KR20110036291A - Pharmaceutical compositions for improving or treating a retinal disease comprising tip60 as an active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing TIP60 is provided to induce transcription activity of a target gene promoter and to relieve or treat retinal diseases. CONSTITUTION: A pharmaceutical composition for relieving or treating retinal diseases contains TIP60 as an active ingredient. TIP60 binds to NRL protein. TIP60 contains chromo domain and histone aceytltransferase domain. The activation of TIP60 is measured by increasing transcription activity of NRL protein.

Description

Pharmaceutical Compositions for Improving or Treating a Retinal Disease Comprising TIP60 as an Active Ingredient}

The present invention relates to a composition for improving or treating retinal disease, including TIP60 as an active ingredient, and a method for screening a therapeutic agent for retinal disease.

The eye projects visual images onto the retina, which acts like a film in the camera. The mammalian retina is a light-sensing tissue located on the inner surface of the eye and is very organic, consisting of six neurons and a glia, and the retina's cells differentiate from retinal progenitor cells and retina It consists of photoreceptor cells (rod cells and corn cells), which correspond to about 70% of the cells. The differentiation of these photoreceptor cells consists of processes that are rearranged and regulated very precisely in time and space, depending on the type of cell. In mammalian embryonic development, the retina and optic nerve are thought to be part of the central nervous system because they originate from the outgrowth of the developing brain.

The retina is a complex, layered structure composed of several layers of neurons connected by synapses, of which only photoreceptor cells are directly sensitive to light. They exist mainly in two forms: rods and cones. Rods function primarily in dim light to provide black and white images, while abstracts are responsible for daytime image and color recognition. As a third photoreceptor, there is a very rare form of photosensitive ganglion cells, which are important in very bright reflexive responses. The molecular mechanisms associated with retinal development are not yet clearly understood.

Retinitis pigmentosa (RP) was first described by Donder in 1855 as an ophthalmologic finding. Pigmentary retinopathy encompasses all retinal dystrophy, represented by the loss of photoreceptors and the deposition of ocular pigments. It is a disease belonging to. RP is a retinal degenerative disease characterized by a large amount of pigment deposits in the peripheral retina and a relatively empty pigment in the central retina. In most RPs, the secondary degeneration of the abstract photoreceptor cones occurs after the primary degeneration of rod photoreceptor rods. Thus, typical RP is also described as rod-cone dystrophy, where rod photoreceptors are more affected than abstractions. The reason why RP patients initially show night blindness and suffer from loss of vision even in the end of the day is due to the sequence of degeneration of the photoreceptors described above (Hamel, C., Orphanet J Rare Dis , 1:40 (2006)). At present, genetic diseases such as retinopathy have not yet been accurately investigated even in the proportion of domestic patients. The incidence rate differs from one country to one in 4,000 in the United States and one in 6,000 in Japan, and it is estimated that there will be patients at similar rates in Korea. The fundamental treatment of this progressive progressive disease, retinal pigmentosa, is preferred for gene therapy to repair gene defects, but it is still attempted at the animal experiment or at the cellular level. Therefore, in order to prevent or treat retinal pigmentosa, the gene defects of each patient must be identified, and as a result, the investigation of gene expression pattern must also be performed.

Many transcriptional regulators are involved in the differentiation of photoreceptors in the retina and normal physiological activity. These transcriptional regulators include CRX, OTX2, retinoblastoma protein (RB), neural retina leucine-zipper (NRL) and thyroid hormone The receptor TRβ2, the simplified-specific orphan nuclear receptor NR2E3, is known. Among them, NRL transcriptional activator was first reported in 1992 by Swaroop et al. (Swarrop et al. , Proc Natl Acad Sci USA , 89: 266-270 (1992)). The NRL gene is conservatively present in vertebrates, specifically expressed in photoreceptors and pineal gland, and belongs to the ZIP (leucine zipper) family of transcriptional regulators (Chau, KY, et al. , J Biol Chem , 275: 37264-37270 (2000); Nishida, A., et al. , Nat Neurosci , 6: 1255-1263 (2003); Zhang, J., et al. , Nat Genet , 36: 351 -360 (2004); Swain, PK, et al. , J Biol Chem , 276: 36824-36830 (2001); and Swain, P., et al. , Mol Vis , 13: 1114-1120 (2007).

