KR20140121088A - Novel ER targeting type I Transmembrane Protein - Google Patents

Abstract

The present invention relates to novel ER-targeting type I transmembrane protein h935 and a use thereof and, more specifically, to novel protein h935; and to a use of the novel protein using molecular mechanisms associated with a endoplasmic reticulum (ER) shape retaining function and a ER stress control function.

Description

The novel envelope membrane permeable protein {Novel ER targeting type I Transmembrane Protein}

The present invention relates to a novel ER-targeting type I transmembrane protein h935 and uses thereof, more particularly to a novel protein h935; And to the use of molecular mechanisms associated with its elastomeric and ER stress control functions.

The endoplasmic reticulum (ER) is one of the organelles found in all eukaryotic cells, and consists of a network of tubules, vesicles, and cisterna. The endoplasmic reticulum is made up of proteins and is delivered to the cells (the prokaryotes do not have any organelles, so they do not have any endoplasmic reticulum). The basic structure and organization of the endoplasmic reticulum is the extension of the nuclear membrane (endomembrane system) Similar to normal cell membranes. The endoplasmic reticulum is composed of proteins (transmembrane receptors or intracellular proteins) to be part of cell membranes, synthesis of proteins to be secreted out of cells (ie digestive enzymes, calcium absorption, synthesis / storage of glycogen and steroids or macromolecules) Structure formation, transport

The lumen of the ER (endoplasmic reticulum) is a specialized cellular environment for post-translational modification and folding of proteins. It is a structure of membrane components that stretches out from the nuclear membrane and is branched. It has a rough endoplasmic reticulum with a ribosome attached, There are two types of reticulate endoplasmic reticulum. Approximately one-third of the intracellular proteins result in post-translational modification from the mRNA to protein in the endoplasmic reticulum (folding and assembly), glycosylation, disulfide bond The protein structure becomes an active form. It is also the synthesis site of lipid and steroid hormones and plays an important role in regulating intracellular calcium concentration by calcium reservoir.

The endoplasmic reticulum plays a number of roles, including protein processing facilitation and transport of the resulting protein reservoirs called sisters. The formation of the secondary structure of the newly formed primary structure protein is carried out by an endoplasmic reticulum protein such as protein disulfide isomerase (PDI), Hsc70 family, calexin, calreticulin, or peptidylpropyl isomerase family . Only correctly formed proteins can be transported from the rough endoplasmic reticulum to the Golgi

If the protein folding and processing of the endoplasmic reticulum is interrupted by viruses, microbial infections, specific cell differentiation, various abiotic stresses or overexpression of exogenously introduced proteins, Proteins that do not undergo proper processing such as addition of sugar chains are accumulated, which is called ER stress (Kaufman, Genes Dev. 13: 1211-1233, 1999). When the stress of the endoplasmic reticulum is generated by various cell environment changes, the cell tries to solve it through various pathways, and when the stress is prolonged, the cell becomes deadly and causes apoptosis.

In the present study, we investigated the effects of stress on endothelial cells, such as ischemia, diabetes, and viral infections, Hyperhomocysteinemia, mutation, and the like.

Therefore, it has always been required to identify molecular mechanisms involved in various reactions in these endoplasmic reticulum, and in particular, there is a constant search for ways to control endoplasmic reticulum stress response.

Therefore, the present inventors first discovered a novel ER-targeting type I transmembrane protein h935 and studied its structure and molecular mechanism. As a result, it was found that a well-conserved TM (transmembrane domain) region of the h935 protein ER targeting function, and h935 protein were also involved in the maintenance of the vesicle shape and the endoplasmic reticulum stress, thus completing the present invention.

1. Ken-ichi Nakajima et al. Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion. EMBO. 2004 June; 23, 3216-3226. 2. Takao lwawaki. Learning more about physiological endoplasmic reticulum stress using the ERAI system. Riken research. 2009 November 13. 3. Simone Fulda, Adrienne M. Gorman, Osamu Hori, and Afshin Samali. Cellular Stress Responses: Cell Survival and Cell Death Int J Cell Biol. 2010 Feb 21 214074.

