KR20220020534A - A Composition for Preventing or Treating Epithelial Barrier Dysfunction Comprising a TRIM40 inhibitor as an Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating epithelial barrier dysfunction and a composition for enhancing epithelial barrier or cell membrane permeability. The present invention efficiently restores a barrier function of the epithelial barrier by restoring a denatured cytoskeleton and an intercellular contact in individuals with reduced epithelial barrier function, thereby eliminating a root cause of etiology away from the conventional symptomatic treatment focused on controlling inflammation, etc. The present invention can also be advantageously used as a drug delivery system which assists in the efficient delivery of pharmacological components by improving the permeability of the epithelial barrier of a target tissue or the cell membrane of a target cell. In addition, the present invention can be applied to a composition for inhibiting cancer metastasis which effectively blocks cancer metastasis by inhibiting the progression of epithelial-mesenchymal transition (EMT) by enhancing the epithelial properties of tumor epithelial cells of primary cancer tissues.

Description

A composition for preventing or treating epithelial barrier insufficiency comprising a TRIM40 inhibitor as an active ingredient {A Composition for Preventing or Treating Epithelial Barrier Dysfunction Comprising a TRIM40 inhibitor as an Active Ingredient}

The present invention relates to a method for restoring the epithelial barrier function by inhibiting the activity or expression of TRIM40 protein.

Epithelial tissue is found in both vertebrates and invertebrates and plays an important role in the physiology of organisms. The epithelial cells that make up the epithelial tissue form a strong intercellular bond by enclosing both the outside and inside of the body's cavity and lumen like a membrane, thereby insulating between the inside and outside pores of the body and preventing the inflow of microorganisms, etc. It forms epithelial barriers that block it. The epithelial barrier functions not only as a defense, but also as a means of regulating the movement of substances so that the body can selectively absorb and excrete specific substances. As such, the epithelial barrier and epithelial cells perform various physiological functions such as chemical absorption and nutrient absorption, intercellular transport, sensory sensing, excretion, and blocking the entry of pathogens. can proceed.

On the other hand, the intestine is an organ in which the host and microorganisms maintain homeostasis, and intestinal homeostasis is achieved through intestinal epithelial cells. The intestinal epithelial barrier formed by interconnecting intestinal epithelial cells through the cytoskeleton prevents excessive immune response by blocking the influx of intestinal microbes into the lamina propria, where the intestinal immune cells are located. A representative disease caused by an excessive immune response in the intestine is inflammatory bowel disease (IBD), and although many studies have suggested risk factors related to inflammatory bowel disease, specific The genetic factors and their mechanisms of action are little known.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosures of the 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.

Non-Patent Document 1. Keita Noguchi er al., J. Immunotoxicol 13(5):738-744

The present inventors have made intensive research efforts to develop an effective and fundamental therapeutic composition for various epithelial diseases caused by skeletal deformation of epithelial cells and consequent loss of the epithelial barrier barrier function. As a result, the TRIM40 protein is highly expressed in individuals with reduced epithelial barrier function, and when the expression or activity of TRIM40 is suppressed, the denatured cytoskeletal structure is restored and the cell-to-cell contact is improved, and the epithelial barrier function is efficiently restored. By discovering, the present invention was completed.

Accordingly, an object of the present invention is to provide a composition for preventing or treating epithelial barrier dysfunction.

Another object of the present invention is to provide a composition for enhancing the epithelial barrier or cell membrane permeability.

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 one aspect of the present invention, the present invention provides a composition for preventing or treating epithelial barrier dysfunction comprising a Tripartite Motif Containing 40 (TRIM40) inhibitor as an active ingredient.

The present inventors have made intensive research efforts to develop an effective and fundamental therapeutic composition for various epithelial diseases caused by skeletal deformation of epithelial cells and consequent loss of the epithelial barrier barrier function. As a result, the TRIM40 protein is highly expressed in individuals with reduced epithelial barrier function, and when the expression or activity of TRIM40 is suppressed, the denatured cytoskeletal structure is restored and the cell-to-cell contact is improved, and the epithelial barrier function is efficiently restored. found

As used herein, the term “epithelial barrier insufficiency” refers to a selective barrier function ( It is meant to encompass a series of diseases caused by loss of the selective barrier function. Loss of epithelial barrier function causes various diseases such as, for example, various infectious diseases, inflammatory diseases, autoimmune diseases due to infiltration of pathogens, skin aging due to decreased contact between epithelial cells, hypertrophic scars and keloids.

As used herein, the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of TRIM40 protein, whereby the activity or expression of TRIM40 becomes undetectable or is present at an insignificant level, as well as cells caused by TRIM40. It refers to a substance that reduces the activity or expression of TRIM40 to the extent that skeletal degeneration and inhibition of intercellular contact can be significantly improved.

As used herein, the term “reduction in expression” may refer to a state in which, for example, the expression level of TRIM40 is reduced by 20% or more compared to the control group, more specifically reduced by 30% or more, and even more specifically reduced by 40% or more. there is.

As used herein, the term “reduction in activity” refers to a significant measurable decrease in the intrinsic function of TRIM40 in vivo compared to a control, specifically, significantly improving or restoring the cytoskeleton and intercellular contact in the subject. It means that the activity of TRIM40 is reduced to the extent possible. A decrease in activity includes not only a simple decrease in function, but also an eventual inhibition of activity due to a decrease in stability.

Inhibitors of TRIM40 include, for example, shRNA, siRNA, miRNA, ribozyme, peptide nucleic acids (PNA), which inhibit the expression of TRIM40 protein at the gene level, whose amino acid sequence and encoding nucleotide sequence are already known in the art; Antisense oligonucleotides, CRISPR systems including guide RNA for recognizing target genes, and antibodies or aptamers that inhibit at the protein level, as well as small molecule compounds, peptides and natural products that inhibit their activity, but are not limited thereto All possible means of inhibition at the gene and protein level can be used.

According to a specific embodiment of the present invention, the TRIM40 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to the TRIM40 protein; or an aptamer that specifically binds to the TRIM40 protein.

According to the present invention, the TRIM40 inhibitor of the present invention may be a TRIM40-specific antibody that inhibits TRIM40 activity at the protein level. The antibody specifically recognizing TRIM40 is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

Antibodies of the present invention can be prepared by methods commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA methods (U.S. Pat. No. 4,816,567). ) or phage antibody library methods (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). . General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984.

As used herein, the term “antigen binding fragment” refers to a portion of a polypeptide capable of binding an antigen in the entire immunoglobulin structure, for example, F(ab')2, Fab', Fab, Fv and scFvs, but are not limited thereto.

As used herein, the term "specifically binding" has the same meaning as "specifically recognizing", and an antigen and an antibody (or a fragment thereof) interact specifically through an immunological reaction. means to do

The present invention can also inhibit its activity by using an aptamer that specifically binds to TRIM40 instead of an antibody. As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target material with high affinity and specificity. For general information on aptamers, see Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

More specifically, the antibody or aptamer specifically binds to the amino acid sequence of SEQ ID NO: 1 .

According to the present invention, the amino acid sequence of SEQ ID NO: 1 is a fragment consisting of 99 consecutive C-terminal residues of the TRIM40 protein.

According to a specific embodiment of the present invention, the TRIM40 inhibitor is a nucleic acid molecule that inhibits the expression of a nucleotide encoding a TRIM40 protein.

As used herein, the term “nucleic acid molecule” has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, are not only natural nucleotides, but also analogs with modified sugar or base sites. (analogue) (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

As used herein, the term “nucleic acid molecule that inhibits expression” specifically recognizes a target gene by including a complementary nucleic acid sequence that can hybridize with the target gene, and may cause a modification in the nucleotide structure that causes a decrease in its function. It refers to a nucleic acid molecule that has, for example, the above-described shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, includes gRNA included in the CRISPR system.

