that in this fraction the p65 protein is degraded in a proteasome-dependent manner. Suggest. To visualize the p65 protein in the nuclear insoluble compartment, cells were stimulated with LPS, then extracted sequentially with detergent, high salt, and DNase I and observed with a fluorescent confocal microscope (FIG. 1e). ). These extractions allowed us to detect insoluble p65 protein as a separated foci in the nucleus. Based on their morphology and previous studies showing that p65 can associate with PML proteins (Wu, WS et al., J. Biol. Chem. 278: 12294-12304 (2003)), these nests are It was thought that it could be PML Nuclear body, an isolated compartment in the nucleus that serves as a proteolytic site. Recent studies have demonstrated that actin is also localized in the nucleus and involved in transcriptional regulation (Torrad M. et al., Invest Ophthalmol Vis Sci. 45: 3955-3963 (2004)). The inventors then tested whether an actin fibril formation inhibitor affects the translocation of p65 to the Nuclear body. Ratrunkrin A, a specific inhibitor of nuclear actin, damaged p65 protein translocation in response to LPS, while cytochalasin D, a general inhibitor of actin formation, or nocodazole, a microtubule inhibitor. Not (FIG. 1f). This implies that p65 intranuclear trafficking to the Nuclear body depends on nuclear actin fibrils.

Example 2: Suppression of p65 transcriptional activity by SLIM binding to p65 and forced expression of SLIM We next sought to search for ubiquitin E3 ligase that mediates ubiquitination of p65 protein in the nucleus. SLIM is a nuclear protein containing a PDZ domain at the N-terminus and a LIM domain at the C-terminus. SLIM was first identified as a nuclear ubiquitin ligase that acts on STAT4 and STAT1 in CD4 ⁺ T cells (Tanaka T. et al, Immunity 22: 729-736 (2005)). We investigated the possibility that SLIM could be a nuclear ubiquitin E3 ligase for p65. SLIM is reported to be expressed at the mRNA level in other types of cells (Tanaka T. et al, Immunity 22: 729-736 (2005)). It was tested whether it was expressed at the protein level in innate immune cells. SLIM was expressed in the nuclei of macrophages, dendritic cells and fibroblasts, but not in the cytoplasm, regardless of LPS stimulation (FIG. 2a). Based on these results, we predicted that SLIM could be a candidate for a nuclear ubiquitin ligase for p65. To test the interaction between SLIM and p65, 293T cells were transiently transfected with an expression plasmid encoding c-myc-tagged SLIM along with p65. Transfectants were either untreated or treated with TNFα for 30 minutes and subjected to co-immunoprecipitation. Even without stimulation, the SLIM and p65 interaction was weakly detected and further enhanced by TNFα stimulation (FIG. 2b). We further investigated the interaction between c-myc-tagged SLIM and endogenous p65. As shown in FIG. 2c, SLIM can associate with p65 but not p50. To elucidate the effect of SLIM on p65-mediated signaling, cells were transfected with a luciferase reporter construct containing an NF-κB binding site from the ELAM1 gene promoter along with the SLIM expression plasmid. Both LPS stimulation or p65 expression plasmid co-transfectants activated reporter activity, whereas SLIM markedly inhibited activation of these reporters. This suggests that SLIM directly inhibits the activity of p65 (FIG. 2d). Furthermore, similar results were obtained when cells were stimulated with TNFα instead of LPS (FIG. 2e).

