KR20150043032A - Pharmaceutical use of STIM1 - Google Patents

Abstract

The present invention relates to a pharmaceutical use of STIM1, wherein a C-terminal of STIM1, which is a SERCA1a-binding region (STIM1-SBR: a 449-671 amino acid sequence region), inhibits activities of a ryanodine receptor calcium channel. Accordingly, by using the STIM1 or a STIM1-SBR inhibitor, the total quantity of STIM1 proteins is adjusted, and a decrease in activities of the ryanodine receptor calcium channel is inhibited. Also, skeletal muscle contraction is promoted, thereby preventing or treating diseases related to hypotonus including skeletal muscle contraction disorders.

Description

The pharmaceutical use of STIM1 {Pharmaceutical use of STIM1}

The present invention relates to the pharmaceutical use of STIM1 which is involved in the relaxation or contraction of skeletal muscles.

Patients with Muscular Hypotonia exhibit a decrease in muscle tone and a decrease in muscle tone. I have a difficulty in moving my arms and legs and I have difficulty in moving my head, I have a rag doll phenomenon. I have difficulty in moving, taking a posture, breathing, and speaking. Developmental delays in children, social competence, language, and overall learning. Muscle relaxation is particularly common in people with Down's syndrome, muscular dystrophy, cerebral palsy, Prader-Willi syndrome, Tay-Sachs disease, And it also appears as a second disease form for cancer, AIDS, and geriatric diseases. Muscle relaxation is a precursor to muscular dystrophy or cerebral palsy and may be an indication of the progression of these diseases.

Accordingly, the present inventors have completed the present invention by identifying the role of STIM1 involved in the contraction and relaxation of skeletal muscle in order to diagnose or treat a disorder in which the skeletal muscle is contracted and relaxed.

Korean Patent Publication No. 2010-0129319 (2010.12.08)

 Skeletal Muscle, 1:16 (2011)

It is an object of the present invention to provide a use for diagnosing or treating a disease in which there is a disorder in skeletal muscle contraction and relaxation by identifying the role of STIM1 involved in contraction and relaxation of skeletal muscle.

In order to achieve the above object, the present invention provides a composition for preventing or treating a Muscular hypotonia-related disease comprising a stromal interaction molecule 1 (STIM1) inhibitor.

The present invention also relates to a method of inhibiting or reducing muscle tone (including, but not limited to, inhibition of muscle relaxation) comprising contacting a stromal interaction molecule 1 (STIM1) gene with a candidate substance outside the human body and determining whether the candidate substance promotes or suppresses expression of the gene A method for screening a medicament for the prevention or treatment of a muscular hypotonia-related disease.

The present invention also relates to a method of treating a subject suffering from muscle tension, comprising contacting a stromal interaction molecule 1 (STIM1) protein with a candidate substance outside the body and determining whether the candidate substance enhances or inhibits the function or activity of the protein There is provided a method for screening a medicament for the prevention or treatment of a disease associated with Muscular hypotonia.

Since the C-terminal fragment (STIM1-SBR) of STIM1 inhibits the activity of the calcium channel of the receptor of the ryanodine receptor, the use of the STIM1 inhibitor, in particular, the STIM1-SBR inhibitor inhibits the degradation of the calcium channel of the ryanodine receptor And promotes skeletal muscle contraction, thereby preventing or treating a muscle tension-lowering disorder having a skeletal muscle contraction disorder.

1 shows the expression of wild-type STIM1 and STIM1-SBR (amino acid residues 449-671) protein in differentiated skeletal muscle cells, wherein (A) shows the STIM1 and STIM1 domains and the scheme for STIM1-SBR used in the present invention, B) is the results of immunocytochemistry showing the expression of wild-type STIM1 (full-length wild-type STIM1) and STIM1-SBR protein externally expressed in differentiated skeletal muscle cells.
FIG. 2 is a graph showing the results of measurement of calcium migration in the cytoplasm of caffeine-treated differentiated skeletal muscle cells expressing wild-type STIM1 or STIM1-SBR (left).
FIG. 3 is a graph showing the results of measuring the amount of intracellular calcium in differentiated skeletal muscle cells expressing wild-type STIM1 or STIM1-SBR in a resting state and a result of treatment of thapsigargin (TG) (B) of the amount of intracellular calcium liberated from the cytoplasmic reticulum and a graph (C) drawn by a bar graph.
FIG. 4 shows the results (A) of the changes in storage-operation external calcium influx (SOCE) in differentiated skeletal muscle cells expressing wild-type STIM1 or STIM1-SBR and the graph (B)

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a composition for preventing or treating a Muscular hypotonia-related disease comprising a stromal interaction molecule 1 (STIM1) inhibitor.