To date, NRL activates the expression of rod-specific target genes such as rhodopsin as transcriptional regulators, and is known to further promote transcriptional activity by binding proteins of NRL such as CRX and NR2E3. In addition, it has been reported that in the retina of mice knocked out of the NRL gene, no differentiation into rod cells occurs, only abstract cells are differentiated. This result shows that NRL plays a very important function in the differentiation of rod and abstract cells, and that the transcriptional activity of NRL directly acts on rod differentiation. Since NRL functions not only in the development and differentiation of the retina, but also in the expression of photoreceptor proteins such as rhodopsin, a study has been reported that the genetic mutation of NRL is associated with retinal disease. This is due to the discovery of mutations in patients with hereditary retinopathy, such as retinitis, in which the 50th serine residue of NRL is substituted with a threonine residue. The site containing the 50th serine residue is a site having the transactivation domain of NRL, and its transcriptional activity is regulated by various post-translation modifications such as phosphorylation and acetylation. Thus, mutations in the NRL transcriptional activity site have been reported to be deeply associated with retinal differentiation and resulting eye diseases.

On the other hand, TIP60 (Tat-interacting protein 60) has been identified as an interaction partner for HIV-1 Tat protein, which increases Tat transactivation of HIV-1 promoter (Kamine, J., et al. , Virology , 216: 35766 (1996). TIP60 with MYST ( M OZ, Y bf2 / Sas3, S AS2 and T IP60) domains belongs to the MYST family of histone acetyltransferases (HAT), which is highly conserved from east to human (Yang, XJ, Nucleic Acids Res. , 32: 959976 (2004). TIP60 functions as a transcriptional coactivator or corepressor depending on the cell or promoter position. For example, TIP60 is HIV-1 Tat (Kamine, J., et al. , Supra), type I nuclear hormone receptors (Brady, ME, et al. , J. Biol. Chem. , 274: 175997604 (1999); Halkidou, K., et al. , Oncogene , 22: 2466477 (2003)) and amyloid-precursor proteins (Cao, X., and Sudhof, TC, Science , 293: 11520 (2001)) in combination with a transcriptional activator. In contrast, TIP60, in combination with the transcription factor CREB (c AMP r esponse e lement- b inding protein) or a transcriptional repression factor ZEB (z inc finger E box- b inding protein) may control the gene expression by phonetic (Gavaravarapu, S., and Kamine, J. Biochem. Biophys. Res. Commun. , 269: 7586624 (2000); Hlubek, F., et al. , J Biochem. , ( Tokyo ) 129: 63541 (2001). It has also recently been reported that TIP60 functions as a coactivator of p53-mediated transcriptional activity (Sykes, SM, et al. , Mol. Cell , 24: 84151 (2006).

However, the possibility of interaction between NRL and TIP60 and molecular regulatory mechanisms in retinal development and normal retinal physiology have not been reported yet.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a method for the treatment of retinal diseases (particularly, retinal pigmentosa). As a result, the present inventors have found that TIP60 (Tat-interacting protein 60) binds to neural retinal leucine zipper (NRL), which is a transcription factor that plays an important role in retinal photoreceptor differentiation, and enhances NRL transcriptional activity, resulting in transcription of NRL target genes. By discovering the increase of, the present invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for improving or treating retinal disease.

Another object of the present invention is to provide a method for screening a therapeutic agent for retinal disease.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition for improving or treating retinal disease, including TIP60 (Tat-interacting protein 60) as an active ingredient.

The present inventors have made efforts to discover biomolecules involved in retinal diseases (particularly, retinal pigmentosa) and use them in medicine development. As a result, the present inventors found that TIP60 binds to NRL, a transcription factor that plays an important role in the differentiation of retinal photoreceptors, and increases the transcriptional activity of NRL to increase transcription of NRL target genes.

The present invention relates to a novel use of the TIP60 gene and protein, and to a novel use as a therapeutic target for TIP60 in connection with retinal disease, in particular retinal pigmentosa. This novel use of the present invention is in accordance with the findings of the present inventors that by regulating the transcriptional activity of NRL protein as a regulatory target of retinal diseases, in particular TIP60, it can ultimately be used in the treatment of retinal diseases.

TIP60, also called HTATIP, is a human gene that belongs to the MYST family of histone acetyltransferases (HATs) and was initially identified as an HIV-1 TAT-interacting protein. HATs play an important role in regulating chromatin remodeling, transcription and other nuclear processes by acetylating histone and non-histone proteins. In addition, HATs are histone acetylases that are involved in DNA repair and apoptosis and are expected to play an important role in signal transduction, and selective splicing of these genes produces a variety of transcriptional variants.

NRL is also a human gene, encoding a basic motif-leucine zipper transcription factor belonging to the Maf family. This protein is very well preserved in vertebrates and plays an important role as an endogenous regulator of photoreceptor development and function. Mutations in this protein are associated with retinitis pigmentosa and retinal degenerative diseases.