The present invention relates to a novel ER-targeting type I transmembrane protein h935 and its use,

The main object of the present invention is to provide a novel envelope membrane permeable protein h935.

Another object of the present invention is to provide a nucleic acid and a transformant which encode the protein h935.

Yet another object of the present invention is to provide a therapeutic use of the above-mentioned protein h935 using a molecular mechanism in the endoplasmic reticulum of an endoplasmic reticulum stress related disease.

In order to solve the above object, the present invention provides a novel ER-targeting type I transmembrane protein h935, which comprises an envelope transmembrane protein consisting of an amino acid sequence represented by SEQ ID NO: 1, 2 or 3, to provide.

In particular, the amino acid sequence is characterized by comprising a TM (transmembrane) domain region consisting of ISVTMILVAWMVIALILFLL, which is a site having the ability to target an endoplasmic reticulum.

The present invention encompasses not only the amino acid sequence of such novel protein but also a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1, 2, or 3, a recombinant expression vector containing the same, and a transformant transformed with the same.

The novel protein of the present invention is present in an endoplasmic reticulum (ER), and the N-terminal of the protein is located on the luminal side of the endoplasmic reticulum. It binds to proteins such as calnexin to maintain the shape of the endoplasmic reticulum .

It also has the ability to regulate ER stress through binding to ATF6 protein.

That is, it also includes the molecular mechanism of the novel protein of the present invention in relation to the function.

Accordingly, the present invention provides, as another embodiment, a composition for treating an endocrine-stress-related disease comprising an expression or activity promoter of an endoplasmic reticulum transmembrane protein comprising an amino acid sequence represented by SEQ ID NO: 1, 2 or 3 as an active ingredient do.

The periplasmic membrane permeability protein expression or activity promoter may be, for example, a recombinant expression vector containing a gene encoding an amino acid sequence represented by SEQ ID NO: 1, 2 or 3. At this time, it is also possible to further include a promoter or enhancer that promotes protein expression or activity.

The endoplasmic reticulum stress-related disease is selected from the group consisting of Type 1 diabetes, Type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, ALS), hemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease Kreutzfeldt-Jakob disease, familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, < RTI ID = 0.0 > St. chapters include disease, colon's disease, ulcerative colitis, breast cancer and prostate cancer.

Thus, the present invention provides novel ER-targeting type I transmembrane proteins h935; And its molecular mechanisms within the endoplasmic reticulum.

The present invention provides novel ER-targeting type I transmembrane proteins h935 and their useful uses that are involved in the various functions of the endoplasmic reticulum and are useful for the maintenance of the shape of the endoplasmic reticulum, In addition, it can be used for the treatment of endoplasmic reticulum stress disease.

Fig. 1 shows the result of confirming the m935 expression pattern in various tissues.
Figure 2 shows the immunohistochemical method And the h935 protein is located in the endoplasmic reticulum.
FIG. 3 is a schematic diagram showing the structure of various mutants prepared to examine the ER retention position in h935 protein.
FIG. 4 shows the result of confirming whether the variants of FIG. 3 were localized in the ER.
5 is a structural schematic diagram of N-linked glycosylation in the N-terminus and C-terminus region of h935.
Figs. 6 and 7 are the h935 positioning results according to Fig.
Figures 8 and 9 show the results of an experiment to determine the association of h935 with the ER chaperone protein.
10 is a schematic diagram related to the ER stress reaction mechanism.

The terms used in the present invention are defined as follows.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .

