KR20210117524A - Use of KAT2B for the diagnosis of ciliary injury disease - Google Patents

Abstract

The present invention relates to a use of KAT2B for diagnosis of ciliary injury disease, and to a preparation for preventing or treating ciliary injury disease comprising KAT2B, which is proposed as a biomarker for diagnosis of ciliary injury disease and provided as a preparation for preventing or treating ciliary injury disease by confirming that KAT2B, a biomarker according to the present invention, promotes a ciliary formation by inducing acetylation of α-tubulin in cilia, and also confirming that a degree of acetylation of α-tubulin decreases, an expression of IFT protein and GLI1 decreases, and a glomerular cyst is formed in KAT2B knockdown and knockout.

Description

Use of KAT2B for the diagnosis of ciliary injury disease

The present invention relates to the use of KAT2B for the diagnosis of ciliary damage disease, and to a preparation for preventing or treating ciliary damage disease comprising KAT2B.

Cilia are organelles of cells that protrude from the cell surface. Primary cilia in all eukaryotic cells are non-motile and provide various senses (sight, smell, hearing, kinesthesia, osmotic pressure, etc.) from the outside. It plays an important role in detection. In addition, as important intracellular signaling processes are known to occur via primary cilia, the importance of primary cilia as a "signaling hub" that integrates cell signaling systems is emerging.

In the kidney, primary cilia are found on the luminal cell surface of all epithelial, glomerular, and glomerular sac cells constituting the tubule except for the intercalated cells of the collecting tubule. The primary cilia respond to fluid flow and chemical factors, and serve as chemical and mechanosensory intracellular organs essential for maintaining the structure and function of the kidney. Ciliary injury diseases in which these primary cilia are damaged include Polycystic Kidney Disease, Nephronophthisis, Joubert syndrome, Bardet-Biedl syndrome, McKell-Gruber syndrome ( Mackel-Grouber syndrome) is known to mainly include rare genetic diseases, but recently, studies have been attempted to reveal the association with diseases with high frequency such as cancer and obesity.

In particular, polycystic kidney disease (PKD) is a genetic disease in which cysts filled with body fluid are formed in the kidneys, thereby inhibiting renal function. Polycystic Kidney Disease 1 (PKD1) and Polycystic Kidney Disease 2 (PKD2) are known as the representative causative genes involved in the development of polycystic kidney. Encrypt. The PC1 and PC2 proteins form a complex together and are located in the cilia present in the renal epithelial cells, and function as a calcium channel. However, the genes involved in the ciliary defect mechanism observed in animal models of polycystic kidney have not been clearly identified.

The present inventors have completed the present invention by confirming the relationship between KAT2B and ciliary injury disease as a new biomarker capable of predicting the prognosis by diagnosing ciliary injury disease in consideration of this point.

The present invention relates to the use of KAT2B for the diagnosis of ciliary damage disease, and to a formulation for preventing or treating ciliary damage disease comprising KAT2B, wherein the biomarker KAT2B according to the present invention is alpha-tubulin (α-tubulin) in cilia. ) was confirmed to promote cilia formation by inducing acetylation, and the degree of acetylation of α-tubulin decreased in KAT2B knockdown and knockout, and IFT proteins and GLI1 By confirming that the expression level is reduced and a glomerular cyst is formed, KAT2B is proposed as a biomarker for the diagnosis of ciliary damage disease, and KAT2B protein is provided as a preventive or therapeutic agent for ciliary damage disease.

The present invention provides a biomarker composition for diagnosing a ciliary injury disease comprising a KAT2B protein or a gene encoding the KAT2B protein as an active ingredient.

In addition, the present invention provides a kit for diagnosing a ciliary injury disease comprising an agent capable of detecting the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein as an active ingredient.

In addition, the present invention provides information for diagnosing ciliary damage disease, comprising comparing the expression level of the KAT2B protein, or the expression level of the KAT2B protein-encoding gene, from a sample isolated from a sample and a normal control. provides

In addition, the present invention provides a biomarker composition for predicting the prognosis of a ciliary injury disease comprising a KAT2B protein or a gene encoding the KAT2B protein as an active ingredient.

In addition, the present invention provides a kit for prognosing the prognosis of ciliary injury disease, which contains an agent capable of detecting the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein as an active ingredient.