Example 3: Nuclear ubiquitination and degradation of p65 by SLIM, and translocation to nuclear insoluble fraction SLIM is reported to mediate polyubiquitination of STAT4 protein and subsequent degradation via the LIM domain. (Tanaka T. et al, Immunity 22: 729-736 (2005)). We examined whether SLIM has a similar activity on the p65 protein. As shown in FIG. 3a, ubiquitination of the p65 protein was minimally detected by co-expression of p65 and histidine-tagged ubiquitin and dramatically enhanced by SLIM co-expression. Deletion of the LIM domain with ubiquitin E3 ligase activity impaired the activity of SLIM to ubiquitinate p65 and the self-ubiquitination activity of SLIM itself (FIG. 3b). We next investigated whether SLIM could induce p65 degradation. The steady state level of p65 was significantly reduced by SLIM only in the nucleus and not in the cytoplasm (FIG. 3c). On the other hand, p65 mRNA level was not changed by co-expression of SLIM (FIG. 3d). Furthermore, immunoblot analysis using the soluble and insoluble fractions of these transfectant nuclei demonstrated that SLIM has the activity of translocating the p65 protein into the nuclear insoluble fraction (FIG. 3e). When these cells were treated with MG132, p65 protein levels recovered only in the insoluble fraction and not in the soluble fraction. Furthermore, treatment of these cells with latrunculin A impaired SLIM-induced nuclear translocation of the p65 protein (FIG. 3f). These data indicate that SLIM has the activity of ubiquitinating p65 proteins in the nucleus and then translocating them into a nuclear insoluble compartment (where subsequent degradation of the p65 protein occurs) in an actin-dependent manner. Imply.

Example 4: Isolation / degradation of p65 by SLIM LIM domain and isolation of p65 by PZZ domain into PML Nuclear body To determine which region of SLIM is responsible for these activities of SLIM, we Next, SLIM mutant expression plasmids lacking the PDZ domain (ΔPDZ) or SLIM mutant expression plasmids lacking the LIM domain (ΔLIM) were made and transfected into SLIM-deficient MEFs with the p65 expression plasmid. As shown in FIG. 4a, ΔPDZ impaired the activity of translocating the p65 protein into the insoluble fraction. This suggests that the PDZ domain is involved in nuclear trafficking of the p65 protein. On the other hand, translocation of p65 by ΔLIM was similar to that of wild type SLIM. This implies that p65 nuclear trafficking is not simply dependent on its ubiquitination. This is because, as shown in FIG. 3b, ΔLIM has an impaired activity to ubiquitinate the p65 protein. However, p65 protein levels in the insoluble fraction of ΔLIM transfectants were significantly enhanced, reflecting the impairment of ΔLIM's E3 ligase activity. Luciferase assay using ELAM1 promoter reporter with SLIM mutant showed that ΔPDZ but not ΔLIM could not inhibit p65-mediated gene activation. This suggests that sequestration of p65 into the insoluble compartment is sufficient to terminate p65 activation (FIG. 4b). In order to further investigate the effect of SLIM variants on the subnuclear localization of p65, we next expressed Flag-p65-IRES-GFP expression in which both Flag-tagged p65 and GFP proteins are expressed. A plasmid was prepared. 293T cells were transfected with this construct along with the SLIM mutant plasmid and subjected to immunofluorescence experiments using confocal microscopy (FIG. 4c). We evaluated the effect of each mutant by calculating the percentage of Flag-p65 and GFP double positive cells / total GFP positive cells (Table 1).

  93% of the control transfectants showed a uniform pattern for nuclear p65 and weak cytoplasmic staining. This is because Flag-p65 can itself translocate into the nucleus by overexpression (FIG. 4c, left panel). On the other hand, in many SLIM transfectants, p65 in the nucleus disappeared (FIG. 4 c, left-middle panel). Interestingly, cytoplasmic p65 expression was not affected in these cells. In ΔPDZ transfectants, the nuclear targeting activity of p65 is partially impaired (FIG. 4c, right panel), whereas ΔLIM transfectants are at the same level as SLIM transfectants, The nuclear translocation of p65 was shown (Fig. 4c, right middle panel). However, in about 17.2% of the double positive cells in ΔLIM transfectants, the p65 protein has a pronounced puncture structure (this is an aggregate of the p65 protein due to insufficient degradation) I think). Furthermore, these structures colocalized with PML and 20S proteasome but not with the splicing factor SC35 (FIG. 4d). These data indicate that SLIM has the activity of enhancing the targeting of p65 to the PML Nuclear body, and that in this fraction, degradation of p65 protein actually occurs by the accumulated proteasome. ing. A recent study reported that SLIM can bind to actin-binding protein α-actinin (Torrad M. et al., Invest Ophthalmol Vis Sci. 45: 3955-3963 (2004)). We further demonstrated that SLIM can associate with α-actinin via its PDZ domain (FIG. 4e). Since α-actinin is also localized in the nucleus, SLIM binds to α-actinin via the PDZ domain, thereby indirectly associating with nuclear actin and leading to intranuclear trafficking of p65 protein along the nuclear actin filaments. It seems to be mediating.