As used herein, the term “complementary” means that a nucleic acid molecule for expression inhibition is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under predetermined annealing or hybridization conditions, and is substantially complementary and completely complementary (perfectly complementary) has a meaning encompassing all things, and preferably means completely complementary. As used herein, the term “substantially complementary sequence” includes not only a completely identical sequence, but also a sequence that is partially mismatched with the sequence to be compared within the range in which sequence-specific hybridization can occur by annealing to a specific sequence. am.

As used herein, the term “shRNA (small hairpin RNA)” is a single strand of 50-70 nucleotides forming a stem-loop structure in vivo . An RNA sequence that creates a tight hairpin structure. Usually, a long RNA of 19-29 nucleotides is complementary to both sides of a loop region of 5-10 nucleotides to form a double-stranded stem. It is transduced and is usually passed on to daughter cells so that suppression of target gene expression is inherited.

As used herein, the term “siRNA” refers to a short double-stranded RNA capable of inducing an RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, blunt ends or cohesive ends Both ends are possible. The structure of the adhesive end can be both a structure in which the three ends protrude and a structure in which the five ends protrude.

As used herein, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is not expressed in cells, has a short stem-loop structure, and inhibits the expression of a target gene through complementary binding to the mRNA of the target gene. do.

As used herein, the term “ribozyme” is a type of RNA and refers to an RNA molecule having the same function as an enzyme that recognizes the base sequence of a specific RNA and cuts it by itself. A ribozyme is a complementary nucleotide sequence of a target mRNA strand and consists of a region that binds with specificity and a region that cuts the target RNA.

As used herein, the term “peptide nucleic acid (PNA)” refers to a molecule capable of complementary binding to DNA or RNA while having both nucleic acid and protein properties. PNA is not found in nature but is artificially synthesized by a chemical method, and forms a double strand through hybridization with a natural nucleic acid of a complementary base sequence to control the expression of a target gene.

As used herein, the term “antisense oligonucleotide” refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and is translated into protein, translocation into the cytoplasm, maturation, or any other It refers to a nucleic acid molecule that inhibits an essential activity for an overall biological function. Antisense oligonucleotides may be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). . The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic sugarscholphonates, and the like.

As used herein, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and specifically cuts the recognized loci by inducing a nuclease. A typical example of such a gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.

The above-described nucleic acid molecule of the present invention can inhibit the expression of TRIM40 at the gene level by expressing it in a subject with epithelial barrier dysfunction.

As used herein, the term “express” means that a gene is transferred into a subject cell by artificially introducing it using a gene delivery system to allow the subject to express an exogenous gene or to increase the natural expression level of an endogenous gene. It means becoming capable of replication as an extrachromosomal factor or by the completion of chromosomal integration. Thus, the term “expression” is synonymous with “transformation”, “transfection” or “transduction”.

As used herein, the term “gene delivery system” refers to any means of delivering a gene into a cell, and gene delivery has the same meaning as intracellular transduction of a gene. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.

More specifically, the nucleic acid molecule specifically binds to the nucleotide sequence of SEQ ID NO:2.

According to the present invention, the nucleotide sequence of SEQ ID NO: 2 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 .

It is clear to those skilled in the art that the nucleotide sequence whose expression is inhibited in the present invention is not limited to the nucleotide sequence shown in the accompanying sequence listing.

A mutation in a nucleotide may not result in a change in the protein. Such nucleic acids include functionally equivalent codons or codons encoding the same amino acid (e.g., due to codon degeneracy, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids It includes nucleic acid molecules that

In consideration of the above-described variation having biological equivalent activity, it is construed that the nucleic acid molecule of the present invention includes a sequence exhibiting substantial identity to the sequence described in the sequence listing. The substantial identity is at least 60% when the sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. means a sequence that exhibits homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx.

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject who has never been diagnosed with a disease or disease, but is likely to have the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, condition or symptom. When the composition of the present invention is administered to a subject, by inhibiting the activity or expression of TRIM40 protein, the denatured cytoskeleton and the cell-to-cell contact are restored, thereby inhibiting the development of symptoms due to loss of epithelial barrier function, eliminating it, or alleviating it do Accordingly, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients and applied as a therapeutic adjuvant for the above diseases. Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

As used herein, the term “administration” or “administering” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.

In the present invention, the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological component in the composition is sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered. prophylactically effective amount”.

As used herein, the term “subject” includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

According to a specific embodiment of the present invention, the epithelial barrier dysfunction is inflammatory bowel disease, pancreatitis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute respiratory distress syndrome (ARDS), chronic bronchitis, chronic rhinitis, It is selected from the group consisting of asthma, skin aging and vesicular exfoliation.

More specifically, the inflammatory bowel disease is selected from the group consisting of crohn's disease, ulcerative colitis and behcet's disease.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and specifically may be administered orally, intravenously, subcutaneously or intraperitoneally.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a functional food composition for improving epithelial barrier dysfunction comprising a TRIM40 inhibitor as an active ingredient.

Since the TRIM40 inhibitor used in the present invention and epithelial barrier insufficiency in which symptoms are improved or alleviated by using the same have already been described above, the description thereof will be omitted to avoid excessive overlap.

When the composition of the present invention is prepared as a food composition, it may include not only the compound of the present invention as an active ingredient, but also carbohydrates, seasonings and flavoring agents that are commonly added during food production. Examples of carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol, but are not limited thereto. As the flavoring agent, natural flavoring agents (taumatine, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, citric acid, high fructose, sugar, glucose, acetic acid, malic acid, fruit juice, licorice root extract, jujube extract, licorice extract, etc. are added in addition to the pine bark extract, which is the active ingredient of the present invention. can be included as

According to another aspect of the present invention, one or more selected from the group consisting of TRIM40 protein or functional partial fragment thereof, nucleotide encoding TRIM40 protein or functional partial fragment thereof, TRIM40 protein activator and ROCK inhibitor is effective It provides a composition for enhancing epithelial barrier or cell membrane permeability comprising as a component.

The pharmacological efficacy of topically administered active ingredients is limited by the barrier function of the epithelial barrier. For most of these active ingredients, particularly hydrophilic pharmacological ingredients administered orally and entering the gastrointestinal tract, the absorption rate at the intestinal epithelial barrier determines the therapeutic effect, and various attempts have been made to improve the epithelial barrier permeability of these active ingredients. The present inventors have found that, since TRIM40 causes destabilization of cortical F-actin and cytoskeletal reorganization and makes cell-to-cell contact flexible, the permeability of the epithelial barrier or cell membrane is improved by enhancing the expression level or activity of TRIM40 in the target tissue, and ultimately It has been found that efficient delivery of pharmacological components can be induced.

As used herein, the term “activator of TRIM40 protein” refers to an active ingredient that enhances the expression level or activity of TRIM40 protein, for example, promotes expression of TRIM40 protein or has intrinsic biological activity or binding affinity with a ligand nucleic acid molecules, peptides, proteins, compounds and natural products that enhance the Accordingly, “TRIM40 protein activator” is used in the same sense as “TRIM40 protein agonist”.

As used herein, the term “ROCK inhibitor” refers to various natural or synthetic molecules that inhibit the expression or activity of rho-associated protein kinase (ROCK), for example, Y-27632, RKI-1447, GSK429286A and Y -30141, but is not limited thereto. Specifically, the ROCK inhibitor used in the present invention is Y-26732.

According to another aspect of the present invention, the present invention provides a composition for preventing or treating metastatic cancer comprising a Tripartite Motif Containing 40 (TRIM40) inhibitor as an active ingredient.

Since the TRIM40 inhibitor used in the present invention has already been described above, the description thereof is omitted to avoid excessive overlap.