In addition, the present invention provides a first step of measuring the expression level of the KAT2B protein, or the expression level of the gene encoding the KAT2B protein in a biological sample isolated from a tissue of a subject; and a second step of comparing the expression level of the protein with a biological sample isolated from a normal tissue; provides an information providing method for predicting the prognosis of ciliary damage disease, including.

In addition, the present invention provides a pharmaceutical composition for preventing or treating ciliary damage disease comprising KAT2B protein or an activity promoter of KAT2B protein as an active ingredient.

In addition, the present invention measures the expression level of the KAT2B protein and the expression level of the KAT2B protein-encoding gene in a biological sample isolated from the tissue of the subject to select the KAT2B protein or the KAT2B protein-encoding gene. the first step; And when the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein increases after treatment with the test substance compared to the disease subject not treated with the test substance, the second determined as a prophylactic or therapeutic agent for ciliary damage disease It provides a screening method of an agent for preventing or treating ciliary damage disease comprising;

According to the present invention, it was confirmed that KAT2B, a biomarker according to the present invention, promotes cilia formation by inducing acetylation of α-tubulin in cilia, and knockdown and knockout of KAT2B In this study, by confirming that the degree of acetylation of α-tubulin decreased, the expression of IFT proteins and GLI1 decreased, and the glomerular cyst was formed, KAT2B was used as a biomarker for the diagnosis of ciliary injury disease. It is proposed, and the KAT2B protein can be provided as an agent for preventing or treating ciliary damage disease.

1 is a photograph showing the Western blot results for analyzing the effect of KAT2B, a biomarker according to the present invention, on the acetylation of α-tubulin, which is important for cilia formation.
2 is a photograph showing the results of immunocytochemistry analyzing the effect of KAT2B, a biomarker according to the present invention, on the acetylation of α-tubulin, which is important for cilia formation.
3 is a photograph showing the results of immunoprecipitation analysis of the interaction of KAT2B, a biomarker according to the present invention, with α-tubulin, which is important for cilia formation.
4 is a diagram showing the structure of the biomarker KAT2B according to the present invention and the structure of the modified KAT2B from which the acetyltransferase (AT) domain has been removed.
5 is a Western blot analyzing the effect of KAT2B, a biomarker according to the present invention, and modified KAT2B from which the acetyltransferase (AT) domain is removed on the acetylation of α-tubulin. A picture showing the results.
6 is a photograph showing the results of immunocytochemistry analyzing the change in the degree of acetylation of α-tubulin according to treatment with Garcinol.
7 is a graph showing the percentage (%) of cilia-forming cells according to treatment with garcinol and a photograph showing the results of Western blot analysis of acetylation of α-tubulin.
8 is a photograph showing the results of Western Blot and Immunocytochemistry analyzing changes in IFT components according to knockdown of KAT2B, a biomarker according to the present invention.
9 is a result of immunocytochemistry analyzing the effect on the acetylation of SMO protein and α-tubulin according to SAG (Smoothened Agonist) treatment, which is an activator of hedgehog signaling pathway. is a picture representing
10 is a Western blot photograph and graph analyzing the mRNA expression of KAT2B and Gli1 according to SAG (Smoothened Agonist) treatment, which is an activator of hedgehog signaling pathway.
11 is a graph analyzing KAT2B mRNA expression in KAT2B knockout mice.
FIG. 12 is a graph analyzing the change in two kidney weight per total body weight in KAT2B knockout mice.
13 is a Western blot photograph analyzing the acetylation level of α-tubulin, the expression level of IFT proteins and GLI1 in KAT2B knockout mice.
14 is a photograph confirming changes in the degree of glomerular cyst and immune cell infiltration in kidney tissue of KAT2B knockout mice.

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. Any numerical limitation given throughout this specification shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation were expressly written.

Hereinafter, the present invention will be described in more detail.

The present invention provides a biomarker composition for diagnosing a ciliary injury disease comprising a KAT2B protein or a gene encoding the KAT2B protein as an active ingredient.

The ciliary damage disease may be a kidney disease, and the kidney disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic renal failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome, Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, or hydronephrosis, but is not limited thereto.

In addition, there is provided a kit for diagnosing ciliary damage disease containing, as an active ingredient, an agent capable of detecting the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein.

The agent capable of measuring the expression level of the protein is an antibody, peptide, aptamer or compound that specifically binds to the protein, and the agent capable of measuring the expression level of the gene is a primer or It may be a probe, but is not limited thereto.