"Coding region" or "coding sequence" refers to a nucleic acid sequence, a complement thereof, or a portion thereof, which encodes a particular gene product or fragment thereof that is required to be expressed, according to a common base pairing and codon usage relationship. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that are joined together by a biochemical machinery of cells to provide mature mRNA. The antisense strand is a complement of the nucleic acid, and the coding sequence can be deduced therefrom. The coding sequence is placed in the relationship of transcriptional regulatory elements and translation initiation and termination codons such that transcripts of appropriate length are generated and translated in the appropriate reading frame to produce the desired functional product

"Transfection, transfection or transfection" refers to the process by which extracellular DNA enters a host cell with and without accompanying entities. "Transfected cell" refers to a cell in which extracellular DNA is introduced into a cell and contains extracellular DNA. DNA can be introduced into the cell and the nucleic acid can be inserted into the chromosome or replicated as an extrachromosomal material. And cells into which external DNA is introduced are referred to as " transformants. &Quot;

The term "vector" refers to any nucleic acid that is inserted into a host cell, recombined with the host cell genome, inserted into it, or contains a competent nucleotide sequence that replicates spontaneously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, and viral vectors.

"Host cell" refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and is understood to refer not only to a particular target cell but also to its progeny or potential progeny. In fact, the offspring are not identical to the parent cell, but are still included within the scope of the term, as used herein, since certain modifications may occur in subsequent generations due to mutations or environmental influences.

"Polypeptide" is intended to encompass a single polypeptide as well as multiple polypeptides and includes chains or chains of one or more amino acids linked together by peptide bonds. The term also includes polypeptides with post-translational modifications.

"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein.

"Accelerator" means a substance that promotes, enhances or increases the expression or activity of a gene of interest. The activation mechanism of the promoter is not particularly limited. Examples include organic or inorganic compounds, proteins, carbohydrates, polymeric compounds such as lipids, and composites of various compounds. For example, 'h935 promoter' may include a substance that promotes, enhances or increases the expression or activity of the h935 protein.

As used herein, an "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an accelerator is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of the relevant disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i. E., Not worsening), slowing or slowing the progression of the disease, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel envelope membrane permeable protein h935.

The novel transmembrane transmembrane protein of the present invention is found in all tissues including the brain and the like, and is characterized by having the following functions as a type I transmembrane protein.

(i) The protein h935 of the present invention is present in an endoplasmic reticulum (ER), and the N-terminal of the protein is located on the luminal side of the endoplasmic reticulum.

Membrane proteins secreted by cells or distributed in the intracellular membrane of proteins biosynthesized in cells are necessarily transferred to the lumen of the endoplasmic reticulum (ER) and subjected to proper folding and processing. Chaperone proteins such as PDI (protein disulfide isomerase), heavy chain binding protein (BiP), calnexin (CNX) and calreticulin (CRT) are present inside the endoplasmic reticulum to allow proteins folded in the endoplasmic reticulum give.

The protein h935 of the present invention is present in such an endoplasmic reticulum, and in particular, the N-terminal region has an important signaling domain, which is located in the lumen of the endoplasmic reticulum.

In addition, the sequence of h935 of the present invention has a well-conserved Transmembrane domain (TM) from invertebrates to mammals, thereby targeting ER.

(ii) The protein h935 binds to various proteins such as calnexin in the endoplasmic reticulum to maintain the endoplasmic reticulum shape.

The endoplasmic reticulum is organized to be suitable for protein folding, saccharification, and transport. The endoplasmic reticulum has an acidic environment suitable for the disulfide bond, a typical chemical bond that causes protein folding. The chaperone protein, a protein that helps folding, and the calcium ion, an auxiliary element of chaperone, It is distributed in abundance.

Calnexin, one of these chaperone proteins, contributes to the folding of proteins in the endoplasmic reticulum by entering the calnexin / calreticulum cycle. The h935 protein of the present invention binds to calnexin and the like to maintain the shape of the endoplasmic reticulum It is possible to do.

(iii) The protein h935 functions to regulate ER stress.

ER stress is the ability of the endoplasmic reticulum to function in response to physiological or pathological conditions. The infiltration of immature proteins into the cell body or the depletion of calcium in the endoplasmic reticulum results in the impairment of endoplasmic reticulum function. Which means that the ER protein folding ability has been exceeded. When an endoplasmic reticulum stress occurs, the cell has a defense mechanism to survive, which is called an ER stress response. The endoplasmic reticulum (ER) response is mediated by three distinct signaling pathways: pancreatic ER kinase (PERK), inositol-requiring 1α (IRE-1α), X-box binding protein 1 (XBP-1), and activating transcription factor Lt; / RTI > A schematic diagram of the ER stress response is shown in Fig.