As used herein, the term “metastatic cancer” refers to a new tumor formed while cancer cells separated from the primary tumor tissue penetrate into surrounding blood vessels or lymphatic vessels and move to other parts of the body through this passage. Since more than 90% of the causes of death of cancer patients are due to metastasis from primary cancer (Nature Reviews Cancer, 2006, 6:449-458), inhibiting cancer metastasis to improve the mortality of cancer patients is the It is as important as treatment.

The mechanism by which cancer cells acquire mobility in the metastasis process is EMT (epithelial to mesenchymal transition), in which tumor epithelial cells acquire the characteristics of mesenchymal cells through genetic mutation, and the reverse process, MET (mesenchymal transition) to epithelial transition) ( J Clin Invest . 2009, 119:1417-1419). In other words, epithelial cells with the characteristics of mesenchymal cells lose their original position due to weakened cell-to-cell bonding and move to the blood vessel, and the cells that migrated through the blood vessel recover their original epithelial characteristics (MET) and return to the primary site. It settles in a distant secondary site and causes the tumor to proliferate.

The present inventors have confirmed from various angles that epithelial properties are restored while the close contact between the cytoskeleton and cells of epithelial cells is strengthened through inhibition of TRIM40 expression or activity. It was found that when it does, it can inhibit the progression of EMT and block the generation of circulating tumor cells (CTCs).

According to another aspect of the present invention, the present invention provides a composition for preventing or treating cancer in which circulating tumor cells (CTC) are generated comprising an activator of Tripartite Motif Containing 40 (TRIM40) as an active ingredient do.

The present inventors have found that it is effective to inhibit the generation of circulating tumor cells that travel through the bloodstream in order to block the progression of EMT, which is the cause of cancer metastasis. We found that artificially persisting the mesenchymal phenotype to prevent engraftment at the secondary site was more effective in inhibiting metastasis. Therefore, when the presence of CTCs in vivo is detected, administration of a TRIM40 activator can inhibit epithelialization and colonization of pre-generated circulating tumor cells and ultimately effectively block the generation of metastatic cancer.

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

(a) The present invention provides a composition for preventing or treating epithelial barrier dysfunction, and a composition for enhancing epithelial barrier or cell membrane permeability.

(b) The present invention provides an effective restoration of the barrier function of the epithelial barrier by restoring the denatured cytoskeleton and intercellular contact in an individual with reduced epithelial barrier function, thereby departing from the conventional symptomatic treatment focused on inflammation control, etc. Phosphorus removal can be achieved.

(c) The present invention can also be usefully used as a drug delivery system that aids in the efficient delivery of pharmacological components by improving the epithelial barrier of the target tissue or the cell membrane permeability of the target cell.

(d) In addition, the present invention can be applied as a composition for inhibiting cancer metastasis that effectively blocks metastasis of cancer by inhibiting the progression of epithelial-mesenchymal transition (EMT) by enhancing the epithelial properties of tumor epithelial cells of primary cancer tissues.

1 is a diagram showing that TRIM40 is involved in the onset of IBD through changes in actin cytoskeletal signaling. 1A is an RNA-seq analysis of UC-related gene expression profiles obtained from published RNA-sequencing (RNA-seq) data sets of UC patients. Green, yellow, blue, brown and black dots represent REG, motility, MMP, chemokine and TRIM protein genes, respectively. Figure 1b is a diagram showing that TRIM40 is increased in UC and CD patients. TRIM40 expression was analyzed by RT-PCR, and quantitative values of TRIM40 mRNA levels were normalized to GAPDH. 1c is a diagram showing that TRIM40 expression is hardly detected in colonic and intestinal epithelial cell lines. SW480 (colon), HT-29 (colon) and Caco-2 (intestine), HeLa (cervical), U937 (lymphocyte), MDA-MB-231 (breast), 293T (embryonic kidney) in various human cell lines. The expression of TRIM40 was analyzed by RT-PCR.
2 is a diagram showing that TRIM40 overexpression promotes expression of inflammatory genes or cytoskeleton-related genes. 2A is a Venn diagram analysis result showing that increased genes and decreased genes significantly overlap as a result of microarray and RNA-seq dataset analysis. 2B is a heat map analysis result showing significant differences in mRNA expression levels of cytokines, chemokines, IFNs or cytoskeleton-related genes in Myc-TRIM40-expressing HT-29 cells. Figure 2c is a diagram showing that TRIM40 overexpression induces mRNA expression of chemokines, cytokines and IFN- or cytoskeleton-related proteins. The mRNA expression of each gene was analyzed by qPCR. *P<0.05, **P<0.01 (Student's t-test), ISP40 (intestine specific protein 40) means TRIM40.
3 is a diagram showing that TRIM40 overexpression inhibits cell-to-cell contact. 3a is It shows a fibrous pattern of TRIM40 in the cytoplasm. HT-29 or Caco-2 cells stably expressing the empty vector or Myc-TRIM40 were stained with anti-Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. Arrowheads indicate fibrous patterns. Figure 3b shows HT-29 or Caco-2 cells expressing the empty vector or Myc-TRIM40 were analyzed by differential interference (DIC). Scale bar, 10 μM. The graph on the right is the result of quantifying the distance between cells. Edge-to-edge distances were measured by obtaining surface-reflection interfering DIC images that clearly indicate cell boundaries. Red, white and yellow lines indicate the boundaries of each cell. **P<0.01 (Student's t-test).
4 is a diagram showing that TRIM40 overexpression promotes destabilization of cortical F-actin. Figure 4a shows that HT-29 and SW480 cells stably expressing empty vector or TRIM40 were tagged differently (Myc, Flag, and tagged N-terminus and C-terminus) and anti-F-actin (phalloidin) or anti- It was stained with Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. Arrow heads indicate cortical actin. Figure 4b is a diagram showing that TRIM40 is involved in the regulation of the F-actin polymerization step. HT-29 cells were treated with Cyto.D and PMA to induce F-actin depolymerization or condensation. 4c is a diagram showing that TRIM40 reduces the F-actin/G-actin ratio. The graph shows the F-actin band intensity in the insoluble fraction normalized to the G-actin band intensity in the soluble fraction. **P<0.01 (Student's t-test). ISP40 stands for TRIM40.
Figure 5 is a diagram showing that TRIM40 promotes the incorporation of AJ and TJ through cortical actin destruction. FIG. 5A shows the results of staining HT-29 cells stably expressing a control empty vector or Myc-TRIM40 with anti-vinculin, anti-E-cadherin, anti-integrin, anti-CD44 or anti-Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. Figure 5b is a diagram showing that TRIM40 overexpression does not affect the protein expression level of AJ and TJ. Empty vector or HT-29 cells expressing Myc-TRIM40 were immunoblotted with the indicated antibodies. ISP40 stands for TRIM40.
6 is a diagram showing that both the RING and C-terminal domains of TRIM40 are important for inhibition of F-actin stabilization and cell-to-cell contact. Figure 6a shows TRIM40 and its RING deletion (TRIM40ΔRING); C-terminal region deletion (TRIM40ACT); coiled-coil and C-terminal domain deletion (TRIM40ΔCC+CT); and B-box, coiled-coil and C-terminal domain deletion (TRIM40ΔBB+CC+CT) mutations. HT-29 cells expressing wild-type TRIM40 or a mutant thereof were stained with anti-phalloidin or anti-Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. Figure 6b shows the result of measuring the edge-to-edge distance by obtaining a surface reflection interference DIC image clearly showing the cell boundary. Red, white and yellow lines indicate the boundaries of each cell. **P<0.01 (Student's t-test). 6c shows the measurement result of the intercellular distance. ISP40 stands for TRIM40.
7 is a diagram showing that TRIM40 regulates the activity of cofilin-1 by blocking phosphorylation. 7A is a diagram showing the levels of propylin-1 and cofilin-1 proteins in control and TRIM40-overexpressing HT-29 cells. Lysates of empty vector or Myc-TRIM40 expressing HT-29 cells were subjected to immunoblotting with anti-propylin-1, anti-cofilin-1, anti-Myc or anti-GAPDH antibodies. Figure 7b is a diagram showing that TRIM40 overexpression triggers the dephosphorylation of cofilin-1. Lysates of empty vector or Myc-TRIM40 expressing HT-29 cells were subjected to immunoblotting with anti-phospho-cofilin-1, anti-cofilin-1, anti-Myc or anti-GAPDH antibodies. Figure 7c is a diagram showing that TRIM40 overexpression rearranges the cytoplasmic fibrous pattern near the nucleus. Empty vector or Myc-TRIM40 expressing HT-29 cells were stained with anti-cofilin-1 or anti-Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. Figure 7d is a diagram showing that TRIM40 directly interacts with cofilin-1. Lysates of empty vector or Myc-TRIM40-expressing HT-29 cells were immunoprecipitated with anti-HA antibody together with HA-cofilin and immunoblotted with anti-Myc or anti-HA antibody. Figure 7e is a diagram showing that both RING and C-terminal domains are essential for binding between cofilin-1 and TRIM40. HA-cofilin-1-expressing HT-29 cells stably expressing wild-type TRIM40 or a deletion mutant thereof were lysed and immunoprecipitated with anti-Myc or anti-HA antibody. ISP40 stands for TRIM40.
8 is a diagram showing that TRIM40 is a novel ubiquitin ligase for RhoA that promotes F-actin destabilization. 8A and 8C show that TRIM40 overexpression did not affect the protein expression level of PP2A/C or LIMK1. Immunoblots were performed on the empty vector or lysates of HT-29 cells expressing Myc-TRIM40 with anti-PP2A/C antibody. 8B is a diagram showing the effect of cofilin-1-PP2A interaction in TRIM40 overexpressing HT-29 cells. HA-cofilin-1-expressing HT-29 cells stably expressing the empty vector or Myc-TRIM40 were lysed, followed by immunoprecipitation with anti-PP2A/C antibody and immunoblotting with anti-Myc or anti-HA. did Fig. 8d is a diagram showing that TRIM40 overexpression reduces LIMK1 phosphorylation, showing cofilin-1-PP2A interaction in TRIM40 overexpressing HT-29 cells. HA-LIMK1-expressing HT-29 cells stably expressing either the empty vector or Flag-TRIM40 were lysed and immunoblotted with anti-phospho-LIMK1, anti-cofilin-1, anti-Myc or anti-tubulin antibodies. . FIG. 8e is a diagram showing F-actin depolymerization and intercellular contact disruption in HT-29 cells treated with Y-26732 and not treated with FRAX597. HT-29 cells treated with ROCK or PAK inhibitor (Y-26732 or FRAX597) were stained with anti-phaloidin or anti-Myc antibody. Nuclei were stained with DAPI (blue). Scale bar, 10 μM. 8D is a diagram showing that TRIM40 degrades RhoA-GTPase. Lysates of empty vector or HT-29 cells expressing Myc-TRIM40 were immunoblotted with anti-RhoA, anti-Myc or anti-tubulin antibodies. Figure 8f is a diagram showing that TRIM40 overexpression affects RhoA levels. ISP40 stands for TRIM40.
9 is a diagram showing an increase in TRIM40 expression in DSS-induced colitis mice. Figure 9a in the mouse The process of inducing colitis with DSS is shown. Colitis was induced by administration of ≥2.5% DSS to control male and female mice for 4 or 8 days. 9B shows the results of measuring body weight and colon length of male or female mice in the DSS-administered control group. 9c and 9d show the results of measurement of body weight and colon length in DSS-administered control group and TRIM40 deficient mice. 9e is a diagram showing that the mRNA expression level of TRIM40 is increased in mice with DSS-induced IBD colitis. The mRNA expression level of TRIM40 was analyzed by qPCR. *P<0.05, **P<0.01 (Student's t-test). FIG. 9f is a diagram showing that the mRNA level of TRIM40, cytoskeleton-related genes, chemokines, cytokines, MMP, REG and motility-related genes significantly increased as a result of RNA-sequencing of DSS-induced colitis mice. ISP40 stands for TRIM40.
Figure 10 is a diagram depicting the predicted pathway by which TRIM40 destabilizes cortical F-actin and lowers intestinal barrier function.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