Example 5: Enhanced reactivity to LPS in SLIM-deficient mice and increased nuclear p65 protein levels We further examined p65-mediated signaling at the individual level using SLIM-deficient mice. To analyze the mouse model for endotoxin shock, mice were injected intraperitoneally with low doses of LPS (1 and 0.1 μg) and D-galactosamine or high dose LPS (600 μg) alone (Table 2).

  Wild type (+ / +) and SLIM deficient (− / −) mice were injected intraperitoneally with LPS alone or both LPS and D-galactosamine at the indicated doses. Survival was monitored and the percentage of lethality was calculated.

  Table 2 shows that SLIM (− / −) mice are more sensitive to the lethal effects of LPS treatment compared to wild type mice. Furthermore, SLIM (− / −) macrophages produced higher amounts of inflammatory cytokines (IL-12p40 and IL-6) in response to LPS stimulation compared to wild type macrophages (FIG. 5a). We next examined the protein level of p65 in SLIM (− / −) cells. The level of nuclear p65 protein in LPS stimulated SLIM (− / −) macrophages was significantly higher than that of wild type cells, but cytoplasmic p65 levels were not affected (FIG. 5b). Similarly, serine phosphorylation of p65 and I-κBα, characteristic of cytoplasmic component activation leading to p65 activation, was normally induced in SLIM (− / −) cells as a response to LPS (FIG. 5c). ). These data suggest that SLIM also functions as an E3 ligase at the individual level and negatively regulates p65-mediated signaling.

Example 6: Binding of HSP90 to SLIM and negative regulation of SLIM's ubiquitin E3 ligase activity by HSP90 Finally, we examined how the activity of SLIM itself is regulated . The expression level of SLIM mRNA was not changed by any stimulus tested by the present inventors. Therefore, the inventors examined the existence of molecules that interact with SLIM and modify its activity. In the process of purification of recombinant SLIM, we have found that chaperones can bind strongly to SLIM. To clarify the role of chaperones in controlling SLIM activity, we first investigated SLIM association with HSP90, one of the major mammalian chaperones. As shown in FIG. 6a (left panel), SLIM can bind to endogenous HSP90. We next examined the effect of HSP90 on SLIM-induced p65 ubiquitination. Treatment of cells with geldanamycin, an inhibitor of HSP90, enhanced both SLIM-induced polyubiquitination of p65 and auto-ubiquitination of SLIM (FIG. 6a, middle and right panels). This suggests that HSP90 negatively regulates SLIM E3 ligase activity. Furthermore, treatment of cells with geldanamycin also enhanced polyubiquitination and subsequent degradation of the endogenous p65 protein (FIG. 6b left and upper right). Moreover, when MG132 was added to the cells, the p65 protein level of the nuclear insoluble fraction was recovered (FIG. 6b lower-right). This suggests that the reduction of nuclear p65 protein levels by geldanamycin is not due to damage of p65 cytoplasmic to nuclear translocation, but due to enhanced proteasome-dependent degradation in the nucleus. To do. We further tested the manner in which HSP90 controls p65-mediated gene activation. Geldanamycin treatment inhibited both LPS-induced and p65-activated ELAM1 reporter activity (FIG. 6c). These data suggest that HSP90 negatively regulates ubiquitination / proteasome-dependent degradation of p65 protein, possibly through inhibition of ubiquitin E3 ligase activity of SLIM.

  Based on the above results, we conclude that SLIM is a nuclear ubiquitin E3 ligase that terminates p65 activation by two mechanisms: p65 ubiquitination via the LIM domain, and PDZ domain. Sequestration of p65 into the PML nucleolus. Several proteins such as p53, Myc and LEF-1 have been reported to be transported into the PML Nuclear body (Zhong S. et al, Nature Cell Biol., 2: E85-90 (2000); Smith KP et al, J. Cell Biochem. 93: 1282-1296 (2004); Sachdev S. et al, Genes & Dev. 15: 3088-3013 (2001)). The function of these factors is regulated either positively or negatively in this compartment. Our data demonstrated that ubiquitinated p65 targeted to this compartment is degraded in a proteasome-dependent manner and then ultimately terminates p65-mediated signaling. Previous studies have shown that overexpression of PML can induce p65 translocation to the PML Nuclear body (Wu, W-S et al., J. Biol. Chem. 278: 12294-12304 (2003)). Our preliminary data indicate that SLIM forms a heterodimer with PML. This suggests that these factors synergistically mediate sequestration of the p65 protein, but further research is required. The pathway that terminates p65 activation revealed by this study may be a useful target for clinical treatment of human inflammatory diseases. In particular, specific inhibition of HSP binding to SLIM can be a good tool for terminating the activation of p65-mediated signaling.