In particular, h935 of the present invention is associated with the ATF6 (activating transcription factor) and participates in the endoplasmic reticulum stress response.

ATF6 is one of the ATF / CREB bZIP DNA-binding protein family of transcription factors present in the membrane of the endoplasmic reticulum. The C terminus, which detects the endoplasmic reticulum stress, is located in the endoplasmic reticulum and contains the bZIP and DNA transcription activation domains Is located in the cytoplasm. In the presence of ER stress, inactive ATF6 (p90) migrates from the endoplasmic reticulum to the Golgi apparatus and becomes proteolytic cleavage by site-1 protease (S1P) and site-2 proteases (S2P) N-terminal ATF6 (p50) migrates into the nucleus and acts as a direct transcription factor, leading to the expression of enzymes and chaperones that are important for protein folding. In addition, active ATF6 may bind to ERSE present in the promoter region of XBP-1 and induce the expression of XBP-1 mRNA. In the case of ER stress, ATF6, which has migrated to the nucleus, induces the expression of XBP-1 mRNA, and the XBP-1 mRNA thus expressed is cleaved by the RNase activity of IRE1 to generate the active XBP-1 protein. In other words, the activity of ATF6 in the event of the stress of the endoplasmic reticulum occurs at a rapid rate because the already formed protein is decomposed and activated, and the activity of XBP-1 occurs relatively slowly because it requires processes such as mRNA transcription, division and protein synthesis. Thus, it can be assumed that the initial ER stress response is immediately attenuated by ATF6, and then the response to sustained ER stress is mediated by the activity of XBP-1

Therefore, h935 of the present invention binds to ATF6 and inhibits its activity, thereby inhibiting the initial stress response.

In one aspect, the present invention relates to a novel transmembrane transmembrane protein h935 having the above-mentioned functions and a method for producing the same.

The h935 protein of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 1, 2 or 3, but includes functional equivalents thereof. Preferably, the amino acid sequence of human-derived SEQ ID NO: 1 is included. SEQ ID NO: 1 shows the human h935 amino acid sequence, SEQ ID NO: 2 shows the mouse-derived h935 amino acid sequence, and SEQ ID NO: 3 shows the h935 amino acid sequence derived from the frog.

SEQ ID NOS: 1 to 3 of the present invention each consist of 100 amino acids. In particular, the sequence includes a similar transmembrane domain (TM), which is well preserved from invertebrates to mammals Area.

The transmembrane domain (TM) has an ER targeting function.

Refers to an amino acid sequence which is at least 60%, preferably 70%, more preferably 80%, 85%, 90% or more homologous to the amino acid sequence of SEQ ID NO: 1, 2 or 3 as a result of addition, substitution or deletion of amino acids. Refers to a polypeptide having 90% or 95% or more sequence homology and exhibiting substantially the same activity as h935 of the present invention. Here, 'substantially homogenous activity' refers to the state of maintaining the vesicle shape and participating in the ER stress response.

Such functional equivalents include, for example, amino acid sequence variants in which some of the amino acids of the amino acid sequence shown in SEQ ID NO: 1, 2 or 3 are substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a site that is not directly involved in the activity of h935 of the present invention. Also included within the scope of such functional equivalents are polypeptide derivatives in which the basic skeleton of h935 and some chemical structures of the polypeptide are modified while maintaining its physiological activity. These include, for example, fusion proteins made by fusion with other proteins while maintaining the structural stability and physiological activity to change the stability, shelf stability, volatility or solubility of the polypeptide of the present invention.

In the present invention, "h935 protein" is interpreted to include such a functional equivalent.

The h935 protein of the present invention can be produced by directly isolating from a tissue such as a mammal, chemically synthesized, or obtained using a gene recombinant technique.