mouse

TRIM40+/- mice of C57BL/6N genetic background were purchased from Cyagen (Taicang City, Jiangsu, China). C57BL/6N male and female mice aged 5-8 weeks were purchased from ORIENT (Seongnam, South Korea). TRIM40 knockout mice were constructed by crossing TRIM40 +/- male and female mice. The genotype of knockout mice was confirmed by PCR. For the experiment, 6-8 week old mice were used, and all mice were maintained under SPF (specific pathogen free) conditions. All animal experiments were performed under the approval of the Animal Experimental Ethics Committee of Yonsei University.

DNA construct

Human TRIM40 fused with N-terminal Myc tag was subcloned into pMSCV vector (Clontech, Mountain View, CA, USA) using the following primers: 5' ATT CTC GAG ATG ATC CCT TTG CAG AAG GAC 3' (XhoI -TRIM40; forward) and 5'-ATT GAA TTC AGA GCT TCT GAG GGG G-3' (EcoRI-TRIM40; reverse). N-terminal Flag-tagged human TRIM40 was subcloned into pMSCV vector using the following primers: 5'-ATT AGA TCT ATG ATC CCT TTG CAG AAG GAC-3' (BglII-TRIM40; forward) and 5'- ATT GAA TTC AGA GCT TCT GAG GGG G-3' (EcoRI-TRIM40; reverse). C-terminal GFP-tagged human TRIM40 was subcloned into pMSCV vector using the following primers: 5'-ATT CTC GAG CCA CCA TGA TCC CTT TGC AGA AGG ACA AC-3' (XhoI-TRIM40; forward) and 5'-AAT GGA TCC GAG CTT CTG AGG GGG CTG AAG-3' (BamHI-TRIM40; reverse). C-terminal Myc-tagged human TRIM40 was subcloned into pMSCV vector using the following primers: 5'-AAT AGA TCT GCC ACC ATG ATC CCT TTG-3' (BglII-TRIM40; forward) and 5'-ATT GTT AAC CTC GAG CTT CTG AGG GGG CTG AA-3' (HpaI-TRIM40; reverse). All human TRIM40 related deletion constructs were tagged with Myc at the C-terminus. The human TRIM40 deletion construct was constructed by PCR and cloned into the pMSCV vector. The human TRIM40 construct with the RING domain deleted (Δ14-66aa) was cloned into a vector using the following primers: 5'-AAT AGA TCT GCC ACCATG ATC CCT TTG CAG AAG GAC AAC CAG GAG GAG GGT GTC CGG AAG CCC TGT TCT GA-3' (BglII-TRIM40; forward) and 5'-ATT GTT AAC CTC GAG CTT CTG AGG GGG CTG AA-3' (HpaI-TRIM40; reverse). The Δ61-258aa deleted human TRIM40 construct was cloned into a vector using the following primers: 5'-AAT AGA TCT GCC ACC ATG ATC CCT TTG-3' (BglII-TRIM40; forward) and 5'-AAT CTC GAG ACA GGG CTT CCG GCA GA-3' (XhoI-TRIM40; reverse). The Δ108-258 deleted human TRIM40 construct was cloned into a vector using the following primers: 5'-AAT AGA TCT GCC ACC ATG ATC CCT TTG-3' (BglII-TRIM40; forward) and 5' AAT CTC GAG AAT GGT CAG TTC ATG ATG AGA C 3' (XhoI-TRIM40; reverse). The Δ153-258aa deleted human TRIM40 construct was cloned into a vector using the following primers: 5'-AAT AGA TCT GCC ACC ATG ATC CCT TTG-3' (BglII-TRIM40; forward) and 5'-AAT CTC GAG AGC CTC CAG CCT GTG GT-3' (XhoI-TRIM40; reverse). Human cofilin-1 cDNA was isolated by PCR using the following primers using the cDNA library of HT-29 cells: 5'-ATT AGA TCT GCC ACC ATG GCC TGG GGT GTG GCT-3'(BglII -TRIM40; forward) and 5'-ATT CTC GAG CAA AGG CTT GCC CTC CAG-3' (XhoI-TRIM40; reverse). Human LIMK1 was obtained from addgene (Plasmid #23511). Human LIMK1 was subcloned into pMSCV vector using the following primers: 5'-CTA AGA TCT GCC ACC ATG AGG TTG ACG CTA CTT TG-3' (Bgl ll-TRIM40; forward) and 5'-ACC GTT AAC GTC GGG GAC CTC AGG-3' (HpaI-TRIM40; reverse). All constructs were verified through sequencing.