The ryanodine receptor calcium channel is a protein that acts as a channel for the calcium release from the sarcoplasmic reticulum (SR) to the cytoplasm during the contractile process of the skeletal muscle. The ryanodine receptor calcium channel, Channel activity is one of the key processes of skeletal muscle contraction. The present inventors have succeeded in differentiating skeletal muscle cells expressing a region (SERCA1a-binding region (SBR)) (hereinafter referred to as 'STIM1-SBR') ranging from the 449th to 671th amino acid sequence present at the C-terminus of human-derived STIM1 Lt; RTI ID = 0.0 > of the < / RTI > ryanodine receptor calcium channel.

In addition, abnormal distribution of calcium was found in differentiated skeletal muscle cells expressing STIM1-SBR. Measuring the calcium concentration of the cytoplasm in the resting state revealed the presence of relatively low calcium in the cytoplasm of the differentiated skeletal muscle cells expressing STIM1-SBR (approximately 20% reduction, Table 2 Reference). In addition, the amount of calcium available to the cytoplasm from the cytoplasmic reticulum was increased. Therefore, in the case of differentiated skeletal muscle cells expressing STIM1-SBR, it is considered that the calcium to be present in the cytoplasm enters into the myofiber cytoplasm, decreases the amount of calcium in the cytoplasm, and increases the amount of calcium in the myofiber cytoplasm. It can be seen that an enemy abnormality occurs.

Next, we found that STIM1 did not change in STIM1-SBR expressing skeletal muscle cells, whereas wild-type STIM1 increased store-operated calcium-entry (SOCE). In other words, STIM1-SBR was found to have no relation with the SOCE parameter, which is a unique role of STIM1.

Accordingly, it has been found that STIM1 is particularly effective for diseases related to lowering of muscle tension such as Down's syndrome, Muscular dystrophy, cerebral palsy, Prader-Willi syndrome, or Tay-Sachs disease , Inhibitors for STIM1-SBR are treated to reduce or inhibit the total amount of STIM1 protein and increase the amount of calcium in the cytoplasm while blocking the change in the external calcium influx process (SOCE) The activity of the anodyne receptor calcium channel is promoted to induce muscle tension, thereby improving muscle tension.

The atrophy of the muscle may be caused by at least one of Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Limb-girdle Muscular Dystrophy, Facioscapulohumeral Muscular Dystrophy ), Or oculopharyngeal muscular dystrophy, and the like.

Wherein said STIM1 inhibitor is an antisense oligonucleotide, siRNA, shRNA, miRNA or vector comprising thereof; Or an antibody.

The STIM1 protein inhibitor may be a peptide or a compound that binds to the STIM1 protein or the SERCA1a-binding region (SBR) of STIM1, which is represented by the amino acid sequence of SEQ ID NO: 1, to decrease the activity of the lysozyme receptor calcium channel. Such an inhibitor may be selected through screening methods exemplified below such as protein structure analysis, and may be designed using methods known in the art.

