KR101075975B1 - Human CIB protein and CIB gene thereof preventing stress-induced cell death - Google Patents

Abstract

The present invention relates to a Calcium and integrin binding protein 1 (CIB1) protein that inhibits apoptosis, a CIB1 gene encoding the protein, and a use thereof, and specifically, to yeast 2-hybrid screening. It relates to the CIB1 protein, the CIB1 gene, and its use in combination with the apoptosis signal-regulating kinase ASK1 (apoptosis signal-regulating kinase 1), which inhibit cell death. The human CIB1 protein or CIB1 gene of the present invention may be used as a selective inhibitor of ASK1-mediated cell death or may be useful for the treatment, prevention, diagnosis or research process of degenerative neurological diseases such as Parkinson's disease and immune system diseases. .

CIB1, ASK1, Calcium, Apoptosis

Description

Human CIB protein and CIB gene affecting stress-induced cell death}

The present invention relates to a calcium-dependent CIB1 (Calcium and integrin binding protein 1) protein, CIB1 gene encoding the protein and its use for inhibiting apoptosis, specifically yeast 2- hybrid screening (yeast 2- The present invention relates to a CIB1 protein that binds to apoptosis signal-regulating kinase ASK1 by hybrid screening and inhibits apoptosis, a CIB1 gene encoding the protein, and its use.

In general, mitogen-activated protein kinase (MAPK) signaling is closely associated with apoptotic cell death. MAPK signaling is known to regulate the expression of numerous genes that are activated by extracellular stimuli and are involved in apoptosis and survival. Protein kinases involved in MAPK signaling are divided into three different components: MAPKs, MAPK kinase (MAPKKs) and MAPK kinase kinase (MAPKKKs). Looking at their mechanism of action, MAPKKKs activated by the kinase or conjugate protein in the upper position of the protein kinase induces the activity by phosphorylating MAPKKs, and thus induced MAPKKs induced to activate MAPKs again. Active MAPKs then move into the nucleus to regulate gene expression or other cellular events by activating various transcription factors or kinase in lower positions. Signaling mechanisms associated with apoptotic apoptosis, such as MAPK signaling, have revealed at least three different signaling pathways in mammalian cells. Extracellular signal-regulated kinase (ERK), c -jun N-terminal kinase / stress-activated protein kinase (c-jun N-terminal kinase / stress-activated protein kinase) and p38 MAPK.

ASK1 is a type of MAPKKK that selectively activates the SEK1-JNK and MKK3 / 6-p38 pathways (Ichijo et al., 1997, Science 275: 90-94) , TNFα- and Fas-induced intracellular signal cascade It is known to play an important role in cytokine-induced apoptosis as a mediator of (signal cascade) (Bijangi-Vishehsaraei et al., 2005, Blood 106 (13): 4124-30; Chang et al., 1998, Science 281: 1860 ). These ASK1-dependent cell signaling is thioredoxin, glutaredoxin, p21, heat shock protein 72, tumor necrosis factor receptor associated factors 2 (TRAF2), Daxx, CIIA, glutathione S-transferase Mu, 14-3-3, and SOCS1. Regulated by a variety of proteins that bind to ASK1 (Chang et al. 1998, Science 281: 1860; Nishitoh et al. 1998, Mol. Cell 2: 389-395; Saitoh et al. 1998, EMBO J. 17: 2596 -2606; Asada et al. 1999, EMBO J. 18: 1223-1234; Cho et al. 2001, J. Biol. Chem. 276: 12749-12755; Park et al. 2002, EMBO J. 20: 446-456 Song et al. 2002, J. Biol. Chem. 277: 46566-46575; Cho et al. 2003, J. Cell Biol. 163: 71-81; Zhang et al. 1999, Proc. Natl. Acad. Sci. 96: 8511-8515; He et al. 2006, J. Biol. Chem. 281: 5559-5566).