The 'expression level of the gene' can be measured using an antisense oligonucleotide, primer pair or probe that specifically binds to the mRNA of the gene, and the agent for measuring the expression of the mRNA is an antisense oligonucleotide specific for the gene. nucleotides, primer pairs, probes, and combinations thereof. That is, the detection of the nucleic acid may be performed by an amplification reaction using one or more oligonucleotide primers hybridized to a nucleic acid molecule encoding a gene or a complement of the nucleic acid molecule, but is not limited thereto. For example, detection of mRNA using a primer may be performed by amplifying a gene sequence using an amplification method such as PCR and then confirming whether amplification is performed by a method known in the art, RT-PCR, competitive RT-PCR, quantitative It may be measured by RT-PCR, RNase protection assay, Northern blot, or DNA chip, but is not limited thereto.

The "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as long as possible to specifically bind to other than mRNA, and is labeled to check the presence or absence of a specific mRNA and the amount of expression. can The probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Suitable probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.

The "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, a short nucleic acid sequence capable of base pairing with a complementary template and serving as a starting point for template strand copying. say The primer is capable of initiating DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art.

The 'expression level of the protein' may be measured using an antibody, peptide or nucleotide that specifically binds to the protein.

The antibodies are polyclonal antibodies, monoclonal antibodies, recombinant antibodies and complete forms having two full-length light chains and two full-length heavy chains, as well as functional fragments of antibody molecules, e.g., Fab, F(ab'). ), F(ab')2 and Fv. Antibody production can be easily prepared using techniques well known in the art to which the present invention pertains, and commercially available antibodies can be used.

In addition, the level of the protein can be measured by an immunoassay or immunostaining method. The method may be implemented in the form of a microchip or automated microarray system capable of detecting the biomarker protein or a fragment thereof in a sample.

The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity purification.

The protein level can be measured using multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high resolution (SWATH), selected reaction monitoring (SRM) or It may also be measured using immuno multiple reaction monitoring (iMRM). MRM is a method to determine the exact fragment of a substance, break it in a mass spectrometer, select a specific ion from among the broken ions once more, and obtain the number using a continuously connected detector.

In addition, the present invention provides information for diagnosing ciliary damage disease, comprising comparing the expression level of the KAT2B protein, or the expression level of the KAT2B protein-encoding gene, from a sample isolated from a sample and a normal control. provides

In addition, the present invention provides a biomarker composition for predicting the prognosis of a ciliary injury disease comprising a KAT2B protein or a gene encoding the KAT2B protein as an active ingredient. The present invention also provides a kit for predicting the prognosis of ciliary injury disease, which contains an agent capable of detecting the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein as an active ingredient.

In addition, the present invention provides a first step of measuring the expression level of the KAT2B protein, or the expression level of the gene encoding the KAT2B protein in a biological sample isolated from a tissue of a subject; and a second step of comparing the expression level of the protein with a biological sample isolated from a normal tissue; provides an information providing method for predicting the prognosis of ciliary damage disease, including.

In addition, the present invention provides a pharmaceutical composition for preventing or treating ciliary damage disease comprising KAT2B protein or an activity promoter of KAT2B protein as an active ingredient.

The KAT2B protein activity promoter is an antibody, peptide, aptamer or compound that specifically binds to the KAT2B protein, and may promote binding of the KAT2B protein and ciliary alpha tubulin, and the KAT2B protein is ciliary alpha tubulin Inducing the acetylation of ciliary damage can show the effect of prevention or treatment.

The pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastas, and cataplasmas.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and diluent for formulation. The pharmaceutically acceptable carriers and diluents include starch, sugar, and excipients such as mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethyl cellulose, hydroxypropyl cellulose, gelatin, alginate, and polyvinyl blood. binders such as rolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol does not The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and is preferably administered orally. In addition, the dosage of the pharmaceutical composition of the present invention varies depending on the subject's body weight, age, sex, health status, diet, administration time, administration method, excretion rate, severity of disease, etc., but is not limited thereto.

In the present invention, the term “prevention” refers to any act of inhibiting or delaying a disease by administering the composition according to the present invention. In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of a disease by administration of the composition according to the present invention.