The STIM1 protein is a protein originating from mammals, preferably humans, mice, rats, rabbits, orangutans, monkeys, hamsters, cats, dolphins and gorillas. In particular, the amino acid sequence of STIM1- Lt; / RTI > For example, human-derived GenBank accession no. NM_003156.3, XP_005253144.1, XP_005253143.1; GenBank accession no. NP_033313.2, NM_009287.4; GenBank accession no. XP_004050545.1; GenBank accession no. From Sumatra Orangutan ( Pongo Abelii ) XP_002822220.1; GenBank accession no. From the squirrel monkey ( Saimiri boliviensis boliviensis ). XP_003923448.1; Hamster ( Cricetulus griseus ) derived from GenBank accession no. XP_003509756.1, ** EGV96289.1, ** ERE79443.1, XP_005074118.1; GenBank accession no. EDM18193.1, NP_001101966.2; Cat ( Felis catus ) derived from GenBank accession no. XP_003992832.1; Himalayan monkey ( Macaca mulatta ) derived from GenBank accession no. NP_001248464.1; Tree rats ( Tupaia Chinensis ) GenBank accession no. ELW71828.1; GenBank accession no. From Oryctolagus cuniculus (xhRL). ** XP_002708775.1; Tursiops truncatus ) derived from GenBank accession no. STIM1 proteins with amino acid sequences such as XP_004319125.1 show 80 to 100% sequence homology to SBR. In the above, the symbol ** indicates 80-89% identity in the amino acid sequence, and the symbol without the mark indicates the identity of 90 or more, and a significant number indicates 100% identity with human-derived STIM1.

The protein inhibitor may be a polyclonal antibody, a monoclonal antibody, a human antibody or a humanized antibody specific to the STIM1 protein or the STIM1-SBR protein described above.

Examples of such antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies (Zapata et al ., Protein Eng . 8 (10): 1057-1062 (1995)); Single chain antibody molecule; And multispecific antibodies formed from antibody fragments and the like.

Upon digestion of the antibody into papain, two identical antigen binding fragments are generated, one for each "Fab" fragment with a single antigen binding site, and the remainder "Fc" fragments. Treatment of pepsin produces an F (ab ') 2 fragment that has two antigen binding sites and is still cross-linked to the antigen. Fv is a minimal antibody fragment containing complete antigen recognition and binding sites. This site is composed of a duplex of one heavy chain and one light chain variable region and is tightly bonded by non-covalent bonds.

Methods for producing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be prepared by injecting the mammal with one or more immunizing agents, if necessary, with an adjuvant. Usually, the immunizing agent and / or adjuvant is injected into the mammal several times by subcutaneous injection or intraperitoneal injection. The immunizing agent may be a protein of the present invention or a fusion protein thereof. It may be effective to inject the immunizing agent with a protein known to be immunogenic to the mammal being immunized.

Monoclonal antibodies according to the invention are described in Kohler et < RTI ID = 0.0 > al ., Nature , 256: 495 (1975)), or may be prepared by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,576). Monoclonal antibodies are also described, for example, in Clackson et al ., Nature , 352: 624-628 (1991) and Marks et al ., J. Mol . Biol ., 222: 581-597 (1991)).

The monoclonal antibody in the present invention specifically means that the part of the heavy chain and / or the light chain is identical with the corresponding sequence of the antibody belonging to the specific antibody class or subclass, Homology, the remainder of the chain (s) includes antibodies derived from other species or antibodies belonging to another antibody class or subclass, or "chimeric" antibodies that are identical or homologous to fragments of such antibodies (Morrison et al ., Proc . Natl . Acad . Sci . USA, 81: 6851-6855 (1984)).

Quot; humanized "forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e. G., Fv, Fab, Fab ', F (ab') 2 or other antigen binding sequences of antibodies). In most cases, humanized antibodies are human immunoglobulins (CDRs) in which residues of the complementarity determining region (CDR) of the recipient are replaced with CDR residues of a non-human species (donor antibody) such as mice, mice or rabbits having the desired specificity, (Acceptor antibody). In some cases, the Fv framework residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, the humanized antibody may comprise an acceptor antibody, or a residue that is not found in the CDR or framework sequences to be introduced. In general, a humanized antibody will comprise substantially all of one or more, typically two or more, variable domains, wherein all or substantially all CDR regions correspond to non-human immunoglobulin regions, and all or substantially all FR regions It corresponds to the region of the human immunoglobulin sequence. Also, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), generally a portion of a human immunoglobulin region (Presta, Curr . Op . Struct . Biol . 2: 593-596 (1992)).

The function or activity inhibitor of the STIM1 protein or STIM1-SBR protein of the present invention may be delivered using liposomes, viruses, gene guns, polymers, ultrasound, or electric shock, but is not limited thereto.