According to a preferred embodiment of the present invention, the composition of the present invention comprises TIP60 (Tat-interacting protein 60) as an active ingredient.

According to a preferred embodiment of the present invention, TIP60 of the present invention binds to neural retina leucine-zipper (NRL) protein.

According to a preferred embodiment of the invention, the binding of the TIP60 and NRL proteins of the invention essentially comprises a chromo domain or a histone acetyltransferase (HAT) domain of the TIP60 protein.

According to a preferred embodiment of the present invention, TIP60 of the present invention increases the transcriptional activity of the NRL protein.

According to a preferred embodiment of the invention, the promoter activated by the NRL protein of the invention comprises a promoter of genes important for retinal development and differentiation, more preferably a promoter of rhodopsin.

According to a preferred embodiment of the present invention, the retinal disease of the present invention is Usher syndrome, Stargardt disease (Bargard-Beed's syndrome), Best disease, choroidal deficiency, gyrate-atrophy, retinal pigmentosa, Retinal macular degeneration, Leber Congenital Amaurosis; Leber's Hereditary Optic Neuropathy disease, most preferably retinitis pigmentosa.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-described TIP60 protein of the present invention; And (b) a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the TIP60 protein.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets, capsules, or gels (eg, hydrogels), and may further include dispersants or stabilizers. .

In the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier, administration method and formulation method, etc., are applied to the contents of the pharmaceutical composition of the present invention. In particular, the pharmaceutical composition of the present invention is preferably parenteral administration, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, transdermal administration or direct administration in tissue (eg, direct administration to the eye). Most preferably by direct administration in tissues. Generally, the dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.

According to another aspect of the present invention, the present invention provides a method for screening a therapeutic agent for retinal disease, comprising the following steps:

(a) treating a test substance to a cell comprising a TIP60 (Tat-interacting protein 60) -encoding nucleotide sequence; And

(b) provides a method of screening for a retinal disease therapeutic agent comprising analyzing the expression or activity of TIP60. When the test substance increases the expression or activity of TIP60, it is considered to be a therapeutic agent for retinal disease.

According to the present invention, as the expression or activity of the TIP60 protein in the cell increases, the transcriptional activity of the NRL protein increases to increase the binding capacity of the NRL target gene to the promoter (eg, rhodopsin promoter), which is the development of a therapeutic agent for retinal disease. Can be applied to

According to the method of the present invention, first, a sample to be analyzed is contacted with a cell containing the nucleotide sequence of TIP60 of the present invention. Cells comprising the nucleotide sequence of the target of the present invention are not particularly limited and are preferably retinal cells. As used to refer to the screening method of the present invention, the term "sample" refers to an unknown substance used in screening to test whether it affects the activity of a TIP60 gene or protein. The sample includes, but is not limited to, chemicals, peptides and natural extracts. The sample analyzed by the screening method of the present invention is a single compound or a mixture of compounds (eg, a cell or tissue culture). Samples can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. USA 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. USA 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994 and the like.

Next, the expression level and activity of the target of the present invention are measured in the cells treated with the sample. The amount of expression and activity may be measured as described below. When the result of the measurement indicates that the expression or activity of the nucleotide sequence of the marker of the present invention is increased, the sample may be determined as a substance for improving or treating retinal disease. Can be.

According to a preferred embodiment of the present invention, the activity analysis of TIP60 in step (b) described above is carried out by measuring the binding of TIP60 to NRL protein, more preferably in the step of watering (b) analysis of activity of TIP60 It is carried out by measuring that TIP60 increases the transcriptional activity of the NRL protein.

According to a preferred embodiment of the invention, the promoter activated by the NRL protein of the invention comprises a promoter of genes important for retinal development and differentiation, more preferably a promoter of rhodopsin.

The screening methods of the present invention can be carried out in a variety of ways, in particular in a high throughput manner according to various binding assays known in the art.

In the screening method of the present invention, the sample or TIP60 gene or protein may be labeled with a detectable label. For example, the detectable label may be a chemical label (eg biotin), an enzyme label (eg horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, β-gal Lactosidase and β-glucosidase), radiolabels (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent labels [eg coumarin, fluorescein, fluoresein Isothiocyanate (FITC), rhodamine 6G ( rhodamine 6G), rhodamine B (rhodamine B), TAMRA (6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl and FAM], luminescent labels, chemiluminescent labels, fluorescence resonance energy transfer (FRET) labels or metal labels (eg gold and silver).