Separation and purification of the h935 protein contained in cells or tissues can be carried out by a number of known methods. Examples of these methods include a method using solubility such as salt precipitation and solvent precipitation, a method using difference in molecular weight such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, a method using ion exchange column chromatography A method using difference in hydrophilicity such as reversed phase high performance liquid chromatography, a method using difference in isoelectric point such as isoelectric focusing electrophoresis, and the like.

The h935 protein can also be chemically synthesized. When they are chemically synthesized, they can be obtained by using a polypeptide synthesis method well known in the art. Peptides may be prepared using conventional stepwise liquid or solid phase synthesis, fractional condensation, F-MOC or T-BOC chemistry. Particularly, a preferred method for producing a polypeptide is to use solid phase syntheses.

The h935 protein can also be obtained using genetic recombination technology. When the recombinant technology is used, a polynucleotide encoding the h935 protein is inserted into an appropriate expression vector, the vector is transformed into a host cell, the host cell is cultured to express the h935 protein, and the h935 protein is recovered from the host cell And the like.

Other forms

The present invention also relates to a nucleic acid encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, 2, or 3 or a functional equivalent thereof, preferably DNA, an expression plasmid (vector) comprising the same, and a transformant; And methods for their manufacture.

The DNA can be cloned using conventional techniques known in the art. The cloning of the DNA can also be performed, for example, by screening a cDNA library derived from a suitable cell line using a suitable portion of the nucleotide sequence shown in SEQ ID NO: 2 herein, as a probe or a PCR primer for hybridization have. For example, a person skilled in the art can easily produce it by using molecular cloning (Molecular Cloning 2nd Ed. Cold Spring Harbor Laboratory Press, 1989).

Such a vector may be an expression vector known to those skilled in the art capable of expressing the h935 protein, and vectors for this purpose, the production of polynucleotides for transformation and the preparation of vectors for transformation are well known to those skilled in the art.

Transformation with genes encoding h935 and its functional equivalents in the present invention can be carried out by transformation techniques known to those skilled in the art. Preferably selected from the group consisting of electroporation, PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, , A vacuum infiltration method, or the like can be used.

Usage

The present invention also relates, in yet another aspect, to the use of the h935 protein to maintain the endoplasmic reticulum and regulate the stress of the endoplasmic reticulum.

That is, the present invention includes a molecular mechanism associated with the function of h935, a novel ER-targeted type I transmembrane protein.

As described above, the h935 protein of the present invention binds to a protein such as calnexin while the N-terminal is located on the luminal side of the ER to maintain an endoplasmic reticulum form, as well as an ATF6 protein associated with ER stress The present invention extends to a genetic approach to up-regulating or down-regulating the expression of genes associated with the expression of h935.

And preferably up-regulates h935 expression to maintain the vesicle shape and inhibit the endoplasmic reticulum response. In general, a common technique used to up-regulate expression may be to increase the number of gene copies or to antagonize inhibitors of gene expression, and down-regulating expression may be accomplished by gene silencing, Genetic means of inducing silencing can be used.

The present invention is based on this h935 action mechanism, and in particular provides therapeutic use for ER stress related diseases.

As a specific example, recombinant polynucleotide constructs (collectively referred to as "expression vector") such as h935 expression or activity promoter, for example, a recombinant expression vector containing h935 expression gene, microorganisms transformed with the expression vector , Compositions for the treatment and prevention of ER stress-related diseases including one of them, and the use thereof.

In order to express h935, a suitable promoter may be operably linked to a DNA / polynucleotide encoding it and, where necessary, additional ' enhancer elements ' may be included in place of or in addition to elements present at the promoter positions described above h935 expression or activity and amplify the activity.

The therapeutic composition of the present invention may be prepared into a suitable preparation including an acceptable carrier depending on the administration mode. Formulations suitable for the mode of administration are known and may include formulations that facilitate passage, typically through the membrane.