The STIM1 or STIM1-SBR gene may be a DNA encoding them or an mRNA transcribed therefrom. Therefore, the inhibitor for the gene may be an inhibitor that binds to the gene itself and interferes with transcription or binds to the mRNA transcribed from the gene, thereby interfering with the detoxification of the mRNA.

Therefore, the inhibitor of the STIM1 or STIM1-SBR gene includes all inhibitors that inhibit the expression of the STIM1 or STIM1-SBR gene. For example, such an inhibitor may be a peptide, a nucleic acid or a compound that binds to the gene. Such inhibitors may be selected through the screening methods illustrated below, such as cell-based screening, and may be designed using methods known in the art.

In one embodiment, the inhibitor may be an antisense-oligonucleotide, siRNA, shRNA, miRNA, or vector comprising the STIM1 or STIM1-SBR gene. Such antisense-oligonucleotides, siRNA, shRNA, miRNA or vectors containing them can be produced using methods known in the art.

As used herein, the term "siRNA" refers to a double-stranded RNA that induces RNA interference through cleavage of the mRNA of a target gene. The term " siRNA " It consists of the RNA strand of the sequence.

The siRNA may include siRNA itself synthesized in vitro or a form expressed by inserting a nucleotide sequence encoding siRNA into an expression vector.

In the present invention, the term "vector" refers to a gene construct containing foreign DNA inserted into the genome encoding the polypeptide.

The vector associated with the present invention is a vector into which a nucleic acid sequence inhibiting the gene is inserted into the genome, and examples thereof include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.

In addition, the antisense may have an entire mRNA sequence transcribed from the STIM1 gene or a fragment thereof, or a sequence complementary to the STIM1-SBR region, and may inhibit the expression of the STIM1 gene or fragment by binding with the mRNA.

In addition, the shRNAi (short hairpin RNAi) can be prepared according to a conventional method by targeting the shRNAi common base sequence region on human or mouse.

In addition, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide But are not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool.

In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline Can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

The composition of the present invention may be administered systemically or locally, and may be formulated into a formulation suitable for such administration by known techniques. For example, upon oral administration, it may be admixed with an inert diluent or edible carrier, sealed in a hard or soft gelatin capsule, or pressed into tablets. For oral administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Various formulations for injection, parenteral administration and the like can be prepared according to techniques known in the art or commonly used techniques. Since STIM1-SBR is dissolved in saline or buffer solution, it is stored in a freeze-dried state and then an effective amount of STIM1-SBR is injected into saline or buffer in a form suitable for intravenous infusion, subcutaneous infusion, muscle infusion, intraperitoneal infusion, In the form of a solution.

An effective amount of the effective ingredient of the pharmaceutical composition of the present invention means an amount required for achieving the preventive, inhibiting or reducing effect of the disease.

Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, Rate of administration, duration of treatment, concurrent medication, and the like. For example, in the case of an adult, 0.1 ng / kg to 10 g / kg when the inhibitor of the present invention is a compound, 0.1 ng / kg when the polypeptide is a protein or an antibody, To 10 g / kg, for antisense oligonucleotides, siRNA, shRNAi and miRNA, in a dose of 0.01 ng / kg to 10 g / kg.

The present invention also provides a method for treating a muscle tension-lowering disorder in an animal comprising administering to a subject a composition for preventing or treating a muscle tension-lowering disorder comprising a pharmaceutically effective amount of a STIM1 or STIM1-SBR inhibitor .

Since the pharmaceutical composition and the administration method used in the method for treating the muscle tension-lowering related diseases have been described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

On the other hand, the individual to which the composition for preventing or treating muscle tension-lowering related diseases can be administered includes all animals. For example, it may be an animal other than a human such as a dog, a cat, or a mouse.

The present invention also relates to a method of inhibiting or reducing muscle tone (including, but not limited to, inhibition of muscle relaxation) comprising contacting a stromal interaction molecule 1 (STIM1) gene with a candidate substance outside the human body and determining whether the candidate substance promotes or suppresses expression of the gene Muscular hypotonia-related diseases. ≪ Desc / Clms Page number 2 >

In addition, the present invention relates to a method for determining whether a candidate substance interacts with a candidate substance, such as a muscle, comprising determining whether the candidate substance enhances or inhibits the function or activity of the protein, comprising contacting a stromal interaction molecule 1 (STIM1) A method for screening a medicament for preventing or treating a disease associated with Muscular hypotonia.