Antibodies and reagents

Anti-Myc (#2276), anti-E-cadherin (#14472), anti-Ezrin/Radixin/Moesin (#3142), anti-Phospho-cofilin (Ser3) (#3313), anti-propylin ( Profilin)1 (#3237), anti-LIMK1 (#3842), anti-Phospho-LIMK1(Thr508)/LIMK2(Thr505) (#3841) and anti-DYKDDDDK tagged (#8146) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-cofilin antibodies were obtained from Abcam (Cambridge, UK), anti-β-catenin (sc-7963), anti-HCAM (sc-7297), anti-integrin β1 (sc-53711), anti-Rho A (sc). -418), anti-Tom20 (sc-11415) and anti-tubulin (sc-23948) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Anti-GAPDH (MA5-15738) and anti-occludin (71-1500) antibodies were purchased from Thermo Fisher Scientific (Fremont, CA). Anti-PP2A C subunit (#05-421), anti-HA or anti-tubulin (#G094) and anti-Flag (G191) antibodies were obtained from Millipore (Billerica, MA, USA), Roche (Basel, Swizerland) or Each was purchased from Applied Biological Materials (Richmond, Canada). Vinculin (V9264, Sigma-Aldrich, St Louis, MO, USA) antibody was provided by Dr. Jae-Seok Roh (Yonsei University, South Korea). AlexaFluor 350-, 488- and 568-conjugated antibodies were purchased from Life Technologies (Carlsbad, CA, USA). AlexaFluor 568 conjugated phalloidin (A12380) and AlexaFluor 594-conjugated deoxyribonuclease I (D12372) were purchased from ThermoFisher Scientific (Fremont, CA). Cytochalasin D (22144-77-0) and PIPES (1,4-piperazindiethanesulfonic acid; P6757) were purchased from SIGMA-Aldrich (St Louis, MO, USA). Y-27632 2HClC (S1049) and FRAX597 (S7271) were purchased from Selleckchem (Houston, TX, USA).

Cell culture and transfection

HT-29 cells (HTB-38, American Type Culture Collection; ATCC, Manassas, VA, USA), a human colonic epithelial cell line, were treated with 10% heat inactivated fetal bovine serum (FBS) and penicillin/streptomycin (HyClone, Logan, UT, USA) containing RPMI. Human intestinal epithelial cell lines Caco-2 (HTB-37, ATCC) and human embryonic kidney (HEK) 293T cells (CRL03216, ATCC) were treated with Dulbecco's modified Eagle (DMEM) supplemented with 10% heat inactivated FBS and penicillin/streptomycin. medium) was cultured. All cells were cultured under a humidified atmosphere of 37° C., 5% CO ₂ . Cells were transfected in serum-free, antibiotic-free DMEM for 36-48 hours using Omicsfect (OmicsBio, Taipei City, Taiwan).

retroviral transfection

HEK293T cells were transfected with a retroviral vector containing the N-terminus or C-terminus of Myc- or Flag-tagged wild-type TRIM40, the C-terminus of the Myc-tagged TRIM40 deletion mutant, cofilin-1-HA or LIMK1-Myc; Together they were transfected with plasmids encoding VSV-G and Gag-Pol. After transfection, the virus particle-containing medium was collected 36-48 hours after transfection and filtered through a 0.45 μm membrane. After centrifugation at 2200 rpm for 45 minutes, the cells were transfected with the virus by culturing in a humidified atmosphere of 5% CO ₂ at 37° C. for 3-4 hours for 3-4 hours, followed by culturing again with fresh culture solution for 24 hours, followed by selection with puromycin.

RT-PCR, real-time RT-PCR and RNA sequencing analysis

Total cellular RNA was extracted using RNA prep kit (GeneAll, Seoul, South Korea), and 1-2 μg of total RNA was extracted using M-MLV (Money Murine Leukemia Virus) reverse transcriptase (Enzynomics, Daejeon, South Korea). Reverse transcription was performed with oligo dT (18mer) and random hexamer primers. RNA samples from UC or CD patients were provided by Professor Cheon-hee Cheon (Severance Biomedical Science Institute, Yonsei University College of Medicine, South Korea). PCR was performed using the appropriate cDNA and the indicated primers, and the PCR product was visualized on an ethidium bromide stained agarose gel. qPCR analysis was performed on a QuantStudio™ Design & Analysis system (Thermo Fisher Scientific) using TOPreal™ qPCR 2xPreMIX (Enzynomics, Daejeon, South Korea). All primer sequences used for RT-PCR and qPCR are shown in Table 1. RNA sequencing analysis was performed by Macrogen (Seoul, South Korea).

Immunofluorescence assay

Cells were fixed in 3.7% formaldehyde in PBS for 5-10 minutes at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 5-10 minutes. After blocking with 2% bovine serum albumin (PBA) in PBS for 30 min–1 h, cells were then incubated with an appropriate primary antibody (1:200–1:400) in 2% PBA for 1 h at room temperature or overnight at 4°C. cultured in The bound antibody was visualized by incubating with Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibody in 2% BSA for 1 hour at room temperature. DAPI (4',6-diamidino-2-phenylindole) was used for counterstaining of nuclei. For immunofluorescence staining of F-actin, cells were stained with Alexa Fluor™ 568 phalloidin (1:200-300) in 2% BSA at room temperature for 30 minutes-1 hour. For visualization of G-actin, cells were incubated with Alexa Fluor 594 labeled Dnase I (1:500) in 2% BSA for 1 hour at room temperature.

Immunoblot analysis

Cells were lysed using 1% nonylphenoxypolyethoxylethanol (NP-40, Sigma) supplemented with protease inhibitor cocktail (Roche) at 4°C for 30 minutes. After centrifugation at 13000 rpm for 10 minutes at 4° C., the supernatant was transferred to a new tube. The lysate was dissolved with 5 x sample buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a PVDF membrane (Milliopore, Bedford, MA). For blocking, the membrane was incubated with 5% skim milk in PBS containing 0.1% tween 20 (BioShop Canada Inc., Canada) at room temperature for 30 minutes-1 hour, and then the membrane was washed 3 times with PBST. The appropriate primary antibody on the membrane was incubated overnight at 4°C or at room temperature for 1 hour. The membrane was washed again 3 times with PBST and incubated in 5% skim milk in PBST with an appropriate horseradish peroxide-conjugated secondary antibody for 1 hour at room temperature. After 1 hour, the membrane was washed 3 times with PBST. Target bands were visualized using enhanced chemiluminescence (ECL) detection reagent (Advansta, Menlo Park, CA, USA).