When using a TIP60 gene, protein or sample labeled with a detectable label, the binding between the TIP60 gene or protein and the sample may be detected by detecting a signal from the label. For example, when alkaline phosphatase is used as a label, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS-B1-phosphate) Signal is detected using a chromogenic reaction substrate such as) and enhanced chemifluorescence (ECF). When hose radish peroxidase is used as a label, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD), and substrates such as naphthol / pyronin to detect the signal.

Alternatively, binding of the sample to the TIP60 gene or protein may be analyzed without labeling the interactants. For example, a microphysiometer can be used to analyze whether a sample binds to a TIP60 gene or protein. Microphysiometers are analytical tools that measure the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). The change in acidification rate can be used as an indicator for binding between the sample and the TIP60 gene or protein (McConnell et al., Science 257: 19061912 (1992)).

The binding capacity of the sample with the TIP60 gene or protein can be analyzed using real-time bimolecular interaction analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63: 23382345 (1991), and Szabo et al. , Curr. Opin.Struct . Biol. 5: 699705 (1995)). BIA is a technique for analyzing specific interactions in real time and can be performed without labeling of the interactions (eg, BIAcore ™). Changes in surface plasmon resonance (SPR) can be used as indicators for real-time reactions between molecules.

In addition, the screening method of the present invention can be carried out according to a two-hybrid analysis or a three-hybrid analysis method (US Pat. No. 5,283,317; Zervos et al. , Cell 72, 223232, 1993; Madura et al. , J. Biol. Chem. 268, 1204612054, 1993; Bartel et al. , BioTechniques 14, 920924, 1993; Iwabuchi et al. , Oncogene 8, 16931696, 1993; and W0 94/10300). In this case, TIP60 protein can be used as a bait protein. According to this method, it is possible to screen substances, in particular proteins that bind to the TIP60 protein. Two-hybrid systems are based on the modular nature of the transcription factors composed of cleavable DNA-binding and activation domains. For simplicity, this assay uses two DNA constructs. For example, in one construct, the TIP60-encoding polynucleotide is fused to the DNA binding domain-encoding polynucleotide of a known transcription factor (eg GAL-4). In another construct, a DNA sequence encoding a protein of interest (“prey” or “sample”) is fused to a polynucleotide encoding the activation domain of the known transcription factor. If bait and prey interact in in vivo to form a complex, the DNA-binding and activation domains of the transcription factors are contiguous, which triggers transcription of the reporter gene (eg, LacZ ). Expression of the reporter gene can be detected, which indicates that the protein of analysis can bind to the TIP60 protein, and consequently, it can be used as a substance for improving or treating retinal disease.

Measurement of the expression level change of the TIP60 gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al. , Molecular and Cellular Methods in Biology and Medicine , 102-108, CRC press), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning.A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al. , Molecular Cloning.A Laboratory Manual , 3rd ed.Cold Spring Harbor Press (2001)).

When performing according to the RT-PCR protocol, first, total RNA is isolated from cells treated with a sample, and then a first-chain cDNA is prepared using oligo dT primers and reverse transcriptase. Subsequently, the first chain cDNA is used as a template, and PCR reaction is performed using a TIP60 gene-specific primer set. TIP60 gene-specific primer sets are described in the Examples below. Then, PCR amplification products are electrophoresed, and the formed bands are analyzed to measure changes in the expression level of granulation factor genes.

In addition, the change in the amount of TIP60 protein can be carried out through various immunoassay methods known in the art. For example, changes in the amount of TIP60 protein may include immunostaining, radioimmunoassay, radioimmunoprecipitation, western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, and sandwich assay. It includes, but is not limited to.

The immunoassay or method of immunostaining is described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the invention is carried out in accordance with radioimmunoassay methods, an antibody labeled with a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) detects a marker molecule of the invention. It can be used to.

When the method of the present invention is carried out by ELISA, the present invention comprises the steps of: (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (Ii) reacting said cell lysate with an antibody to a target as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase, β-galactosidase, hose Radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as (phenzaine methosulfate) may be used. All.

When the method of the invention is carried out in a capture-ELISA mode, certain embodiments of the invention comprise (i) coating an antibody against a target of the invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with the cell sample; (Iii) reacting the product of step (ii) with a detecting antibody that has a label that generates a signal and that specifically reacts with TIP60 protein; And (iv) measuring a signal originating from said label.

The detection antibody carries a label which generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent, and fluorescence resonance energy transfer (FRET). Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of such signals allows for qualitative or quantitative analysis of the targets of the present invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

By analyzing the final signal intensity by the above-described immunoassay process, the test substance is identified and treated for disease treatment, preferably retinal disease treatment, for example, Usher syndrome, Stagart disease, Bark-Bead syndrome, Best disease, choroid Lack, chorioretinal atrophy, retinitis pigmentosa, retinal macular degeneration, leber congenital cataract, blue-cone monochromacy (BCM), interretinal separation, MA (Mlattia Leventinese), Oguchi's disease, and Lef's disease Diseases, and most preferably for retinal pigmentosa.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a composition for improving or treating retinal disease comprising TIP60 as an active ingredient, and a method for screening a retinal disease therapeutic agent.