According to the screening method of the present invention, a candidate substance to be analyzed may be firstly brought into contact with a muscle tension-lowering disease cell comprising the gene or protein.

The candidate substance may be a substance which promotes or inhibits the transcription or translation of mRNA, protein from STIM1 or STIM1-SBR gene sequence or a substance which promotes or inhibits the function or activity of STIM1 or STIM1-SBR protein according to a conventional selection method Proteins, peptides, other extracts or natural products, compounds, etc., which are presumed to have the potential as a candidate, or randomly selected.

Thereafter, the amount of expression of the gene, the amount of the protein, or the activity of the protein can be measured in the cells treated with the candidate substance. As a result of the measurement, the expression amount of the gene, the amount of the protein or the activity of the protein is increased or decreased The candidate substance can be judged as a substance capable of treating or preventing a muscle tension-lowering-related disease.

The method of measuring the expression level of the gene, the amount of the protein, or the activity of the protein may be carried out through various methods known in the art, including, but not limited to, reverse transcription polymerase chain reaction transcriptase-polymerase chain reaction, real time-polymerase chain reaction, Western blot, Northern blot, ELISA, radioimmunoassay (RIA), radioimmunodiffusion And an immunoprecipitation assay.

The candidate substance obtained by the screening method of the present invention which shows the activity of suppressing gene expression or decreasing the function of protein may be a candidate substance for treating a muscle tension reducing disease.

Such candidates for the treatment of muscle tension-lowering-related diseases act as a leading compound in the development of therapeutic agents for muscle tension-related diseases, and the leading substances function as STIM1 or STIM1-SBR genes or proteins expressed therefrom By modifying and optimizing the structure so as to exhibit the promoting or inhibiting effect, a new therapeutic agent for muscle tension-related diseases can be developed.

(2001)) and Frederick et al. (Frederick et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Production and Expression of cDNA for STIM1-SBR

PCR was performed using the human primer STIM1 cDNA (GenBank accession number: NM_003156.3) as a sample and the PCR primers shown in Table 1. The obtained PCR product was inserted into the pMO91 vector (vector used for expressing the protein in mammalian cells) using restriction enzymes Xho I and Bam H1, and the nucleotide sequence of the prepared cDNA was confirmed again by gene sequencing.

The PCR primer sequence used in the cloning of STIM1-SBR PCR primer Restriction enzyme STIM1-SBR Forward direction 5'-CCGCTCGAGATGTCACTGGTGGCTGCCCTCAAC-3 ' Xho I Reverse 5'-GGGGATCCGTCCGGCCTGGGCTGGAGTCTGTTTC-3 Bam HI

(Method of Culturing and Differentiation of Skeletal Muscle Cells)

Primary skeletal muscle cells were isolated from skeletal muscle of mice and cultured in a cell culture (F10 Nutrient Mixture Composition: 20% FBS, 100 units / mL penicillin, 100 ㎍ / mL streptomycin, 2 mM L-glutamine, 20 nM basic fibroblast growth factor) and cultured in a 5% CO ₂ incubator at 37 ° C. As a used culture dish, a 10-cm or 96-well plate was used depending on the application, and all dishes were coated with Matrigel.

Cell differentiation was induced when the primary skeletal muscle cells grew to about 70% of the culture dish. (Cell differentiation composition: 5% heat-inactivated horse in 20% FBS and F-10 Nutrient Mixture instead of F- serum and low-glucose DMEM were used, bFGF was not added, and 18% CO ₂ incubator was used). Differentiated cells were called differentiated skeletal muscle cells (myotube), and images of differentiated skeletal muscle cells were obtained using an optical microscope.

(Expression of wild-type STIM1 and STIM1-SBR in differentiated skeletal muscle cells)

In the case of skeletal muscle cells, cDNAs for wild-type STIM1 and STIM1-SBR were transfected into skeletal muscle immature myotubes, which were three days after differentiation induction, for 4 hours (cDNA transfection: each cDNA was pMO91 CFP vector, and 30 μl of FuGENE 6, 20 μg of cDNA were treated in the case of cells cultured in a 10-cm culture dish). Cells transfected with cDNA induced further differentiation for 36 hours. The cells in this state are differentiated cells that express wild-type STIM1 and STIM1-SBR, and have been used for the experiments described below.