Meanwhile, CIB1 (calcium and integrin binding protein 1), also known as calmyrin, is a 22kDa protein, showing sequence similarity with calmodulin, calcineurin, and calcium regulatory protein groups in neurons. CIB1 consists of two calcium-bound EF-hands at the C-terminus and two EF-hands that do not function at the N-terminus (Gentry et al. 2005, J. Biol. Chem. 280: 8407 -8415; Blamey et al. 2005, Protein Sci. 14: 1214-1221). First known in platelets as proteins that bind to the cytoplasm of Integrin α _IIb (Naik et al. 1997, J. Biol. Chem. 272: 4651.4654), the current protein is presenilin 2 (Stabler et al. 1999, J. Cell Biol) 145: 1277-1292) , DNA-dependent protein kinase (Wu and Lieber 1997, Mutat. Res. 385: 13-20), the polo-like kinases Fnk and Snk (Kauselmann et al. 1999, EMBO J. 18: 5528-5539), Rac3 (Haataja et al. 2002, J. Biol. Chem. 277: 8321-8328), and Pax3 (Hollenbach et al. 2002, Biochim. Biophys. Acta 1574: 321-328) It is combined with these. Furthermore, it has been reported that CIB1 binds to calcium, resulting in conformational changes leading to changes in binding capacity with proteins that bind to this protein, such as integrin, presenilin 2, and PAK (Yamniuk et al. 2004). , Biochemistry 43: 2558-2568). CIB1 expressed in various tissues is expected to perform a variety of functions in cells, but it is necessary to elucidate specific biological functions (Shock et al. 1999, Biochem. J. 342: 729.735; Whitehouse et al. 2002, Eur. J. Biochem. 269: 538-545).

It is an object of the present invention to provide a novel substance capable of modulating the activity of ASK1, which affects apoptosis activity induced by various kinds of stress. In order to achieve the above object, the present invention provides a human CIB1 protein and cDNA encoding the same. The present invention also provides a gene therapy agent comprising a gene construct containing a human CIB1 gene as an active ingredient. Finally, the present invention provides an inhibitor of apoptosis activity comprising human CIB1 protein and a therapeutic agent for apoptosis-related diseases induced by various stresses.

In order to achieve the above object, the present invention provides a composition for preventing or treating apoptosis-related diseases comprising human CIB1 (Calcium and integrin binding protein 1) protein as an active ingredient.

In one embodiment of the invention, the protein comprises all of the known CIB1 protein and its functional fragments having the function of the protein, preferably characterized by having the amino acid sequence set forth in SEQ ID NO: 1. In addition, in the amino acid sequence of SEQ ID NO: 1, deletion, substitution and addition of at least one mutation of one or more amino acids are introduced within a range in which the characteristics of the CIB1 protein indicated by the protein having these amino acid sequences are not impaired. Variant CIB1 is also included in CIB1 according to the present invention.

In addition, the present invention includes a CIB1 gene encoding a CIB1 protein having the amino acid sequence of SEQ ID NO: 1, and the gene sequence thereof may be represented by SEQ ID NO: 2. In addition, the mutant CIB1 gene encoding the mutant CIB1 protein obtained by mutating the nucleotide sequences of SEQ ID NO: 2 is also included in the CIB1 gene according to the present invention.

In one embodiment of the invention, the protein preferably interacts with calcium, the protein is characterized in that it inhibits ASK1 (apoptosis signal-regulating kinase 1) activity, but is not limited thereto.

In one embodiment of the present invention, the cells are preferably neurons, cancer cells or immune cells, but is not limited thereto.

In another preferred embodiment of the present invention, the cell related disease is preferably a neurological disease, cancer or immune disease.

In one embodiment of the present invention, the cerebral neurological disease is preferably selected from the group consisting of Lou Gehrig's disease, Alzheimer's dementia, Parkinson's disease, Huntington's disease, stroke, traumatic brain injury and traumatic spinal cord injury, but is not limited thereto.

In another embodiment of the present invention, the cancer is breast cancer, liver cancer, lung cancer, gastric cancer, cerebrospinal tumor, head and neck cancer, thymoma, mesothelioma, esophageal cancer, colon cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer Cancer, selected from the group consisting of germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.

In another embodiment of the present invention, the immune disease is preferably selected from the group consisting of rheumatoid arthritis, psoriasis and asthma, but is not limited thereto.

Meanwhile, the composition for preventing or treating apoptosis-related diseases of the present invention may further include a pharmaceutically acceptable excipient, carrier, diluent, and the like.

Carriers usable in the present invention include macromolecules that are slowly metabolized, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers and inert virus particles. Also, for example, salts of inorganic acids such as hydrochloride, hydrobromide, phosphate and sulfate; Pharmaceutically acceptable salts such as salts of organic acids such as acetates, propionates, malonates and benzoates; Liquids such as water, saline, glycerol and ethanol, and auxiliary substances such as wetting agents, emulsifiers or pH buffering materials can be used.