(Ii) The present invention is the first report to investigate the mutual binding of NRL and TIP60 and transcriptional activation of NRL protein through retinal disease.

(Iii) TIP60 of the present invention can be used to improve or treat retinal disease by directly binding to NRL and increasing the transcriptional activity of the NRL protein to induce transcriptional activation of a promoter of the target gene (eg, a rhodopsin promoter).

(Iii) The present invention also provides basic data on the development and treatment of new drugs through the regulation of TIP60 protein in the treatment of retinal diseases (especially retinal pigmentosa).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Materials and Experimental Methods

Cell Line Cultures and Transfection

Human embryonic kidney (HEK) 293 cell line was purchased from the American Type Culture Collection (Manassas, VA) containing 10% fetal bovine serum (FBS; Invitrogen, USA) and penicillin-streptomycin (50 units / ml). Incubated for 12 hours prior to transfection in DMEM (Dulbecco's modified Eagle's medium, Invitrogen, USA). The cells were washed with DMEM medium containing no FBS and subjected to transient transfection with Lipofectamine (Lipofectamine, Invitrogen, USA) according to the manufacturer's protocol to perform EcoR I of pEGFP-NRL (pEGFP vector (clonetech)). Expression plasmids of NRL gene insertion at site) and pcDNA-FLAG-TIP60 (TIP60 gene insertion at EcoR I and Xba I sites of pcDNA-FLAG vector (clonetech)) were transfected into HEK293 cells.

The control group was injected with the empty pcDNA-FLAG vector. Transfected cells were incubated in a 37 ° C. incubator for 24 hours.

Plasmid

Full length NRL was amplified from E18 mouse eye cDNA using reverse transcriptase (Intron, Korea). NRL cDNAs amplified by polymerase chain reaction (PCR) using the following primers were inserted into the pCRII-TOPO vector to obtain pCRII-TOPO-NRL: NRL F primer (forward), 5'-ATGGCTTTCCCTCCCAGTCCC-3 '; And NRL R primer (reverse direction), 5'-TCAGAGGAAGAGGTGTGTGTG-3 '). Full length NRL was amplified from pCRII-TOPO-NRL by PCR.

The region encoding the full-length hTIP60 was amplified from the hTIP60 cDNA present in the human brain library (Clontech) using PCR and inserted into the pCRII-TOPO vector (Invitrogen) to obtain pCRII-TOPO-TIP60. The resulting hTIP60 clone was confirmed by sequencing. Full length TIP60 was amplified from pCRII-TOPO-TIP60 by PCR reaction, and the primers used were as follows: TIP60 FL-F (forward), 5'-GAATTCATGGCGGAGGTGGGG-3 '; TIP60 FL-R (reverse), 5'-TCTAGATCACCACTTCCCCCT-3 '. In addition, fragment mutations of TIP60, TIP60-chromo, TIP60-Zn and TIP60-HAT constructs, were constructed using PCR from full-length TIP60 using the following primers: TIP60-chromo F (forward), 5 '. -GAATTCATGGCGGAGGTGGGG-3 ', TIP60-chromo R (reverse), 5'-TCTAGAGAGGACATGTGT-3'; TIP60-Zn F (forward), 5'-GAATTCACCCCCACTAAGAAC-3 ', TIP60-Zn R (reverse), 5'-TCTAGAATTGTAGTCTTCCGT-3'; And TIP60-HAT F (forward), 5'-GAATTCGTGGCCTGCATCCTA-3 ', TIP60-HAT R (reverse), 5'-TCTAGATCACCACTTCCCCCT-3. These PCR products were inserted into the pCRII-TOPO vector, subcloned into the EcoR I and Xba I positions of the pcDNA -HA vector and confirmed by sequencing.

NRL Polyclonal Antibody Preparation

Polyclonal antibodies of the NRL protein were prepared from rabbits using affinity-purified GST-NRL as antigen. Prior to immunization, pre-immune sera from the same rabbit was obtained. Each serum was tested by western blotting in purified GST-NRL and 4 week mouse eye lysates. Specificity of the purified antibodies was further analyzed by Western blotting using serially diluted NRL antibodies.