(Immunocytochemical method)

In order to perform immunocytochemistry on differentiated skeletal muscle cells, first, the cells were fixed with cold methanol for 30 minutes, followed by addition of anti-CFP antibody (1: 500) and FITC-conjugated anti- Antibody (1: 500, Sigma). Images of differentiated skeletal muscle cells and the degree of expression of wild-type STIM1 and STIM1-SBR were confirmed by optical microscopy and fluorescence microscopy.

FIG. 1 (A) shows the amino acid sequence of the STIM1 protein and its domains and shows the position of the STIM1-SBR used in the present invention. The numbers represent amino acid sequences.

In order to confirm the successful expression of wild-type STIM1 and STIM1-SBR in skeletal muscle cells differentiated by cDNA transfection, immunocytochemistry was performed using a CFP antibody used as a tag As a result, as shown in Fig. 1 (B), successful expression of wild type STIM1 and STIM1-SBR in skeletal muscle differentiated cells was confirmed.

<Example 2> Measurement of intracellular calcium migration

(Measurement of calcium migration in single cells)

When combined with calcium before and calcium binding is the differentiated skeletal muscle cells expressing the wild-type STIM1 and STIM1-SBR to prepare the fluo-4 (5 μM) of other fluorescent calcium fluorescent dye (Ca ^{2 +} dye) that the Incubated for 45 min at 37 &lt; 0 &gt; C. At this time, the differentiated cells were treated with an imidation solution (125 mM NaCl, 5 mM KCl, 2 mM KH ₂ PO ₄ , 2 mM CaCl ₂ , 25 mM HEPES, 6 mM glucose, 1.2 mM MgSO ₄ , 0.05% BSA ) Have been processed. Fluorescence microscopy (Nikon x40 oil-immersion objective, NA 1.30, ECLIPSE Ti, Nikon) was used to measure intracellular calcium mobilization. Fura-2 was used instead of fluo-4 to measure the absolute calcium concentration change by intracellular calcium mobilization. Fluorescence changes of calcium fluorescent dye were transferred to a computer using a 75-watt Xenon lamp (FSM150Xe, Bentham Instruments, Ltd) connected to a fluorescence microscope and a 12-bit CCD camera (DVC-340M-OO-CL, Digital Video Camera Company) InCyt Im1 image acquisition and analysis software (v5.29, Intracellular Imaging Inc) was used for data using fluo-4 and High-Speed InCyt Im2 image acquisition and analysis was used for data using fura-2. analysis software (v5.29, Intracellular Imaging Inc). Cells were treated with 8-channel perfusion pipette (AutoMate Scientific). To measure the amount of calcium that can be liberated from myocyte retinosis, cells were treated with thapsigargin (TG) in the absence of external calcium, and the cytoplasmic calcium released from the myocyte reticulum was treated with fluo-4 Respectively.

(Storage - operating external calcium influx (SOCE) measurement)

For storage-operated external calcium influx measurement experiments, the calcium-free imaging solution without calcium was treated with cells for 5 minutes, followed by thapsigargin (TG) to induce depletion of calcium in SR, Then, the normal imaging solution was reprocessed into the cells to measure the migration of calcium into the interior from the outside of the cell, that is, the SOCE. TG Me ₂ SO (<0.05%) and treated with cells according to the manufacturer's instructions, indicating no change in cellular calcium migration due to Me ₂ SO (<0.05%) itself. The results of TG treatment and SOCE measurement with relatively long measurement time (more than 20 minutes) were statistically processed for the area of calcium migration peak.

 (Statistical analysis)

All data are expressed as ± SEM by combining the data obtained through a number of experiments. The value obtained in the control was regarded as 1 and expressed in a normalized ratio manner in which the relative change was expressed. Significant differences were determined using paired t- test (GraphPad InStat, v2.04) and significant differences were marked with an asterisk for P <0.05. Graphing was done using Origin v7 program.