In addition, the present invention measures the expression level of the KAT2B protein and the expression level of the KAT2B protein-encoding gene in a biological sample isolated from the tissue of the subject to select the KAT2B protein or the KAT2B protein-encoding gene. the first step; And when the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein increases after treatment with the test substance compared to the disease subject not treated with the test substance, the second determined as a prophylactic or therapeutic agent for ciliary damage disease It provides a screening method of an agent for preventing or treating ciliary damage disease comprising;

Hereinafter, experimental examples and examples will be described in detail to help the understanding of the present invention. However, the following experimental examples and examples are only to illustrate the content of the present invention, the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example> Experimental material and method

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell Culture

In the experimental example of the present invention, mouse embryonic fibroblasts (NIH/3T3, CRL-1658TM, ATCC) were used. NIH/3T3 cells were cultured in DMEM medium (Dulbecco's Modified Eagle's medium, WELGENE, LM 001-05) supplemented with 10% fetal bovine serum (FBS, Fetal Bovine Serum, Gibco, #26140-079) at 37° C., 5% CO ₂ cultured under conditions. All culture media were supplemented with 1% penicillin-streptomycin (P/S, WELGENE, LS 202-02).

2. Antibodies and Drugs

The information of the primary antibody used for Western Blot, Immunocytochemistry, and Immunoprecipitation is as follows: mouse anti-PCAF (Kat2b) (Santa cruz, sc- 13124; 1:500-1:1,000 for WB; for IP), rabbit anti-IFT20 (Proteintech, 13615-1-AP; 1:500-1:1,000 for WB), rabbit anti-IFT25 (Proteintech, 15732-1) -AP; 1:500 for WB, 1:200 for ICC/IF), rabbit anti-IFT46 (Abcam, ab122422; 1:1,000 for WB), rabbit anti-IFT52 (Proteintech, 17534-1-AP; 1:1,000 for WB, 1:500 for ICC/IF), rabbit anti-IFT88 (Santa cruz, sc-84318; 1:1,000 for WB), rabbit anti-IFT140 (Proteintech, 17460-1-AP; 1:1,000 for WB, 1:500 for ICC/IF), rabbit anti-IFT172 (Santa cruz, sc-138962; 1:1,000 for WB, 1:25 for ICC/IF), mouse anti-IFT172 (Santa cruz, sc-398393, 1: 10 for ICC/IF) rabbit anti-GLI1 (Cell Signaling Technologies, #2534, 1:500 for WB), mouse anti-SMO (Santa cruz, sc-166685; 1:50 for ICC/IF), mouse anti-Acetylated -alpha-tubulin (Sigma, T6793; 1:1,000 for WB and ICC/IF), rabbit anti-Acetylated-alpha-tubulin (Cell Sig naling Technologies, #5335; 1:1,000 for WB), mouse anti-gamma-tubulin (Sigma, T6557; 1:1,000 for ICC/IF), mouse anti-alpha tubulin (Cell Signaling Technologies, #3873; 1:1,000 for WB), mouse anti- alpha-tubulin (Santa cruz, sc-5286; for IP), rabbit anti-Pericentrin (Abcam, ab4448; 1:1,000 for WB and ICC/IF), rabbit cytoskeletal actin antibody (Bethyl Laboratories, A300-491A; 1:14,000) used as loading control for WB), normal mouse anti-IgG (EMD millipore, 12-371; for IP), and rabbit anti-Flag (Sigma, F7425; 1:2,000 for WB).

The secondary antibody information used is as follows: goat anti-mouse IgG (Enzo Life Sciences, ADI-SAB-100J; 1:2,500 for WB), goat anti-rabbit IgG (Enzo Life Sciences, ADI- SAB-300J; 1:2,500 for WB), anti-biotin HRP-linked antibody (Cell Signaling Technologies, #7075S; 1:2,500 for WB) and Alexa Flour™ 488 goat anti-mouse (Invitrogen, #A11029; 1:1,000) for ICC/IF).

Garcinol (Garcinol, provided by Catholic University of Korea) was used at a concentration of 500 nM, and SAG (Abcam, ab142160) was used at a concentration of 400 nM.

3. siRNA Transfection

To control gene expression, Control siRNA (Santa Cruz, sc-37007) and PCAF (Kat2b) siRNA (Santa cruz, sc-36199) at a concentration of 30-40 nM were used, and Lipofectamine RNAiMax transfection reagent ( Invitrogen, #13778150) was used. For plasmid transformation, FuGENE HD Transfection Reagent (Promega) was used according to the manufacturer's protocol.