co-immunoprecipitation

For cofilin 1-TRIM40 interaction, cells were incubated for 48 h and immediately sonicated in PBS supplemented with protease inhibitor cocktail. Cells were sonicated for 9 seconds on ice at 1 pulse/sec. The sonicated cells were centrifuged at 13000 rpm at 4°C for 10 minutes, and the supernatant was transferred to a new tube. An appropriate primary antibody was added to the supernatant and incubated at 4°C overnight, followed by protein G-Sepharose (Sigma-Aldrich) incubated at 4°C for 1 hour. After 1 hour, the supernatant containing protein G-Sepharose was centrifuged at 5000 rpm for 1 minute. The supernatant was discarded and the remaining protein G-sepharose was washed three times with PBS. Immunoprecipitated proteins were incubated in 1 x denaturing buffer (50 mM Tris-HCl [pH6.8], 5% β-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) at 95 °C for 7-10 min. It was eluted by boiling, and the eluted sample was separated by SDS-PAGE.

mitochondrial purification

Mitochondria and cytoplasmic fractions were isolated using the Mitochondria Isolation Kit for Cultured cells (#89874, Thermo Scientific, Rockford, IL, USA). Briefly, the collected 5 x 10 ⁶ cells were centrifuged and mitochondrial isolation reagent A was added to the pellet. Cells were vortexed for 5 seconds at medium speed and then incubated on ice for 2 minutes. Mitochondrial isolation reagent B was added and vortexed for 5 seconds at maximum speed, followed by incubation for 5 minutes on ice with vortexing every minute. Mitochondrial isolation reagent C was added and the tube was inverted several times to mix. After centrifugation at 3200 rpm at 4°C for 10 minutes, the supernatant was transferred to a new tube and centrifuged at 13000 rpm at 4°C for 15 minutes. The supernatant containing the cytoplasmic fraction was transferred to a new tube. Mitochondrial isolation reagent C was added to the pellet containing the mitochondrial fraction, centrifuged at 13000 rpm at 4°C for 5 minutes, and then the supernatant was discarded. The pellet was resuspended in 1x sample buffer and analyzed by immunoblotting.

Cytoskeletal fractionation

Cells were washed twice with PBS and incubated for 1 minute with cytoskeletal stabilization buffer (4M glycerol, 25mM PIPES (pH6.9), 1 mM EGTA, 1 mM MgCl ₂ ) at 4°C. Cells were lysed with cytoskeletal stabilization buffer containing 0.1% Triton X-100 and centrifuged at 7500 g for 5 minutes at 4°C, and then the supernatant (soluble actin) was transferred to a new tube. The pellet (insoluble actin) was resuspended in cytoskeletal stabilization buffer containing 0.1% TritonX-100. The supernatant and pellet were analyzed by immunoblotting.

statistical analysis

All experiments were repeated at least 3 times, and the data are expressed as mean and standard deviation (sd). Statistical differences between the two means were evaluated by two-tailed independent sample Student's t-test or Mantel-Cox log-rank test. The P value of the Venn diagram was calculated as a hypergeometric distribution. If the P value difference was less than 0.05, it was considered to be statistically significant. No sample was excluded from the analysis, the data were normally distributed, and similar variances were observed between groups. No statistical method was used to pre-determine the sample size, but rather based on previous experience and experimental variables.

Experiment result

TRIM40 is involved in IBD pathogenesis through changes in actin cytoskeletal signaling.

In previous studies ^3,4,31 of 200 gene loci or abnormal gene expression in inflammatory bowel disease (IBD), which consists of two major diseases, Crohn's disease (CD) and ulcerative colitis (UC), the The mechanism of action has not been clearly elucidated. Therefore, we reanalyzed the UC-related gene expression profile using a known RNA-sequencing (RNA-seq) dataset of UC patients. >79% of these reads aligned and >40,030 genes were included. As previously reported, the expression of the Reg (regenerating gene) family protein MMP (metalloprotease), chemokines or cytokines was greatly increased, whereas the expression of mucin (MUC) and adhesion-related proteins was decreased. Interestingly, the mRNA levels of several TRIM proteins (TRIM22, TRIM29, TRIM40 or ISO69) or cytoskeleton-related proteins (tubulin polymerization-promoting protein (TPPP), TLN (talin) or TUBAL3 (tubulin alpha like 3)) were significantly Since it has changed, the relative ranking of the genes corresponding to these RNAs was investigated (FIG. 1a). Several subsets of TRIM have been diversified to adapt to changing environmental conditions, such as the gut microbiome genome, and since TRIM40 is associated with gastrointestinal cancer, we focused on the role of TRIM40 in the pathogenesis of IBD. To validate the RNA-seq results, we investigated the expression level of TRIM40 mRNA in control and CD or UC patient samples. Compared to normal small intestine (ileum) tissue, CD and UC patients showed high TRIM40 mRNA levels (~56% higher than controls, FIG. 1B ). Likewise, human intestinal cell lines HT-29, Caco-2 and SW480 all showed very low TRIM40 expression (Fig. 1c). These results suggest that an increase in TRIM40 is involved in the pathogenesis of IBD.

To determine the effect of TRIM40 overexpression in the gut on pathogenesis, TRIM40 was performed by RNA-seq and microarray analysis in HT-29 cells stably expressing N-terminal Myc-tagged TRIM40 (Myc-TRIM40). The effect of gain of function on cellular mRNA properties was investigated. As a result of Venn diagram analysis, a significant overlap of increasing genes and decreasing genes was observed ( FIG. 2A ). As a result of KEGG (Kyoto Encyclopedia of Genes and Genomes) amplification analysis, it was found that significant changes in mRNA expression level were related to interferon (IFN) signal, interleukin (IL)-17 signal and chemokine signal, all of which are involved in the pathogenesis of IBD. factors (Fig. 2a, left graph). Interestingly, as a result of KEGG analysis, TRIM40 overexpression and PI3K (phosphoinositide 3 kinase) signal, STAT (signal transducer and activator of transcription) signal and Ras-related signal were found to have significant associations, which are factors deeply related to cytoskeletal reorganization. are (Fig. 2a, left graph). Furthermore, TRIM40 overexpression was significantly associated with the progression of IBD and several human cancers including gastric or colorectal cancer ( FIG. 2A , right graph). In particular, as a result of heat map analysis, Myc-TRIM40-expressing HT-29 cells showed that cytokines and chemokines [e.g., chemokines CXC motif ligands (CXCL) and chemokine ligands (CCL)], IFN-related genes (e.g., OAS ( oligoadenylate synthases), interferon-inducible proteins (IFI), interferon-stimulated genes (ISG) or interferon regulatory factor (IRF) or actin cytoskeleton-associated genes (e.g. TPPP3, troponin C1 (TNNC1), Rho-associated genes (ROCK1P1) Coiled-coil containing protein kinase 1) or MYL9 (myosin light chain 9)] and STAT1 (signal transducer and activator of transcription 1) showed significant differences (Fig. 2b).To confirm these results, TRIM40-expressing HT- As a result of examining the mRNA expression levels of STAT1 and IFI6 in 29 cells, these mRNA expression levels also significantly increased with TRIM40 overexpression ( FIG. 2c ).

To further investigate the biological function of TRIM40, the subcellular localization of TRIM40 in HT-29 and Caco-2 cells was analyzed. As a result of immunofluorescence assay (IFA), it was found that TRIM40 showed a fibrous pattern in the cytoplasm (FIG. 3a). Unlike the control HT-29 cells that tightly aggregated with each other, the TRIM40-expressing HT-29 cells exhibited intercellular repulsion, and the gap between neighboring cells widened and eventually dispersed ( FIG. 3b ). The average edge-to-edge distance between Myc-TRIM40-expressing cells was significantly greater than that of control HT-29 cells (Fig. 3b, right graph). As described above, cortical actin filaments are important for maintaining cell shape and maintaining the integrity of the intestinal epithelium, and cortical actin dysfunction is associated with the pathogenesis of IBD. In addition, it has recently been reported that the actin cytoskeleton plays an important role in innate immunity and cellular self-defense by detecting infectious microorganisms and operating a mechanism to eliminate them. Taken together, TRIM40 overexpression acts as a cause of IBD pathogenesis by increasing mRNA expression of IFN, cytoskeleton-related genes, and STAT1 and inhibiting cell-to-cell contact.