As a result of treating caffeine (caffeine, an activator of the caffeine, the calcium necessary for the skeletal muscle contraction in the cytoplasm of cytoplasmic reticulum) as a wild-type STIM1 or STIM1-SBR expressing skeletal muscle cell, As compared to the negative control (vector control), the wild-type STIM1 did not cause any change, whereas the STIM-SBR significantly lowered the calcium release compared to the negative control. The results on the left are plotted on the bar graph on the right. (*) When there was a significant difference compared to the negative control group.

That is, STIM1-SBR, unlike wild-type STIM1, plays a role in lowering the intracellular calcium migration required for skeletal muscle contraction.

FIG. 3 (A) shows that in the dormant state, the skeletal muscle differentiated cells expressing STIM1-SBR have low calcium in the cytoplasm (approximately 20% reduced compared to the negative control group or the differentiated skeletal muscle cells expressing the wild-type STIM1 ) (See Table 2 for the absolute calcium amount).

Figure 3 (B) is to measure the amount of calcium that can be liberated from muscle cytoplasmic reticulum (releasable Ca ^{2 +} from the SR), process the tapsi with the cells in the absence of external calcium state, the glass in the near cytoplasmic reticulum As a result, STIM1-SBR-expressing skeletal muscle-differentiated cells showed that the amount of calcium in the cytoplasm was increased from the mycotic cytoplasm to the cytoplasm (about 38%).

In other words, it was confirmed that STIM1-SBR expression decreased the amount of calcium in the dormant state and the amount of calcium that could be liberated in the myocyte reticulum.

To determine whether there is a change in storage-activity external calcium influx (SOCE) by STIM1-SBR, single cell calcium imaging was performed in single cells. After pretreatment with TG, the cells were depleted of calcium in the cytoplasmic reticulum of the cells and treated with 2 mM calcium to induce SOCE.

As shown in FIG. 4, relatively high external calcium influx (SOCE) was confirmed (about 47% increase) in the differentiated skeletal muscle cells expressing wild-type STIM1, but STIM1-SBR showed no change in SOCE It did not happen. The bar graph on the right represents SOCE changes in a standardized ratio fashion based on the SOCE of a control skeletal muscle cell (control vector) that does not express anything. That is, invariance of SOCE by STIM1-SBR expression was confirmed.

Comparison of the characteristics of differentiation skeletal muscle cells expressing wild-type STIM1 and STIM1-SBR Control group Wild type STIM1 STIM1-SBR Caffeine reaction 1.00 + - 0.15
(108) 1.04 + 0.11
(129) 0.69 ± 0.13 ^*
(151) In the resting state, cytoplasmic [Ca ^{2 +} ] (nM) 86.48 + - 7.93
(159) 84.00 ± 8.69
(153) 67.60 + - 7.09 ^*
(187) The Ca ^{2 +} 1.00 + - 0.11
(181) 0.97 + 0.14
(189) 1.38 + - 0.20 ^*
(194) SOCE 1.00 0.20
(111) 1.47 ± 0.17 ^*
(136) 1.06 ± 0.16
(168) Data of differentiated skeletal muscle cells expressing wild-type STIM1 or STIM1-SBR are expressed in a normalized ratio manner based on data obtained from a control vector (vector-infected) differentiation control skeletal muscle cells (Control Vector). The numbers in parentheses are the number of cells used for experiments and analysis.

From these results, the present inventors have found that the activity of the lysozyme receptor calcium channel in the differentiated skeletal muscle cells expressing STIM1-SBR is lowered.

In addition, abnormal distribution of calcium was found in differentiated skeletal muscle cells expressing STIM1-SBR. As a result of measuring the cytoplasmic calcium concentration in the dormant state, the presence of relatively low calcium in the cytoplasm of skeletal muscle differentiating cells expressing STIM1-SBR was found. In addition, the amount of calcium available to the cytoplasm from the cytoplasmic reticulum was increased. Thus, in the case of skeletal muscle differentiated cells that found STIM1-SBR, the distribution of intracellular calcium was overall abnormal. In other words, the activity of SERCA1a is increased by STIM1-SBR, and the increased amount of SERCA1a activity causes the calcium to be present in the cytoplasmic reticulum to decrease in the amount of calcium in the cytoplasm and increase in the amount of calcium in the cytoplasmic reticulum.