FIGS. 1 a-1 f show ubiquitination of nuclear p65 and translocation of nuclear p65 to the Nuclear body in an actin-dependent manner. RAW264.7 cells, PM (peritoneal macrophages) and MEF (mouse fetal fibroblasts) were stimulated with 1 μg / ml LPS for 3 hours, followed by treatment with 10 μM MG132 for 1 hour. Whole cell extracts were subjected to immunoprecipitation with anti-p65 antibody and immunoblot with anti-Ub or anti-p65 antibody. RAW264.7 cells were stimulated with 1 μM CpG, 50 μg / ml poly (I: C) or 1 μg / ml LPS for 3 hours, followed by treatment with 10 μM MG132 for 1 hour. Whole cell extracts were subjected to immunoprecipitation with anti-p65 antibody and immunoblot with anti-Ub or anti-p65 antibody. Cytoplasmic / nuclear extract was prepared from RAW264.7 cells stimulated as in FIG. 1a and subjected to immunoprecipitation with anti-p65 antibody and immunoblotting with anti-Ub or anti-p65 antibody. MEF was stimulated with 10 ng / ml LPS for 1 hour, washed to remove LPS and then left untreated or treated with 10 μM MG132 for 2 hours. A soluble and insoluble fraction of the nucleus was prepared and immunoblotted with anti-p65 antibody. Fractions of each extract were confirmed using antibodies against Sp1 (protein in the nuclear soluble fraction) and lamin B (protein in the nuclear insoluble fraction). MEF is stimulated with 1 μg / ml LPS for 1 hour and left untreated or continuously using CSK buffer (surfactant), extraction buffer (high salt), and DNase I buffer (DNase) Extracted and stained with anti-p65 antibody followed by AlexaFluor 594 labeled anti-rabbit IgG and subjected to indirect immunofluorescence. MEF was pretreated with 2 μg / ml latrunculin A (Lat. A), 10 μM cytochalasin D (Cyt. D), or 10 μM nocodazole (NCZ) for 1 hour followed by stimulation with 100 ng / ml LPS for 30 minutes. . Soluble and insoluble fractions of nuclei were prepared and immunoblotted with the indicated antibodies. Figures 2a-e show SLIM binding to p65 and inhibition of p65-mediated transactivation by SLIM. Cytoplasm / nuclear extract was prepared from peritoneal macrophages (PM) or fetal fibroblasts (MEF) and immunoblotted with anti-SLIM antiserum. The purity of each extract was checked using anti-PKC or HSP90 (for cytoplasm) and anti-Sp1 (for nuclear) antibodies. COS7 cells were transfected with an expression plasmid of c-myc-SLIM (WT) or frameshift (FS) mutant along with Flag tag p65. Whole cell extracts that were untreated or treated with human TNFα (20 ng / ml) for 30 minutes were immunoprecipitated with anti-c-Myc antibody and immunoblotted with anti-Flag antibody. 293T cells were transfected with c-myc-SLIM (WT) or frameshift (FS) mutants. Whole cell extracts were immunoprecipitated with anti-c-Myc antibody and immunoblotted with anti-p65 or anti-p50 antibody. MEFs were transfected with or with the ELAM-1 reporter construct without or with the SLIM expression plasmid (left panel). MEFs were transfected with the ELAM-1 reporter construct without or with p65 and without or with the SLIM expression plasmid (right panel). Luciferase activity was measured without LPS stimulation (right panel) or with LPS stimulation (100 ng / ml, 5 hours) (left panel). RAW264.7 cells were transfected with the ELAM-1 luciferase reporter without or with the SLIM expression plasmid. Luciferase activity was measured with or without TNFα stimulation (10 ng / ml, 5 hours) or with TNFα stimulation (10 ng / ml, 5 hours). Figures 3a-f show the promotion of p65 ubiquitination, nuclear translocation and degradation by SLIM. 293T cells were transfected with expression plasmids of His-ubiquitin, p65 and SLIM (WT) or frameshift (FS) mutants. The histidine-labeled protein was purified with Ni-NTA beads and immunoblotted with anti-p65 antibody. 293T cells were transfected with expression plasmids for His-ubiquitin, p65 and SLIM (WT), frameshift (FS) or LIM domain deletion (ΔLIM) mutants. Histidine-labeled proteins were purified with Ni-NTA beads and immunoblotted with anti-p65 (left panel) or anti-c-Myc antibody (right panel). 293T cells were transfected with Flag-tagged p65 and SLIM (WT) or frameshift (FS) mutant expression plasmids. The cytoplasm / nuclear extract was subjected to immunoblotting with the indicated antibodies. Total RNA was extracted from the same transfectants as in FIG. cDNA was prepared and subjected to PCR using the indicated primer pairs. MEFs were transfected with p65 and SLIM (WT) or frameshift (FS) mutant expression plasmids. Soluble and insoluble fractions of nuclei untreated or treated with MG132 (10 μM) for 5 hours were extracted and subjected to immunoblotting with anti-Flag or anti-c-Myc antibody. MEFs were transfected with p65 and SLIM (WT) or frameshift (SF) mutant expression plasmids. Soluble and insoluble fractions of nuclei untreated or treated with latrunculin A (Lat. A) (0.2 μM) for 20 hours were extracted and subjected to immunoblotting with the indicated Ab. 4a to 4e are diagrams illustrating the roles of the SLIM LIM domain and the PDZ domain. 