In Vivo Binding Assay, Western Blotting and Immunoprecipitation

HEK 293 cells were dispensed in 100-mm plates at a concentration of 2 × 10 ⁶ cells / well and incubated for 12 hours. Cells were transfected with each plasmid and further incubated for 24 hours. Cell cultures were treated with 1% Triton X-100 (Sigma), 150 mM NaCl (Sigma), 50 mM Tris-HCl (pH 7.5) (Sigma), 0.1% SDS (Sigma), 1% Nonidet P-40 (Calbiochem) And 1 mM phenylmethanesulfonyl fluoride (PMSF, Sigma). The cell suspension was allowed to react on ice for 20 minutes and then centrifuged at 4 ° C. for 20 minutes at 12,000 rpm. For immunoprecipitation assays, the supernatant was pre-cleaned with 20 μl of Protein A-agarose beads (50% slurry; Millipore, USA) and then 30 μl of fresh Protein A in the presence of each antibody. Reaction was carried out overnight at 4 ° C. in agarose beads. The beads were washed three times with PBS and then resuspended in SDS sample buffer and boiled for 10 minutes. After the samples were separated using SDS-PAGE, the separated proteins were transferred to nitrocellulose membrane (Whatman, protran NC paper). The membranes were blocked for 12 hours in 0.05% skim milk, and then samples were analyzed by Western blotting using each antibody. The antibodies used were as follows: Polyclonal antibodies against HA (sc-805, 1: 1,000 dilution) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.) And GFP (GFP-1814 460, 1: 5,000 dilution) and monoclonal antibodies against FLAG-M2 (1: 10,000 dilution) were purchased from Roche Diagnostics and Sigma-Aldrich, respectively.

Luciferase Assay

After culturing HEK 293 cells in a 60-mm petri dish, the firefly luciferase rhodopsin reporter gene (0.1 μg) and pCMV-β-galactosidase (0.1 μg) were added to the GFP empty vector, GFP-NRL, HA-TIP60- Transfected using Liporectamine 2000 (Invitrogen) in combination with vectors expressing FL, HA-chromo, HA-Zn and HA-HAT. After 36 hours of transfection, cells were lysed in Reporter Lysis Buffer (Promega). Cell extracts were analyzed by a luciferase reporter assay system using a luminometer (Luminometer, Lumat LB 9501 Berthold, Germany). Luciferase activity of the rhodopsin-luciferase vector was normalized based on the activity of co-transfected β-galactosidase.

Chromatin Immunoprecipitation (ChIP)

ChlP assays were performed according to the protocol provided by Upstate Biotechnology, Inc. (Lake Placid, NY). Briefly, the ocular tissues were extracted into small pieces (1-3 mm ³ ) by the time of mouse development, and then the tissues were cross-linked for 15 minutes at 37 ° C. with 1% formaldehyde (Sigma). linked). Cells were then washed with ice-cold PBS and resuspended in 200 μl SDS-sample buffer containing protease inhibitor complex (Sigma). The suspension was sonicated three times with ice at ice for 1 minute per 10 seconds of pulses on ice, followed by 20 μl of protein A-agarose beads blocked with sonicated salmon sperm DNA. Pre-washed at 30 ° C. for 30 minutes. The beads were removed and the chromatin solution of each experimental group was treated with anti-NRL (antibody prepared in the present invention), anti-TIP60 (sc-5725, SantaCruz), Ac-H3 (1766-1, Epitomics, USA), Ac-H4 (1796-1, Epitomics, USA) and immunoprecipitated overnight at 4 ° C. with anti-polymerase II (2245-s, Epitomics, USA), then at 4 ° C. with 40 μl of protein A-agarose beads. It was further reacted for a time. Immune complexes were separated with 100 μl of elution buffer (1% SDS and 0.1 M NaHCO ₃ ) followed by separation of formaldehyde cross-links by heating at 65 ° C. for 6 hours. Proteinase K (Sigma) was added to the reaction complex and reacted at 45 ° C for 1 hour. Immunoprecipitate DNA and control input DNA were purified by phenol / chloroform extraction method and analyzed according to the normal PCR method using rhodopsin promoter-specific primers. The primer sequences used were as follows: rhodopsin promoter F primer (forward), 5'-TCAAGCCGGAGGTCAACAAC-3 '; And rhodopsin promoter R primer (reverse), 5'-TCTTGGACACGGTAGCAGAG-3 '.

Statistical analysis

Statistical analysis of the variation between the two different groups was carried out using a comparison test of Turkey's post hoc using SPSS (Version 11.5). Transcriptional assays were repeated at least three times.