Pharmaceutically acceptable carriers are described in Remingtion's Pharmaceutical Sciences, Mack Publishing Company, 1991.

The prophylactic and therapeutic agents can be administered orally, by injection (eg, intramuscular injection, intraperitoneal injection, intravenous injection, oral or parenteral), depending on the condition and the condition of the individual to be treated during clinical administration. Parenteral injection administration is preferred, and may be formulated into a suitable dosage unit formulation comprising a pharmaceutically acceptable carrier, excipient, vehicle, conventionally used and non-toxic, depending on the route of administration. Depot formulations that may be included are also within the scope of the present invention.

The effective dose of the protein of the present invention and its mutant protein is 0.01 to 100 mg / kg body weight daily, preferably 0.1 to 10 mg / kg body weight can be administered once or divided into several times a day. However, the actual dosage of the active ingredient is dependent on a number of related factors such as the disease to be prevented or treated, the severity of the disease, the route of administration, the patient's weight, age and sex, combination of the drug, sensitivity to the reaction and resistance / response to treatment. It is to be understood that it is to be determined, and therefore, that dosage does not limit the scope of the invention in any aspect.

CIB1 of the present invention may also be administered by gene therapy. To this end, the CIB1 of the present invention must be introduced into a patient according to conventional gene therapies in the art so that it can be expressed from the DNA sequence and combined in situ .

 Hereinafter, the present invention will be described.

The present inventors have studied to find a protein that can physically bind to ASK1, and identified ASK1-binding protein CIB1 (Calcium and integrin binding protein 1). The present inventors completed the present invention by confirming that CIB1 directly binds with ASK1 to inhibit stress- or cytokine-induced ASK1 activity, thereby inhibiting cell death by ASK1-induced apoptosis. Therefore, the human CIB1 protein or CIB1 gene of the present invention can be used as a selective inhibitor against ASK1-mediated apoptosis or useful for treating, preventing, diagnosing or researching degenerative brain neurological diseases such as Parkinson's disease, immune system diseases, and the like. Available.

Hereinafter, a gene therapy method using the CIB1 gene for treating or preventing apoptosis caused by stress of the present invention will be described in more detail.

In the present invention, ASK1 activation is a major cause of apoptosis in the apoptotic apoptosis process by TNF-α and 6-OHDA, and this process is effectively apoptosis by CIB1 protein and gene active ingredients whose properties are controlled by calcium. Is based on the amazing fact that it is suppressed. Moreover, the fact that the biological function of CIB1 protein and gene, which has not yet been revealed to date, has been presented for the first time, it is another significance of the present invention.

That is, when the expression of the CIB1 gene is increased as in the present invention, diseases related to apoptosis, for example, degenerative neurological diseases such as Parkinson's disease, immune system diseases, etc. can be effectively treated or prevented. However, the gene therapy according to the present invention is applicable to any disease associated with apoptotic apoptosis due to stress, and the scope of the present invention is not limited to the above-described degenerative neurological disease, cancer, immune system disease or inflammation.

The human CIB1 protein of the present invention and the gene encoding it selectively bind to ASK1 as a selective inhibitor of apoptosis, which is regulated by calcium and effectively inhibits apoptotic apoptosis, so that the protein selectively inhibits ASK1 protein or cells The gene may be usefully used in treating diseases involving death-related processes, and the gene may be used in gene therapy for degenerative neurological diseases, such as Parkinson's disease, and immune system diseases.

Hereinafter, the following examples and the like will be described in order to describe the present invention in more detail. However, embodiments according to the present invention can be modified in many different forms and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided by way of example in order to facilitate a specific understanding of the present invention.