4. Quantitative real time PCR (qRT-PCR)

Extract mRNA from cells using NucleoSpin® DNA/RNA/Protein kit (Macherey-Nagel) according to the manufacturer's instructions, and extract 2 μg mRNA M-MLV Reverse Transcriptase (Promega, M170B), RNasin® Ribonuclease inhibitor (Promega, N211A), 100 nM oligo-dT (C1101, Promega) and 2.5 nM dNTP mixture (Promega, U1205, U1225, U1215, U1235) were used to reverse transcribe into cDNA.

RT-PCR was performed using SYBR Green qPCR Master Mix (PB20.15-05, PCR Biosystem, London, UK) and LightCycler® 96 System (Roche).

5. Western Blot

Proteins were extracted from cells or mouse kidneys using the NucleoSpin® RNA/Protein kit (Macherey-Nagel) according to the manufacturer's protocol. Protein concentration was quantified with bicinchoninic acid (BCA) solution (Sigma, B9643) and copper (II) sulfate sodium (Sigma, C2284).

Protein samples were separated by gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (ATTO, AE-6667-P). The PVDF membrane was blocked with 5% skim milk in PBS (PBST) containing 0.1% Tween® 20 at room temperature for 1 hour, and reacted overnight with a primary antibody diluted with 1% skim milk for probe. Then, the PVDF membrane was washed 3 times for 10 minutes with PBST buffer, and reacted with the HRP-conjugate-secondary antibody and anti-HRP antibody at room temperature for 2 hours. Afterwards, the PVDF membrane was washed 3 times for 10 minutes with PBST buffer, and the results were obtained with LAS-3000, LAS-4000 (Fujifilm), and Amersham Imager 600 (GE Healthcare) devices using the chemiluminescence reagent EzWestLumWe Plus (ATTO, WSE-7120). was secured.

6. Immunocytochemistry/Immunofluorescence (ICC/IF)

After culturing the cells on 18 x 18 mm coverslips, they were washed with PBS buffer to remove the remaining medium. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes, or at 20°C with cold methanol for 5 minutes. Fixed cells were blocked and treated with PBS containing 0.1% Triton X-100 and 1% Bovine Serum Albumin (BSA). Thereafter, the diluted primary antibody was reacted at 4° C. overnight, and washed 3 times with PBS. The secondary antibody was reacted at room temperature for 2 hours and stained with DAPI for 5 minutes. It was washed three times with PBS and fixed with a mounting solution (DAKO, S3023). Cells were imaged using confocal microscopy (Carl Zeiss, LSM-700) and Zen software (Carl Zeiss).

7. Co-Immunoprecipitation (Co-IP)

NIH/3T3 cells were grown identically, and the cells were cultured in serum-free medium for 6 hours to induce the formation of primary cilia. Cells were washed with PBS, containing 25 mM HEPES, 150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, Protease inhibitor (Roche, cOmplete™ Protease inhibitor cocktail, 11697498001), and Phosphatase inhibitor (Roche, PhosSTOP™, 4906837001) was dissolved with a buffer solution. Proteins were quantified by the BCA method. 500-1000 μg of protein was treated with PCAF (Kat2b), alpha tubulin, or mouse IgG antibody overnight at 4° C. using a rotator. Magnetic beads were added and the mixture was treated at 4° C. using a rotator for 4 hours. The samples were then washed three times with a buffer containing 150 mM NaCl, 50 mM Tris-Cl, a protease inhibitor, and a phosphatase inhibitor. The samples were then boiled at 98° C. for 5 minutes and analyzed by Western Blot.

Example 1. Evaluation of the effect of KAT2B on alpha-tubulin as a biomarker in ciliary injury disease

In order to confirm that the KAT2B protein and the gene encoding the KAT2B protein according to the present invention are biomarkers for diagnosing ciliary damage disease, the effect of KAT2B on the acetylation of α-tubulin in cilia was analyzed.