293T cells were transfected with p65 and SLIM (WT), frameshift (FS), LIM domain deletion (ΔLIM) or PDZ domain deletion (ΔPDZ) mutant expression plasmids. The soluble and insoluble fraction of the nuclei, untreated or treated with MG132 (10 μM) for 5 hours, was extracted and subjected to immunoblotting with the indicated Ab. SLIM (− / −) MEFs were transfected with ELAM-1 luciferase reporter plasmid and SLIM (WT), frameshift (FS), LIM domain deletion (ΔLIM) or PDZ domain deletion (ΔPDZ) mutants. Luciferase activity was measured with or without stimulation with 100 ng / ml LPS for 5 hours. 293T cells were transfected with Flag-p65-IRES-GFP and SLIM (WT), frameshift (FS), LIM domain deletion (ΔLIM) or PDZ domain deletion (ΔPDZ) mutant expression plasmids. Cells were stained with biotinylated anti-Flag antibody followed by AlexaFluor 594 labeled streptavidin (upper panel). The middle panel shows GFP expression and the lower panel shows DNA staining with DAPI. 293T cells were transfected with p65 and SLIM LIM domain deletion (ΔLIM) mutant expression plasmids. Cells were stained with biotin anti-Flag antibody followed by AlexaFluor 594 labeled streptavidin and anti-PML, SC35 or 20S proteasome antibody followed by AlexaFluor 488 labeled anti-mouse IgG. 293T cells were transfected with SLIM (WT), frameshift (FS), LIM domain deletion (ΔLIM) or PDZ domain deletion (ΔPDZ) mutant expression plasmids. Whole cell extracts were prepared and subjected to immunoprecipitation with anti-c-myc antibody and immunoblotting with anti-α-actinin antibody. Figures 5a-c show enhanced cytokine production and increased nuclear p65 protein levels in SLIM-deficient macrophages. The resident peritoneal macrophages used in the experiments shown in FIGS. 5a to 5c were prepared from wild type (+ / +) and SLIM deficient (− / −) mice. Cells were stimulated for 24 hours without or with LPS as indicated. IL-12p40 and IL-6 production in the culture supernatant was measured by ELISA. Cells were stimulated for 2 hours with or without LPS as indicated. A cytoplasm / nuclear extract was prepared and subjected to immunoblotting with anti-p65 antibody. Fractions were confirmed with anti-HSP90 (for cytoplasm) or anti-Sp1 (for nuclear) antibodies. Cells were stimulated for 2 hours with or without LPS as indicated. A cytoplasmic extract was prepared and subjected to immunoblotting with the indicated antibodies. Figures 6a-c show negative regulation of SLIM-mediated p65 ubiquitination and degradation by HSP90. (Left panel) 293T cells were transfected with SLIM (WT) or frameshift (FS) mutants and subjected to immunoprecipitation with anti-c-myc antibody and immunoblotting with anti-HSP Ab. (Center and right panel) 293T cells were transfected with His-ubiquitin, p65 and SLIM (WT) or frameshift (FS) mutant expression plasmids. From untreated or geldanamycin (10 μM, 5 hours) treated cells, histidine-labeled proteins were purified with Ni-NTA beads and immunoblotted with anti-p65 or anti-c-Myc antibodies. Left panel; RAW264.7 cells were treated without treatment or with geldanamycin (GA) (10 μM) and / or MG132 (10 μM) for 6 hours. Whole cell extracts were prepared and subjected to immunoprecipitation with anti-p65 antibody and immunoblotting with anti-ubiquitin or anti-p65 antibody. Right panel; MEF pre-treated with geldanamycin (GA) (10 μM) for 6 hours in the absence (right, top) and presence (right, bottom) of MG132 (10 μM), then LPS (100 ng / ml) For 30 minutes. Nuclear soluble (top-right) and insoluble (bottom-right) fractions were immunoblotted with anti-p65 antibody. Left panel; MEFs were transfected with ELAM-1 luciferase reporter plasmid. Transfectants were stimulated with or without LPS in the absence or presence of geldanamycin (GA) (10 μM, 6 hours) or luciferase activity with LPS (200 ng / ml, 4 hours) Was measured. Right panel; MEFs were transfected with or with the ELAM-1 luciferase reporter plasmid without or with the p65 expression vector. Luciferase activity was measured untreated or treated with geldanamycin (GA) (10 μM) for 6 hours.