In addition, the therapeutic composition of the present invention can be used in the form of a general pharmaceutical preparation. The parenteral preparation may be in the form of a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion or a lyophilized preparation, or a tablet, troch, capsule, elixir, suspension, syrup or wafer in the case of oral administration. May be formulated in unit dosage ampoules or multiple doses. In addition, the therapeutic composition of the present invention may be administered together with a pharmaceutically acceptable carrier. For oral administration, for example, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring matters or fragrances may be used. In the case of injections, buffering agents, preservatives, An isotonic agent, an isotonic agent, a stabilizer, etc. may be used in combination. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used.

In addition, the method for treating the stress related to the endoplasmic reticulum using the therapeutic composition of the present invention may include administration through a general route in which a predetermined substance is introduced into a patient by an appropriate method. Such administration methods include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration. However, upon oral administration, the cells may be digested, so that the oral composition is preferably formulated to coat the active agent or protect it from being decomposed from above.

In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell. Preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular or drip injectable. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (for example, oleic acid), an alcohol (for example, ethanol, benzyl alcohol, propylene glycol or glycerin) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor Preservatives (for example, mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.). Preferably, the method for treating a brain or neurological disease using the therapeutic composition of the present invention comprises administering a therapeutically effective amount of the therapeutic composition of the present invention. The pharmaceutically effective amount may be appropriately selected depending on the kind of the disease, the age, body weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, administration frequency, And can be readily determined by those skilled in the art depending on the factors.

In the present invention, the disease to be treated is a disease associated with ER stress response.

The endoplasmic reticulum stress-related disease is selected from the group consisting of Type 1 diabetes, Type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis, ALS), hemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease, Creutzfeldt-Jakob disease Kreutzfeldt-Jakob disease, familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Wolcott-Rallison syndrome, Wolfram syndrome, < RTI ID = 0.0 > St. chapters include disease, colon's disease, ulcerative colitis, breast cancer and prostate cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

The techniques and procedures used in the experiments are generally well understood by those skilled in the art and can be carried out according to conventional methods, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, the process involving the use of commercially available kits and reagents is generally performed according to protocols and / or parameters defined by the manufacturer, unless otherwise noted.

< Example  1>

h935  Isolation and Expression of Proteins

h935 was cloned by PCR using human HeLa cDNA (h935 RT-S: ggg cgg attaacatc gtc aa, h935-RT-A: ttt ggt cgt gca agg tgt ta) h935 coding region, Sequencing was performed to confirm the nucleotide sequence.

The amino acid sequence of the isolated protein was analyzed. As a result, the domain of TM (transmembrane) domain was well preserved as described below.

1-1. Expression in various tissues

(m935-S: 5'-tgg gcg ggt tta aat agt tg-3 ', m935-A: 5'-agt gcc tgg tcg tct tca gt-3') which is known as c4orf34 is expressed in various tissues of mice Respectively. CDNA was synthesized from each tissue (heart, thymus, cortex, hippocampus, cerebellum).

As a result, as shown in Fig. 1, it was confirmed that all of them were expressed in the heart, thymus, cerebral cortex, hippocampus, and cerebellum of a mouse.

1-2. ER localization of h935 in HEK293T cells

Immunocytochemistry using HEK (Human Embryonic Kidney) 293T cells The presence of h935 protein in the endoplasmic reticulum was confirmed microscopically (Fig. 2). For this purpose, h935 was subcloned into pEGFP-N3 vector with HindIII / EcoRI site and 3xFLAG tag was inserted into the c-terminal of h935 to construct pcDNA3.1-h935-3xFLAG. This was stained with ER marker, Sec61-EGFP or calnexin. As a result, it was confirmed that EGFP-h935 and h935-3xFLAG were located in the ER membrane. This suggests that h935 is a membrane protein located in the ER.

1-3. Identification of ER retention sequence

Various mutants as shown in Fig. 3 were constructed to examine the ER retention positions in the h935 protein. The h935 WT and variants were then transfected into HEK 293T.

As a result, h935 (C, PPN) -GFP and h935 (N, NGI) were not located in the ER, although several h935 constructs were located in the ER, as shown in Fig. This implies that the TM region (transmembrane domain) of h935 is involved in ER targeting.