It has been reported that acetylation of α-tubulin increases the flexibility of microtubules, relieves their mechanical stress, and plays a role in extending lifespan. Acetylated α-tubulin is used as a marker to recognize the presence and activity of cilia, and if KAT2B induces acetylation of α-tubulin, it induces the generation and activity of primary cilia it can be understood that

As shown in FIG. 1 , when KAT2B was knocked down with KAT2B siRNA, the amount of total α-tubulin was not changed, but the level of acetylated α-tubulin was decreased. appear. In addition, as shown in FIG. 2 , acetylated α-tubulin was decreased in KAT2B-depleted cells, but was reduced when the KAT2B overexpressing plasmid was treated. ) was found to recover. Also, as shown in Fig. 3, it was confirmed that KAT2B strongly interacts with acetylated α-tubulin.

Referring to the above results, KAT2B induces acetylation of α-tubulin, which is important for cilia formation, and a decrease in KAT2B reduces acetylation of α-tubulin, so cilia are damaged. Therefore, it is demonstrated that the KAT2B protein or the gene encoding the KAT2B protein can be used as a diagnostic biomarker for ciliary damage disease.

In addition, in order to confirm that KAT2B induces acetylation of α-tubulin, a modified KAT2B in which the acetyltransferase (AT) domain is removed was prepared. KAT2B and acetyltransferase (acetyltransferase; AT) domain to handle the removed strain KAT2B the KAT2B-deficient cells, and the results verified the acetylation level of alpha-tubulin (α-tubulin), the strain KAT2B As shown in Figure 5 alpha The acetylation of α-tubulin was reduced. In addition, when NIH3T3 cells were treated with DMSO as a control and garcinol, which inhibits the acetylation function of KAT2B, as shown in FIGS. 6 and 7 , acetylation of α-tubulin was reduced. .

The above results confirm that KAT2B directly acetylates α-tubulin, and when the acetylation ability of KAT2B is inhibited, the acetylation of α-tubulin is also reduced, so the KAT2B protein is As a substance that induces acetylation of α-tubulin, it can be understood that it induces the generation and activity of primary cilia, and KAT2B protein or an activity enhancer of KAT2B protein is a prophylactic or therapeutic agent for ciliary damage disease. prove that it can be provided.

Example 2. Effect of KAT2B as a biomarker on IFT components and hedgehog signals in ciliary injury disease

In order to confirm that the KAT2B protein and the gene encoding the KAT2B protein according to the present invention are biomarkers for diagnosing ciliary damage disease, changes in IFT components and hedgehog signaling pathway according to KAT2B deficiency were evaluated.

As shown in FIG. 8 , when KAT2B was knocked down in NIH/3T3 cells, the levels of IFT components, IFT46, IFT52, IFT172 and IFT140, were significantly reduced (A) at 6 hours, which is the initial stage of cilia formation. When KAT2B was knocked down in the focal microscopy data, the fluorescence intensities of IFT25, IFT52, and IFT172 were significantly reduced (C). In addition, the level of the IFT component remained reduced at 24 hours, the cilia maturation stage, and the fluorescence intensities of IFT25, IFT52 and IFT172, which were strongly stained in the basal body, were significantly reduced in KAT2B knockdown.

The above results demonstrate that KAT2B deficiency reduces the level of IFT components that affect cilia function, thereby impairing cilia function.

In addition, as shown in FIG. 9 , when KAT2B was knocked down in NIH/3T3 cells, smoothened agonist (SAG), an activator of hedgehog signaling pathway, was treated, but hedgehog signaling (Hedgehog signaling). The fluorescence intensity of the Smo protein involved in the signaling pathway was decreased compared to the control. In addition, as shown in FIG. 10 , when KAT2B was knocked down in NIH/3T3 cells, smoothened agonist (SAG), an activator of hedgehog signaling pathway, was treated, but hedgehog signaling (Hedgehog signaling). signaling pathway) and decreased mRNA expression level and protein expression level of Gli1.

The above results demonstrate that the process by which KAT2B induces acetylation of α-tubulin is related to IFT components and hedgehog signaling pathway.

Example 3. Evaluation of ciliary damage disease development through KAT2B knockout in an animal model

In order to confirm that the KAT2B protein and the gene encoding the KAT2B protein according to the present invention are biomarkers for diagnosing ciliary damage disease, KAT2B was knocked out in an animal model and glomerular cyst, a ciliary damage disease, was confirmed. .

11 to 13 , it was confirmed that the mRNA expression of KAT2B was reduced in KAT2B knockout mice, and the expression levels of IFT components and GLI1 were reduced. In addition, as shown in FIG. 14 , it was confirmed that glomerular cysts were formed in mice in which KAT2B was knocked out.