Claims (16)

  A modulator of p65-mediated signaling comprising a substance that modulates SLIM expression or function.   The agent according to claim 1, wherein the substance that modulates SLIM expression or function is an inhibitor of p65-mediated signal transduction, which is a substance that promotes SLIM expression or function.   The agent according to claim 2, wherein the substance that promotes the expression or function of SLIM is a SLIM expression vector or a substance that suppresses the expression or function of HSP.   The agent according to claim 2, wherein the inhibitor of p65-mediated signal transduction is a medicament for a disease selected from the group consisting of inflammatory diseases and autoimmune diseases.   The agent according to claim 1, wherein the substance that modulates SLIM expression or function is an activator of p65-mediated signal transduction, which is a substance that suppresses SLIM expression or function.   The agent according to claim 5, wherein the substance that suppresses the expression or function of SLIM is an HSP expression vector.   The agent according to claim 6, wherein the HSP is HSP90.   The agent according to claim 5, wherein the activator of p65-mediated signal transduction is a medicament for a disease selected from the group consisting of infectious diseases and malignant tumors.   A SLIM-deficient non-human animal to which a TLR ligand has been administered.   SLIM deficient cells treated with TLR ligands.   A method for screening for a substance capable of modulating p65-mediated signal transduction, comprising evaluating whether a test substance can modulate SLIM expression or function.   1) Measurement of SLIM expression using a cell capable of measuring SLIM expression 2) Measurement of SLIM function using a reconstitution system capable of measuring SLIM function 3) Cell capable of measuring SLIM function The screening method according to claim 11, wherein the screening method is performed by measuring SLIM function using a system, or 4) measuring SLIM expression or function using an animal.   The assessment is based on an assessment of whether the subject has the ability to modulate p65 ubiquitination by SLIM, or whether it has the ability to regulate sequestration of p65 to PML Nuclear Body by SLIM. 11. The method according to 11.   The assessment is based on an assessment of whether the subject modulates the ability to form a complex comprising SLIM and HSP90, or whether the subject regulates the ability to form a complex comprising SLIM and actinin. The screening method according to claim 11.   12. The method of claim 11, wherein the method is performed under TLR ligand stimulation.   A complex comprising SLIM and HSP90.