&Lt; Example 2 >

Identification of h935 protein

In order to characterize the h935 protein, mutations were artificially made to make N-linked glycosylation in the N-terminal and C-terminal regions of h935 (h935 (Ngly, N ) -GFP and h935 (Ngly, C) -GFP) (Figure 5). N-linked glycosylation occurs only in the lumen of the endoplasmic reticulum, and upon modification, protein electrophoresis is performed to increase the molecular weight. As a result of introducing the mutant using this property, it can be seen that the molecular weight is increased only when a mutant is formed at the N-terminus. As a result, it was confirmed that h935 is a type I transmembrane protein located at the endoplasmic reticulum of the endoplasmic reticulum.

&Lt; Example 3 >

Functional analysis of h935 protein

To investigate the ER function of h935, we investigated the association with ER chaperone protein. For this, 3xFLAG-hCaM and h935-EGFP were cotransfected in HEK293T. Twenty-four hours after transfection, FLAG co-IP was performed using M2-agarose (Sigma), an antibody capable of recognizing FLAG peptide.

In the 3xFLAG-hCaM co-IP experiment using lysate of HEK293T cells, each gene was introduced into HEK293T cells using lipofectamine 2000 (Invitrogen) to express the protein. (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% triton-X 100, protease inhibitor cocktails (Roche), 2 mM CaCl ₂ (or 5 mM EGTA) ] And the cells were disassembled. The cytoplasmic sites were separated and the intracellular proteins were quantitated using the BCA method. Protein-containing cytoplasmic solution was mixed with 30 μl of M2-agarose for 2 hours at 4 ^° C. The beads were then washed three times with the same lysis solution and 30 μl of the extract solution (5 mg / ml of 3xFLAG peptide dissolved in the binding solution) was added. Then, the supernatant was taken, and 5X SDS sample buffer was added, followed by protein electrophoresis and Western blotting using GFP antibody and FLAG antibody.

As a result, it was confirmed that 3xFLAG-h935 interacted with h935-EGFP and endogenous calnexin (Fig. 8).

In addition, 3xFLAG-hCaM and BIP-myc were coexpressed in HEK293T, confirming that 3xFLAG-h935 also interacted with BIP (WT) and BIP mt (T37G)

Thus, it was confirmed that the h935 protein of the present invention has a deep association with the ER chaperone protein.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Kyungpook National University <120> Novel ER targeting type I Transmembrane Protein <130> pN1209-452 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 99 <212> PRT <213> human h935 amino acid sequence <400> 1 Met Ala Glu Gly Gly Phe Asp Pro Cys Glu Cys Val Cys Ser His Glu   1 5 10 15 His Ala Met Arg Arg Leu Ile Asn Leu Leu Arg Gln Ser Gln Ser Tyr              20 25 30 Cys Thr Asp Thr Glu Cys Leu Gln Glu Leu Pro Gly Pro Ser Gly Asp          35 40 45 Asn Gly Ile Ser Val Thr Met Ile Leu Val Ala Trp Met Val Ile Ala      50 55 60 Leu Ile Leu Phe Leu Leu Arg Pro Pro Asn Leu Arg Gly Ser Ser Leu  65 70 75 80 Pro Gly Lys Pro Thr Ser Pro His Asn Gly Gln Asp Pro Pro Ala Pro                  85 90 95 Pro Val Asp             <210> 2 <211> 98 <212> PRT <213> mus h935 amino acid sequence <400> 2 Met Ala Glu Gly Gly Phe Asp Pro Cys Glu Cys Val Cys Ser His Glu   1 5 10 15 His Ala Met Arg Arg Leu Ile Asn Leu Leu Arg Gln Ser Gln Ser Tyr              20 25 30 Cys Thr Asp Thr Glu Cys Leu Gln Glu Leu Pro Gly Pro Ser Ser Asp          35 40 45 Ser Gly Ile Ser Ile Thr Val Met Ile Leu Met Ala Trp Met Val Ile      50 55 60 Ala Met Leu Leu Phe Leu Leu Arg Pro Pro Asn Leu Arg Gly Ser Ser  65 70 75 80 Leu Pro Gly Lys Pro Ser Ser Pro His Ser Asp Pro Pro Ala Pro Pro                  85 90 95 Val Asp         <210> 3 <211> 103 <212> PRT <213> xenopus h935 amino acid sequence <400> 3 Met Ala Glu Gly Asn Phe Asp Pro Cys Glu Cys Ile Cys Ser His Glu   1 5 10 15 His Ala Met Arg Arg Leu Ile Asn Leu Leu Arg Gln Ser Gln Ser Tyr              20 25 30 Cys Thr Asp Asn Glu Cys Phe Gln Glu Leu Pro Gly Pro Asn Ser Ser          35 40 45 Thr Asp Gly Gly Phe Asn Ile Ala Met Ile Ala Met Ala Trp Leu Val      50 55 60 Ile Gly Val Thr Leu Tyr Leu Leu Arg Pro Arg Ser Leu Arg Gly Asn  65 70 75 80 Gly Pro Ser Ala Lys Pro Asn Ser Pro His Ser Asn Gln Gly Pro Glu                  85 90 95 Pro Ala Pro Pro Val Asp             100