Example 1 In Vitro Binding Assay of CIB1 and ASK1

We have prepared GST-fusion recombinant proteins of various ASK1 deletion mutants to investigate where in the ASK1 protein the CIB1 protein binds. Specifically, ASK1 (1-377) including amino acids of ASK1-N-terminals 1 to 377, ASK1 (378-648) containing amino acids of ASK1-N-terminals 378 to 648 Recombination of ASK1 (649-938) comprising amino acids of ASK1-N-terminals 649-938, ASK1 (939-1374) comprising amino acids of ASK1-N-terminals 939-1374 Proteins were prepared and labeled with 35 S-methionine and then in vitro transcribed using the TNT reticulocyte lysate system (Promega). The recombinant proteins isolated and purified as described above were fixed on glutathione-agarose beads, and then mutant recombinant proteins of [35 S] methionine-labeled ASK1 or ASK1 in vitro transcribed. Each CIB1 GST-fusion protein in buffer (composed of 50 mM Tris, pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40 and 5 mg / ml BSA) The reaction was carried out for a time. After completion of the reaction, wash three times with wash buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Tween 20) and 35 S from each glutathione-agarose beads. -Labeled proteins were eluted and analyzed by SDS-polyamide gel electrophoresis.

As a result, it was confirmed that the CIB1 GST fusion protein directly binds to ASK1 (378-648) containing amino acids of the ASK1-N-terminal part 378 to 648 (Fig. 1a). As previously reported, it suggests that the regulatory domain constituting the N-terminus of ASK1 binds directly to CIB1.

Order in the cell to investigate the binding of ASK1 and CIB1 after performing immunoprecipitated by ASK1 antibody in 293T cell line was confirmed that the result of ASK1 and CIB1 performing a Western blot combine the precipitate with CIB1 antibody, H ₂ O It can be seen that the binding does not change due to stress such as ₂ and TNF-α (FIG. 1B).

Example 2 H of CIB1 ₂ O ₂  And inhibition of ASK1 activation by TNF-α

Previous reports have shown that ASK1 is activated by external stimuli, such as H ₂ O ₂ and TNF-α, which can cause free radicals (Saitoh et al. 1998, EMBO J. 17: 2596-2606; Liu et al. 2000, Mol. Cell. Biol. 20: 2198-2208; Takeda et al. 2008, Annu. Rev. Pharmacol. Toxicol. 48: 199-225).

Therefore, the present inventors performed an in vitro kinase assay to investigate whether ASK1 activation by H ₂ O ₂ and TNF-α can inhibit CIB1. Mammalian cell lines 293 cells were transfected with or simultaneously with Flag-CIB1 and HA-ASK1, and then treated with H ₂ O ₂ and TNF-α to induce ASK1 activity. The transfected cells were treated with buffer A (20 mM Tris-HCl [pH 7.4], 150 mM sodium chloride, 1% Triton X-100, 1% deoxycholate, 12 mM β-glycerphosphate, 10 mM sodium florids, 5 mM EGTA and 1). Hemolysis using mM PMSF) was followed by centrifugation at 12,000 g for 15 minutes at 4 ° C. The precipitate was removed after centrifugation and the lysate obtained was immunoprecipitation using anti-HA antibody. Protein G-Sepharose TM beads (Protein G sepharose TM bead, Pharmacia) were added to the reaction solution, and then carefully mixed at 4 ° C. for 1 hour. The immunocomplexes obtained therefrom were subjected to immunocomplex kinase assay to analyze ASK1 activity.

ASK1 activated by H ₂ O ₂ and TNF-α was confirmed that the activity is significantly reduced when CIB1 is expressed at the same time (Fig. 2a).

In order to investigate the endogenous function of CIB1 that regulates the activity of ASK1, CIB1 siRNA and control siRNA (control siRNA) were injected into HeLa cells to reduce intracellular CIB1 expression, and then ASK1 activity was measured. The activity of ASK1 activated by H ₂ O ₂ and TNF-α was further increased by injection of CIB1 siRNA (FIG. 2B), and the ASK1-JNK / p38 MAPK activity mediated by ASK1 was also increased by injection of CIB1 siRNA. Showed a tendency (FIG. 2C). Thus the endogenous function of CIB1 suggests the possibility of acting as a negative regulator of ASK1 activated by H ₂ O ₂ and TNF-α.

Example 3 Inhibition of ASK1 Activation of CIB1

Three mechanisms of ASK1 activation have been reported to date: first, homo-oligomer formation between ASK1 and binding of TRAF protein to ASK1, and finally autophosphorylation of threonine groups of ASK1 kinase domain. ASK1 activation has been reported (Gotoh and Cooper 1998, J. Biol. Chem. 273: 17477-17482; Liu et al. 2000, Mol. Cell. Biol. 20: 2198-2208; Tobiume) et al. 2002, J. Cell. Physiology. 191: 95-104; Noguchi et al. 2005, J. Biol. Chem. 280: 37033-37040; Fujino et al. 2007, Mol. Cell. Biol. 27: 8152 -8163).