Next, we found that there was no SOCE change in skeletal muscle differentiated cells expressing STIM1-SBR. Unlike wild-type STIM1, which increased SOCE, skeletal muscle differentiated cells expressing STIM1-SBR had no change in SOCE. In other words, SBR (449-671) of STIM1 was found to have no relation with the SOCE parameter, which is a unique role of STIM1.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Pharmaceutical use of STIM1 <130> P131058 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 223 <212> PRT <213> Homo sapiens stromal interaction molecule 1 (STIM1) C-terminus <400> 1 Ser Leu Val Ala Leu Asn Ile Asp Pro Ser Trp Met Gly Ser Thr   1 5 10 15 Arg Pro Asn Pro Ala His Phe Ile Met Thr Asp Asp Val Asp Asp Met              20 25 30 Asp Glu Ile Val Ser Pro Leu Ser Met Gln Ser Pro Ser Leu Gln          35 40 45 Ser Ser Val Arg Gln Arg Leu Thr Glu Pro Gln His Gly Leu Gly Ser      50 55 60 Gln Arg Asp Leu Thr His Ser Asp Ser Glu Ser Ser Leu His Met Ser  65 70 75 80 Asp Arg Gln Arg Val Ala Pro Lys Pro Pro Gln Met Ser Arg Ala Ala                  85 90 95 Asp Glu Ala Leu Asn Ala Met Thr Ser Asn Gly Ser His Arg Leu Ile             100 105 110 Glu Gly Val His Pro Gly Ser Leu Val Glu Lys Leu Pro Asp Ser Pro         115 120 125 Ala Leu Ala Lys Lys Ala Leu Leu Ala Leu Asn His Gly Leu Asp Lys     130 135 140 Ala His Ser Leu Met Glu Leu Ser Pro Ser Ala Pro Pro Gly Gly Ser 145 150 155 160 Pro His Leu Asp Ser Ser Arg Ser His Ser Ser Ser Ser Pro Asp Pro                 165 170 175 Asp Thr Pro Ser Pro Val Gly Asp Ser Arg Ala Leu Gln Ala Ser Arg             180 185 190 Asn Thr Arg Ile Pro His Leu Ala Gly Lys Lys Ala Val Ala Glu Glu         195 200 205 Asp Asn Gly Ser Ile Gly Glu Glu Thr Asp Ser Ser Pro Gly Arg     210 215 220

Claims (8)

A composition for preventing or treating a Muscular hypotonia-related disease comprising a stromal interaction molecule 1 (STIM1) inhibitor.
The method according to claim 1,
Wherein the STIM1 inhibitor inhibits the SERCA1a-binding region (SBR) of STIM1 represented by the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 1.
3. The method according to claim 1 or 2,
STIM1 is a composition for preventing or treating a muscle tension-lowering disease, which is derived from human, mouse, rat, rabbit, orangutan, monkey, hamster, cat, dolphin or gorilla.

3. The method according to claim 1 or 2,
STIM1 inhibitors include antisense-oligonucleotides, siRNA, shRNA, miRNA or vectors containing the same; Or an antibody for preventing or treating muscle tension-related diseases.
3. The method according to claim 1 or 2,
Wherein the STIM1 inhibitor inhibits the deactivation of the ryanodine receptor calcium channel by STIM1 or STIM1-SBR, thereby promoting skeletal muscle contraction.
The method according to claim 1,
The muscle tension-related diseases are related to muscle tone depression including Down syndrome, Muscular dystrophy, cerebral palsy, Prader-Willi syndrome, or Tay-Sachs disease. A composition for preventing or treating diseases.
A stromal interaction molecule 1 (STIM1) gene is contacted with a candidate substance outside the human body,
And determining whether the candidate substance promotes or suppresses the expression of the gene. The method for screening a medicament for preventing or treating a Muscular hypotonia-related disease.
A stromal interaction molecule 1 (STIM1) protein is contacted with a candidate substance outside the human body,
And judging whether the candidate substance enhances or inhibits the function or activity of the protein. The method for screening a medicament for preventing or treating a Muscular hypotonia-related disease.

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