TRIM40 promotes the incorporation of AJ and TJ by disrupting cortical actin.

Since the conversion between G-actin monomer and fibrillar F-actin is involved in cell-to-cell contact and cell signaling pathways, the effect of TRIM40 on actin kinetics was investigated. Cortical F-actin was investigated through phalloidin staining in HT-29 cells expressing Mock or Myc-TRIM40. As expected, in control HT-29 cells, F-actin was significantly increased as a layer of the cytoskeletal network below the cell surface membrane (Fig. 4a, arrowhead). On the other hand, in TRIM40-overexpressing HT-29 cells, F-actin retreated from the membrane and accumulated near the nucleus, thereby promoting cortical actin decay and inhibiting cell-to-cell contact (FIG. 4a, second panel). No differences in F-actin stability due to different tags of TRIM40 (Myc, Flag and tagged N-terminal and C-terminal regions) were observed, suggesting that this was not due to tagging specificity or location (Fig. 4a, 3, 4 and 5 panels). Interestingly, HT-29 treated with cytochalasin D (Cyto. D) and PMA (phobol 12-myristate 13-acetate, activator of protein kinase C) induces F-actin depolymerization and contraction, respectively. It was similar to the pattern observed in cells, and it can be seen that TRIM40 was involved in regulating the F-actin polymerization process (FIG. 4b). As in the experiment using HT-29 cells, this pattern was also found in SW480 cells expressing TRIM40 (Fig. 4a, 6th panel). To confirm TRIM40-mediated depolymerization of F-actin, the G-actin/F-actin ratio was measured from the G- and F-actin fractions in control or Myc-TRIM40 expressing cells. As a result, the ratio was significantly reduced in TRIM40-expressing HT-29 cells (Fig. 4c). Through this, it was found that TRIM40 promotes F-actin depolymerization and inhibits cell-to-cell contact.

Cortical actin filaments are densely organized into dense cross-linked reticulum composed of numerous ABPs, which are essential for intestinal barrier function as they maintain the cell surface targeting AJ and TJ. Accordingly, the present inventors investigated the effect of TRIM40 overexpression on the localization of AJ and TJ in the plasma membrane. In Myc-TRIM40-expressing HT-29 cells, cell surface expression of AJ and TJ proteins including vinculin, E-cadherin, integrin β1 and CD44 was significantly reduced ( FIG. 5A ). Furthermore, intracellular plaques containing these AJs and TJs were clearly detected in TRIM40-overexpressing HT-29 cells (Fig. 5a, arrow). However, there was no difference between the control and TRIM40-overexpressing HT-29 cells in the total protein levels of these AJs and TJs (Fig. 5b). Taken together, these results suggest that TRIM40 promotes the incorporation of AJ and TJ from the cell surface through the breakdown of cortical actin mediated by F-actin depolymerization.

Both the RING and C-terminal domains of TRIM40 are important for inhibiting F-actin stability and cell-to-cell contact.

To identify the major domain of TRIM40 that inhibits F-actin polymerization, a TRIM40 mutant in which RING is deleted (TRIM40ΔRING); TRIM40 mutant with deletion of the C-terminal region (TRIM40ΔCT); TRIM40 mutant (TRIM40ΔCC+CT) with deletion of coiled-coil and C-terminal domains; and a TRIM40 mutant (TRIM40ΔBB+CC+CT) in which B-box, coiled-coil, and C-terminal domains are deleted, respectively (FIG. 6a). In TRIM40ΔRING-expressing cells, intercellular contact was partially impaired, but F-actin stabilization was not ( FIG. 6b , 3rd panel). Interestingly, in HT-29 cells expressing TRIM40ΔCT, TRIM40ΔCC+CT, or TRIM40ΔBB+CC+CT, in contrast to the wild-type, significant damage was observed in both cortical actin structure and cell-to-cell contact (Fig. 6b, 4th, 5th and 6th panels). ). As a result of measuring the distance between the corners of each mutant-expressing HT-29 cells, the average distance between the corners between the TRIM40ΔRING-expressing HT-29 cells was much larger than that of the control HT-29 cells, similar to the wild-type TRIM40 expressing cells ( FIG. 6c ). Taken together, it can be seen that the C-terminal variable region of TRIM40 is essential for promoting F-actin destabilization and inhibiting cell-to-cell contact, and the RING domain is involved in TRIM40-mediated cell-to-cell contact.

TRIM40 regulates its activity by blocking phosphorylation of cofilin-1.

To investigate the specific molecular mechanism by which F-actin is destabilized and cell-to-cell contact is inhibited by TRIM40, we focused on propylin-1 and cofilin-1 involved in F-actin polymerization and depolymerization, respectively. it was To this end, as a result of measuring the protein levels of propylin-1 and cofilin-1 in control and TRIM40-overexpressing HT-29 cells, no significant difference was found ( FIG. 7a ). However, since cofilin-1 is regulated by phosphorylation of serine 3, it was investigated whether TRIM40 affects cofilin-1 phosphorylation. Although total cofilin-1 levels were similar, phosphorylated cofilin-1 was hardly detected in TRIM40-overexpressing HT-29 cells, unlike control HT-29 cells ( FIG. 7B ). In addition, cofilin-1 ^8,23-25 distributed throughout the cytoplasm was redistributed in a cytoplasmic fibrous pattern near the nucleus, similar to the subcellular distribution of TRIM40 in TRIM40-overexpressing HT-29 cells (Fig. 7c). . This suggests the possibility that these two proteins interact with each other, and as a result of co-immunoprecipitation analysis, it was confirmed that TRIM40 actually strongly interacts with cofilin-1 (FIG. 7d). Interestingly, both the RING and C-terminal domains were important for interaction with cofilin-1, which reaffirms the IFA results showing that the TRIM40 deficient mutant does not properly inhibit F-actin depolymerization and intercellular contact. to confirm (FIGS. 7e and 6b). Through this, it can be seen that TRIM40 makes cofilin-1 a dephosphorylated active step and subsequently promotes F-actin depolymerization, and that TRIM40 RING and C-terminal domains are essential for binding to cofilin-1.

TRIM40 is a novel ubiquitin ligase for Rho A.

Since cofilin-1 phosphorylation at serine 3 is known to be dephosphorylated by PP2A ^19,32,33 , the present inventors attempted to investigate whether TRIM40 affects PP2A expression or PP2A-cofilin-1 interaction. To this end, immunoblotting using an anti-PP2AC subunit antibody was performed to measure endogenous PP2A expression, but no difference in PP2A expression level or PP2A-cofilin-1 interaction was observed between control and TRIM40-expressing HT-29 cells. did not (Figs. 8a and 8b). These results suggest the possibility that TRIM40 induces F-actin destabilization by being involved in the regulation of LIMK1, a key kinase that phosphorylates cofilin-1. Although the difference in LIMK1 expression level was insignificant, a significant decrease in phosphorylated LIMK1 was observed in TRIM40-expressing HT-29 cells, where TRIM40 inactivates LIMK1, resulting in increased dephosphorylated cofilin-1 and F-actin destabilization. It was found that (Figs. 8c and 8d).

Additionally, we investigated whether TRIM40 affects upstream signaling kinases ROCK and PAK. To this end, the pattern of F-actin destabilization in HT-29 cells treated with ROCK or PAK inhibitor (Y-26732 or FRAX597) was investigated. As expected, Y-26732-treated HT-29 cells showed clear F-actin depolymerization and cell-to-cell contact inhibition in a pattern similar to that of TRIM40-expressing HT-29 cells, but not in FRAX597-treated cells ( FIG. 8E ). This suggests that TRIM40 may be involved in regulating the activation of ROCK1 or its major upstream kinase, GTPase RhoA. Next, the effect of TRIM40 on the activity and stability of RhoA was investigated.