The above results confirmed in an animal model that KAT2B deficiency reduces the levels of IFT components and GLI1, and forms renal glomerular cysts. prove that it can be used.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (18)

A biomarker composition for diagnosing ciliary damage disease comprising a KAT2B protein or a gene encoding the KAT2B protein as an active ingredient. The diagnostic biomarker composition according to claim 1, wherein the ciliary injury disease is a kidney disease. According to claim 2, wherein the kidney disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic kidney failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome, A diagnostic biomarker composition, characterized in that at least one selected from the group consisting of Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, and hydronephrosis. A kit for diagnosing ciliary damage disease comprising, as an active ingredient, an agent capable of detecting the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein. According to claim 4, wherein the agent capable of measuring the expression level of the protein is an antibody, peptide, aptamer or compound that specifically binds to the protein; The agent capable of measuring the expression level of the gene is specific for the gene. A kit for diagnosing cilia damage disease, characterized in that it is a primer or probe that binds positively. A method for providing information for diagnosing a ciliary injury disease, comprising comparing the expression level of the KAT2B protein, or the expression level of the KAT2B protein-encoding gene, from a sample isolated from a specimen and a normal control. A biomarker composition for prognosing the prognosis of ciliary injury disease comprising a KAT2B protein or a gene encoding the KAT2B protein as an active ingredient. The method of claim 7, wherein the ciliary injury disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic renal failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome , Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, and hydronephrosis, characterized in that at least one selected from the group consisting of a biomarker composition for predicting the prognosis of cilia damage disease. A kit for prognosing the prognosis of cilia damage disease, comprising as an active ingredient an agent capable of detecting the expression level of the KAT2B protein or the expression level of the gene encoding the KAT2B protein. 10. The method of claim 9, wherein the ciliary damage disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic kidney failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome , Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, and hydronephrosis kit for prognosis prediction of ciliary damage disease, characterized in that at least one selected from the group consisting of. A first step of measuring the expression level of the KAT2B protein, or the expression level of the gene encoding the KAT2B protein in the biological sample isolated from the tissue of the subject; And A second step of comparing the expression level of the protein with a biological sample isolated from a normal tissue; Information providing method for predicting the prognosis of cilia damage disease comprising a. The method of claim 11, wherein the ciliary injury disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic renal failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome , Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, and hydronephrosis, characterized in that at least one selected from the group consisting of an information providing method for predicting the prognosis of ciliary injury disease. A pharmaceutical composition for preventing or treating cilia damage disease, comprising KAT2B protein or an activity promoter of KAT2B protein as an active ingredient. The method of claim 13, wherein the ciliary injury disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic kidney failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome , Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, and a pharmaceutical composition for the prevention or treatment of ciliary damage disease, characterized in that at least one selected from the group consisting of hydronephrosis. The cilia damage disease according to claim 13, wherein the KAT2B protein activity promoter is an antibody, peptide, aptamer or compound that specifically binds to the KAT2B protein, and promotes the binding of the KAT2B protein and ciliary alpha-tubulin. A pharmaceutical composition for the prevention or treatment of [Claim 14] The pharmaceutical composition for preventing or treating cilia damage disease according to claim 13, wherein the KAT2B protein induces acetylation of cilia alpha-tubulin. A first step of selecting a disease subject deficient in expression of the KAT2B protein or the gene encoding the KAT2B protein by measuring the expression level of the KAT2B protein and the expression level of the gene encoding the KAT2B protein in the biological sample isolated from the tissue of the subject; And When the expression level of the KAT2B protein, or the expression level of the gene encoding the KAT2B protein increases after treatment with the test substance compared to the disease subject not treated with the test substance, the second judged as a prophylactic or therapeutic agent for ciliary damage disease A screening method of an agent for the prevention or treatment of ciliary damage disease, comprising a step. 18. The method of claim 17, wherein the ciliary injury disease is polycystic kidney, glomerular cyst, nephrotic syndrome, kidney cancer, chronic renal failure, diabetic kidney disease, acute pyelonephritis, acute renal failure, hypertensive kidney disease, Reye's syndrome, gout, Sjogren's syndrome , Behcet's disease, lupus, candidiasis, nephrotic hemorrhagic fever, leptospirosis, legionellosis, and hydronephrosis, characterized in that at least one selected from the group consisting of ciliary damage disease screening method for preventing or treating agents.

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