The present inventors investigated each case to find the portion of the above-mentioned ASK1 activation mechanism regulated by CIB1.

In order to investigate the effect of CIB1 on the formation of ASK1 homopolymers, immunoprecipitation was performed. Next, to investigate the effect of CIB1 on the binding capacity of ASK1 and TRAF2 proteins, immunoprecipitation was performed after transfection of 293 cells with the combination shown in FIG. 3A with Flag-CIB1, Flag-TRAF2, and HA-ASK1. Transfection of Flag-TRAF2 and HA-ASK1 together increased binding between the two proteins, and high expression of CIB1 decreased significantly the binding between the two proteins (FIG. 3a). In addition, CIB1 siRNA (SEQ ID NO: 3) and control siRNA (control siRNA; SEQ ID NO: 4) were injected into HeLa cells to inhibit intracellular CIB1 expression, and H ₂ O ₂ was treated to increase the binding capacity between ASK1 and TRAF2 proteins. The impact of the following CIB1 was investigated. Increased binding capacity between the two proteins by H2O2 increased the binding capacity by decreasing the expression of CIB1 with siRNA (FIG. 3B).

To investigate the role of CIB1 in the process of autophosphorylation, which is the final stage of the ASK1 activation mechanism, immunoprecipitation was performed with anti-ASK1 antibody after transfection as shown in FIG. 3A and Western blot was performed with anti-phospho ASK1 antibody (pASK1). As a result, transfection of HA-ASK1 undergoes phosphorylation of ASK1, but over-expression of Flag-CIB1 and HA-ASK1 simultaneously reduces this phosphorylation. In addition, this phenomenon is more clearly observed by treating an environment capable of raising ASK1 activity, for example, H ₂ O ₂ (FIG. 3C). Then, as FIG. 3b is injected CIB1 siRNA and control siRNA (control siRNA) in HeLa cells inhibit the intracellular CIB1 expression by treatment with H ₂ O ₂ was examined whether phosphorylation of ASK1. As a result, injection of CIB1 siRNA further increased ASK1 phosphorylation by H ₂ O ₂ (FIG. 3D). In other words, the inhibition of ASK1 activity by CIB1 is suggested by controlling the binding capacity between TRAF2 and ASK protein and the process of ASK1 autophosphorylation.

Example 4 Influence of CIB1 Expression Inhibition on ASK1 Activation by 6-OHDA and TNF-α and Apoptotic Apoptosis Induced by ASK1

6-Hydroxydopamine (6-OHDA), a well known drug that induces Parkinson's disease, produces free radicals and kills dopaminergic neurons, and it has been reported that ASK1 mediates this fixation (Ouyang and Shen 2006, J. Neurochem. 97: 234-244; Pan et al. 2007, Molecular Pharmacology. 72: 1607-1618).

We injected CIB1 siRNA and control siRNA (control siRNA) into the SH-SY5Y neuronal cell line to limit CIB1 expression and then treated with 6-OHDA to measure ASK1 activity and neuronal cell death.

When the ASK1 activity was measured by the method mentioned in FIG. 2B, when the expression of CIB1 was restricted by the injection of CIB1 siRNA, the ASK1 activity increased by 6-OHDA in the control group was further increased, and apoptotic neurons were flow cytometry method. Quantification of apoptosis, similar to the results seen in ASK1 activity, increased apoptosis by 6-OHDA in the group that restricted CIB1 expression (FIG. 4A). Next, in the MCF7 cells, which are human breast cancer cell lines, the intracellular expression of CIB1 was suppressed in the same manner as in FIG. 4A, and then treated with TNF-α to measure the activity and apoptosis of ASK1. Similar to the results of FIG. 4A, ASK1 activity increased by TNF-α was further increased when CIB1 expression was suppressed, and MCF7 apoptosis was also increased by TNF-α (FIG. 4B). In other words, CIB1 suggests that apoptosis can be selectively controlled by various cell deaths mediated by ASK1, for example, dopaminergic neuronal cell death by 6-OHDA and TNF-α.