Experiment result

Binding between NRL protein and TIP60 protein

To confirm the interaction between NRL protein and TIP60 protein, we examined co-precipitation of full length NRL and FLAG-TIP60 in transient transfected HEK 293 cells. HEK 293 cells were cultured according to the method described above. Cultured HEK 293 cells were transfected with FLAG empty vector and FLAG-TIP60 individually or with GFP-NRL expression vector and the cells were lysed 24 hours later. The lysed extract was reacted with anti-FLAG antibody and then subjected to western blot with anti-GFP and anti-FLAG.

As can be seen in Figure 1, compared to cells transfected with FLAG empty vector and GFP-NRL (control, lane 1), the protein was extracted from proteins transfected with FLAG-TIP60 and GFP-NRL transfected cells. Western blotting results confirmed that NRLs co- precipitated in combination with TIP60. This shows that two proteins (NRL and TIP60) bind to each other in HEK 293 cells.

TIP60 protein site important for binding to NRL protein

To investigate the binding site of the TIP60 protein to NRL, a fragment mutant construct of the TIP60 protein was constructed using PCR. These mutant constructs (HA-chromo, HA-Zn (Zinc finger) and HA-HAT) were used to identify the mutual binding sites of TIP60 and NRL.

HEK 293 cells were combined with full-length TIP60 (HA-TIP60-FL) and GFP-NRL expression vectors, or TIP60 fragment mutant expression vectors (HA-chromo, HA-Zn and HA-HAT) and GFP-NRL expression vectors. Cells were incubated for 24 hours after transfection. For immunoprecipitation assays, the supernatant was pre-washed with 20 μl of protein A-agarose beads (50% slurry) and then at 4 ° C. in 30 μl fresh protein A-agarose beads in the presence of each antibody. The reaction was overnight. Cell lysates were immunoprecipitated with anti-HA antibody followed by Western blotting with anti-GFP antibody.

As can be seen in Figure 2, it was confirmed that the NRL is bound to HA-TIP60-FL (lane 1), TIP60 fragment mutations (lane 2, HA-chromo; lane 4, HA-HAT). This indicates that, among the domains in the TIP60 protein, the chromo domain and the HAT domain are important for binding to NRL and the Zn domain is not important for binding to NRL.

Increasing Transcriptional Activity of NRL Protein by TIP60 Protein

In order to determine whether the TIP60 protein can regulate the transcriptional activity of the NRL protein through binding to the NRL, a rusperase assay was performed. To this end, the promoter region of the rhodopsin gene, a target gene of NRL, was cloned with the luciferase expression vector. After culturing HEK 293 cells in a 60-mm petri dish, the rhodopsin-luciferase expression vector was rephorec in combination with a GFP empty vector, GFP-NRL, HA-TIP60-FL, HA-chromo, HA-Zn and HA-HAT. Transfection with Tamine 2000 (Invitrogen). After 36 hours of transfection, cells were lysed in Reporter Lysis Buffer (Promega). Cell extracts were analyzed by a luciferase reporter assay system using a luminometer (Luminometer, Lumat LB 9501 Berthold, Germany). Luciferase activity was normalized using the GFP empty vector used as a negative control.

As can be seen in Figure 3, the TIP60 construct capable of binding to NRL was co-transfected with the NRL construct (lanes 3, 4 and 6; HA-TIP60-FL, HA-chromo and HA- HAT) was found to increase the transcriptional activity of NRL, TIP60 construct that can not bind NRL (lane 5; HA-Zn) showed a similar transcription activity to the GFP-NRL alone treatment group. This means that the transcriptional activity of NRL protein is increased through the combination of TIP60 protein and NRL protein.

The interaction of TIP60 and NRL proteins in retinal extracts and an increase in the level of transcription of the rhodopsin gene suggests the possibility that NRL proteins activate transcription of the rhodopsin gene by recruiting TIP60 to the target sequence to promote histone acetylation. That is, rhodopsin transcription can be promoted by binding of NRL protein on the promoter and recruiting of HAT and Ac-H3 / Ac-H4 during photoreceptor development. To test this, it was examined whether the histone acetyltransferase TIP60 is present on the promoter of the NRL target gene (eg rhodopsin). Eyes were extracted from the time of development of mice (embryonic day (E15) to postnatal day 28, P28)] and NRL, TIP60, histone protein, and active polymerase II present therein. The change pattern of was tested by ChIP (Chromatin immunoprecipitation).