Claims (15)

An endoplasmic reticulum membrane protein consisting of an amino acid sequence represented by SEQ ID NO: 1, 2 or 3; The method according to claim 1,
Wherein the amino acid sequence comprises a TM (transmembrane) domain region consisting of ISVTMILVAWMVIALILFLL. 3. The method of claim 2,
Wherein the TM (transmembrane) domain region has endoplasmic reticulum targeting ability. The method according to claim 1,
Wherein said transmembrane transmembrane protein is present in an endoplasmic reticulum (ER) and wherein the N-terminus of said protein is located on the luminal side of the endoplasmic reticulum. The method according to claim 1,
Wherein said transmembrane transmembrane protein has a function of maintaining the shape of an endoplasmic reticulum. 6. The method of claim 5,
Wherein said transmembrane transmembrane protein retains the shape of the endoplasmic reticulum by binding to calnexin. The method according to claim 1,
Wherein said transmembrane transmembrane protein has a function of regulating ER stress. 8. The method of claim 7,
Wherein the ER stress regulation is through binding to the ATF6 protein. A nucleic acid encoding an amino acid sequence represented by SEQ ID NO: 1, 2, or 3; A recombinant expression vector containing a nucleic acid encoding an amino acid sequence represented by SEQ ID NO: 1, 2, or 3. A transformant transformed with a recombinant expression vector containing a nucleic acid encoding an amino acid sequence represented by SEQ ID NO: 1, 2, or 3. A composition for treating an endometrial cancer-associated disease comprising an expression or activity-promoting agent for an endoplasmic reticulum permeability protein comprising an amino acid sequence represented by SEQ ID NO: 1, 2, or 3 as an active ingredient. 13. The method of claim 12,
Wherein said endoplasmic reticulum transmembrane protein expression or activity promoter is a recombinant expression vector containing a gene encoding an amino acid sequence represented by SEQ ID NO: 1, 2 or 3. 14. The method of claim 13,
Wherein said recombinant expression vector comprises a promoter or enhancer promoting the expression or activity of an envelope transmembrane protein comprising an amino acid sequence represented by SEQ ID NO: 1, 2 or 3. 13. The method of claim 12,
The endoplasmic reticulum stress-
Type 2 diabetes, Alzheimer's disease, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis (ALS), hemodialysis Amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia, Huntington ' s disease, Kreutzfeldt-Jakob disease, Familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses, Ulcott-Ralisss syndrome Wolcott-Rallison syndrome, Wolfram syndrome, inflammatory bowel disease, coronary disease, ulcerative colitis &Lt; RTI ID = 0.0 &gt; prostaglandin, &lt; / RTI &gt; salt, breast cancer and prostate cancer.

Images