Surprisingly, TRIM40 reduced RhoA protein levels (Fig. 8f). Taken together, it can be seen that TRIM40 promotes RhoA degradation to reduce LIMK1 phosphorylation, thereby destabilizing F-actin. As a result, intercellular contact of intestinal epithelial cells is ultimately inhibited, resulting in the onset of IBD.

TRIM40 expression is significantly increased in DSS-induced colitis mice.

To investigate the effect of TRIM40 on F-actin destabilization-mediated cortical actin decay in vivo , a dextran sodium sulfate (DSS)-induced colitis mouse model was established. In summary, colitis was induced by administration of ≥2.5% DSS to control male or female mice for 4 or 8 days ( FIG. 9B ). As expected, control mice exhibited approximately 30% weight loss and decreased colon length, characteristic of IBD (Fig. 9b). When 3% DSS was added to drinking water and administered for 4 days, the TRIM40-deficient mice had a slight decrease in colon length, but there was no significant difference in body weight compared to the control group ( FIGS. 9c and 9d ). Interestingly, the mRNA level of TRIM40 was significantly increased in DSS-treated wild-type mice (Fig. 9e). Similarly, in the RNA-seq dataset of DSS-induced colitis mice, mRNA expression of TRIM40 and cytoskeleton-related genes significantly increased and decreased, respectively ( FIG. 9f ). Taken together, the results of the in vitro and in vivo studies of the present invention show that TRIM40 acts as a pathogenesis of IBD by promoting intestinal epithelial barrier disorders.

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

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A Composition for Preventing or Treating Epithelial Barrier Dysfunction Comprising a TRIM40 inhibitor as an Active Ingredient <130> HPC-9442 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 99 <212> PRT <213> Homo sapiens <400> 1 Gly Pro Glu Ser Gln His Gln Thr Arg Glu Gln Leu Gly Ala Leu Pro 1 5 10 15 Gln Gln Trp Leu Gly Gly Gln Leu Glu His Met Pro Ala Glu Ala Ala Arg 20 25 30 Ile Leu Asp Ile Ser Arg Ala Val Thr Gln Leu Arg Ser Leu Val Ile 35 40 45 Asp Leu Glu Arg Thr Ala Lys Glu Leu Asp Thr Asn Thr Leu Lys Asn 50 55 60 Ala Gly Asp Leu Leu Asn Arg Ser Ala Pro Gln Lys Leu Glu Val Ile 65 70 75 80 Tyr Pro Gln Leu Glu Lys Gly Val Ser Glu Leu Leu Leu Gln Pro Pro 85 90 95 Gln Lys Leu <210> 2 <211> 300 <212> DNA <213> Homo sapiens <400> 2 gggccggaga gccagcacca aaccagggaa cagctgggtg ccctccctca gcagtggctg 60 ggccagctgg agcacatgcc agcagaagcg gccagaatcc ttgacatctc cagggcagta 120 acacagctca gaagcctggt cattgatctg gaaaggacgg ccaaggaatt agacaccaac 180 acactgaaga atgctggtga cttactgaac aggagtgctc cacagaaatt agaggttatt 240 tatccccagt tggagaaagg agtcagtgaa ttgcttcttc agccccctca gaagctctga 300 300 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> N-term Myc-tagged TRIM 40 F primer <400> 3 attctcgaga tgatcccttt gcagaaggac 30 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> N-term Myc-tagged TRIM 40 R primer <400> 4 attgaattca gagcttctga ggggg 25 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> N-term Flag-tagged TRIM 40 F primer <400> 5 attagatcta tgatcccttt gcagaaggac 30 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> N-term Flag-tagged TRIM 40 R primer <400> 6 attgaattca gagcttctga ggggg 25 <210> 7 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> C-term GFP-tagged TRIM 40 F primer <400> 7 attctcgagc caccatgatc cctttgcaga aggacaac 38 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C-term GFP-tagged TRIM 40 R primer <400> 8 aatggatccg agcttctgag ggggctgaag 30 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> C-term Myc-tagged TRIM 40 F primer <400> 9 aatagatctg ccaccatgat ccctttg 27 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> C-term Myc-tagged TRIM 40 R primer <400> 10 attgttaacc tcgagcttct gagggggctg aa 32 <210> 11 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> RING-del-TRIM 40 F primer <400> 11 aatagatctg ccaccatgat ccctttgcag aaggacaacc aggaggaggg tgtccggaag 60 ccctgttctg a 71 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> RING-del-TRIM 40 R primer <400> 12 attgttaacc tcgagcttct gagggggctg aa 32 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 61-258-del-TRIM 40 F primer <400> 13 aatagatctg ccaccatgat ccctttg 27 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 61-258-del-TRIM 40 R primer <400> 14 aatctcgaga cagggcttcc ggcaga 26 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 108-258-del-TRIM 40 F primer <400> 15 aatagatctg ccaccatgat ccctttg 27 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> 108-258-del-TRIM 40 R primer <400> 16 aatctcgaga atggtcagtt catgatgaga 30 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 153-258-del-TRIM 40 F primer <400> 17 aatagatctg ccaccatgat ccctttg 27 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 153-258-del-TRIM 40 R primer <400> 18 aatctcgaga gcctccagcc tgtggt 26 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> cofilin-1 F primer <400> 19 attagatctg ccaccatggc ctggggtgtg gct 33 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> cofilin R primer <400> 20 attctcgagc aaaggcttgc cctccag 27 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> LIMK1 F primer <400> 21 ctaagatctg ccaccatgag gttgacgcta ctttg 35 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LIMK1 R primer <400> 22 accgttaacg tcggggacct cagg 24

Claims (13)

A composition for preventing or treating epithelial barrier dysfunction comprising a Tripartite Motif Containing 40 (TRIM40) inhibitor as an active ingredient.
The method of claim 1, wherein the TRIM40 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to the TRIM40 protein; Or a composition characterized in that it is an aptamer that specifically binds to the TRIM40 protein.
The composition according to claim 2, wherein the antibody or aptamer specifically binds to the amino acid sequence of SEQ ID NO: 1.
The composition of claim 1, wherein the TRIM40 inhibitor is a nucleic acid molecule that inhibits the expression of a nucleotide encoding a TRIM40 protein.
The composition according to claim 4, wherein the nucleic acid molecule specifically binds to the nucleotide sequence of SEQ ID NO:2.
According to claim 1, wherein the epithelial barrier dysfunction is inflammatory bowel disease, pancreatitis, cystic fibrosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute respiratory distress syndrome (ARDS), chronic bronchitis, chronic rhinitis, asthma, skin A composition selected from the group consisting of aging and vesicular exfoliation vesicles.
The composition of claim 6, wherein the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis and Behcet's disease.
A functional food composition for improving epithelial barrier dysfunction comprising a TRIM40 inhibitor as an active ingredient.
A composition for enhancing epithelial barrier or cell membrane permeability comprising at least one selected from the group consisting of TRIM40 protein or a functional fragment thereof, a nucleotide encoding a TRIM40 protein or a functional fragment thereof, an activator of TRIM40 protein, and a ROCK inhibitor as an active ingredient .
10. The composition of claim 9, wherein the functional partial fragment of the TRIM40 protein comprises the amino acid sequence of SEQ ID NO: 1.
10. The composition of claim 9, wherein the ROCK inhibitor is Y-26732.
A composition for preventing or treating metastatic cancer comprising a Tripartite Motif Containing 40 (TRIM40) inhibitor as an active ingredient.
The composition according to claim 12, wherein the composition inhibits epithelial-mesenchymal transition.

Images