As can be seen in FIG. 4, NRL is located at the rhodopsin promoter (phosphorylated NRL is located at promoter at P0; results are not shown), then TIP60 is recruited to the promoter (P2), then AcH3 / AcH4 and phosphorylated Pol II bound to TIP60. These results demonstrate that the binding of each regulator to the rhodopsin high promoter site in retinal development occurs sequentially: (a) binding of NRL; (b) binding of leucrute and acetylated histone proteins (AcH3 / AcH4) of TIP60; And (c) an activated form of RNA polymerase II (C-terminal domain comprising phosphorylated Ser5; p-Pol II).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a result showing the mutual binding of NRL protein and TIP60 protein. GFP-fusion NRL expression vectors were transfected into HEK 293 cells with pFLAG-CMV2 covector (lane 1) or pFLAG-CMV2-TIP60 expression vector. Total cell lysates were immunoprecipitated with anti-FLAG antibody (M2) and then western blotted with the corresponding antibodies (anti-GFP and anti-FLAG).

2 shows the results of identifying important domains of the TIP60 protein in binding to the NRL protein. The TIP60 protein encodes 513 amino acids, with each domain as follows: N-terminal chromo domain, zinc finger domain and C-terminal conserved MYST domain. The MYST domain consists of a conservative HAT (histone acetyltransferase) domain with catalytic activity in a short sequence of 367-386 residues. Three domain constructs were constructed to identify sites of TIP60 protein important for binding to NRL protein: HA-attached chromo domain (HA-chromo), zinc finger domain (HA-Zn) And HAT domains (HA-HAT). GFP-NRL expression vectors were co-transfected into HEK 293 cells in combination with three different TIP domain constructs, respectively. Total cell lysates were immunoprecipitated with anti-FLAG antibody (M2) followed by western blotting with anti-GFP antibody.

3 is a graph showing the increase in transcriptional activity of NRL protein through interaction with TIP60 protein for NRL protein. The rhodopsin promoter was combined with HEK 293 cells by combining a GFP empty vector, or GFP-NRL and HA-attached TIP60 constructs (HA-TIP60-FL, HA-Chromo, HA-Zn and HA-HAT) expression vectors. Co-transfection with the containing luciferase reporter plasmid. Luciferase activity was measured 24 hours after transfection according to the methods described in Experimental Materials and Experimental Methods.

4 is a ChlP assay result showing proteins recruited on the NRL target gene promoter in retinogenesis. Antibodies against NRL, TIP60, Ac-H4, Ac-H3 and p-Pol II were used to immunoprecipitate bound chromatin fragments from the developing mouse retina. The detected bands indicate that the transcription factors recognized by the immunoprecipitating antibody are bound to the promoter region of the target gene. In the present invention, the target gene is a promoter of rhodopsin. Total fragment sequences are detected by PCR using target gene primers specific as input controls.

Claims (11)

Pharmaceutical composition for improving or treating retinal disease, including TIP60 (Tat-interacting protein 60) as an active ingredient. The composition of claim 1, wherein the TIP60 binds to a neural retina leucine-zipper (NRL) protein. The composition of claim 2, wherein the TIP60 essentially comprises a chromo domain or a histone acetyltransferase (HAT) domain involved in binding to the NRL protein. The composition of claim 1, wherein the TIP60 increases the transcriptional activity of the NRL protein. The composition of claim 2, wherein the promoter activated by the NRL protein is a promoter of a rhodopsin gene. The method of claim 1, wherein the retinal disease is Usher syndrome, Stargardt disease, Barth-Bead syndrome, Best's disease, choroidal deficiency, choroid-atrophy, retinal pigmentosa, retinal macular degeneration Leber Congenital Amaurosis; Leber's Hereditary Optic Neuropathy; Blue-cone monochromacy; BCM; Composition. (a) treating a test substance to a cell comprising a TIP60 (Tat-interacting protein 60) -encoding nucleotide sequence; And (b) analyzing the expression or activity of TIP60. The method for screening a retinal disease therapeutic agent, wherein the test substance increases the expression or activity of TIP60 and is determined to be a retinal disease therapeutic agent. 8. The method of claim 7, wherein the activity analysis of TIP60 in step (b) is performed by measuring the binding of TIP60 to NRL protein. 8. The method of claim 7, wherein the activity analysis of TIP60 in step (b) is performed by measuring that TIP60 increases the transcriptional activity of the NRL protein. 10. The method of claim 8 or 9, wherein the promoter activated by the NRL protein is a promoter of a rhodopsin gene. 8. The method of claim 7, wherein the retinal disease is Usher syndrome, Stagart's disease, Barth-Bead syndrome, Best's disease, choroidal deficiency, choroidal atrophy, retinal pigmentosa, retinal macular degeneration, leber congenital cataract, BCM (Blue) -cone monochromacy), interretinal separation, ML (Malattia Leventinese), Oguchi disease or Lefsome disease.

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