Example 5 Effect of Calcium on Inhibitory Effect of CIB1 on ASK1 Activation and Apoptotic Apoptosis by 6-OHDA

CIB1 proteins as a Ca ²⁺ binding protein that has two functional EF-hand motif, and Ca ²⁺ is when combined produces a structural change (change confromational) reported (Hwang and Vogel 2000, J. Mol . Recognit 13: 83-92; Yamniuk et al. 2004, Biochemistry 43: 2558-2568).

Therefore, the present inventors treated with 6-OHDA alone or influx of intracellular Ca ²⁺ to SH-SY5Y cells, which are neuron-derived cell lines, to investigate the effect of intracellular influx of Ca ^{2+ on} the regulation of ASK1 activation through CIB1. KCl was treated together to induce. BAPTA-AM was treated together to determine whether this phenomenon was inhibited by Ca ²⁺ chelator (see FIG. 5A). After immunoprecipitation with ASK1 antibody under the above conditions, the precipitated lysates were subjected to Western blot using CIB1 antibody or TRAF2 antibody, respectively.

The physical binding of ASK1 and CIB1 by 6-OHDA treatment was inhibited by the influx of intracellular Ca ²⁺ due to the change of membrane potential by KCl, and this phenomenon was recovered by Ca ²⁺ chelator called BAPTA-AM. In addition, as a result of investigating the binding of TRAF2 protein in the ASK1 activity regulation mechanism of CIB1 shown in FIG. 3 under the treatment conditions shown in FIG. 5A, physical binding of ASK1 and TRAF2 by 6-OHDA treatment was performed by intracellular Ca ²⁺ Influx was further increased, which was inhibited by a Ca ²⁺ chelator called BAPTA-AM.

Flag-CIB1 (D127N) caused by HA-ASK1 and Flag-CIB1 or point mutations in SH-SY5Y cells to confirm whether the phenomenon shown in 5a regulates the physical binding capacity of CIB1 and ASK1 due to Ca ²⁺ influx. / E172Q), respectively, and then treated with the combinations shown in FIG. 5B. Flag-CIB1 (D127N / E172Q), produced by point mutations, is characterized by impaired Ca ²⁺ binding capacity of CIB1 EF-hand. Then, to investigate the changes in the physical binding capacity of Flag-CIB1 and HA-ASK1, immunoprecipitation was performed with Flag antibody and Western blot was performed with HA antibody. In the group transfected with Flag-CIB1 and HA-ASK1 with normal Ca ²⁺ binding capacity, the physical binding of ASK1 and CIB1 by 6-OHDA treatment was inhibited by the influx of intracellular Ca ²⁺ due to the membrane potential change by KCl. This phenomenon was recovered by a Ca ²⁺ chelator called BAPTA-AM (see FIG. 5B). On the other hand, in the group transfected with Flag-CIB1 (D127N / E172Q) and HA-ASK1, which had impaired Ca ²⁺ binding ability, the physical binding of ASK1 and CIB1 by 6-OHDA treatment was related to intracellular Ca ² due to the change in membrane potential by KCl. ^{Although +} was introduced, there was no effect on the physical binding between the two proteins, and this phenomenon did not change even after treatment with Ca ²⁺ chelator called BAPTA-AM (see FIG. 5B). These experimental results support the fact that intracellular Ca ²⁺ influx can regulate the physical binding of CIB1 and ASK1.

Inhibition of intracellular CIB1 protein expression in neuronal cell-derived SH-SY5Y cells to investigate the possibility that intracellular Ca ²⁺ influx regulates the activity of ASK1 induced by 6-OHDA and apoptotic apoptosis by ASK1 After transfection of CIB1 siRNA or a control (control siRNA), the drug treatment was performed in the combination shown in FIG. 5C, and then the activity of ASK1 was quantified by an in vitro kinase assay method by the same method used in FIG. 2A. In the group injected with control siRNA, ASK1 activated by 6-OHDA was further activated by the influx of intracellular Ca ²⁺ , and this phenomenon was dramatically suppressed by BAPTA-AM. The degree of activation of ASK1 according to CIB1 siRNA was activated by 6-OHDA, but there was no change in the influx of intracellular Ca ²⁺ and BAPTA-AM treatment for inhibiting it (see FIG. 5C). Apoptosis was quantified by flow cytometry, but apoptosis was increased in CIB1 siRNA-injected cells compared to the control group, but the effect of intracellular Ca ²⁺ on BAPTA-AM treatment was significantly affected by apoptosis. Did not look (see FIG. 5D). In the control group, 6-OHDA-induced apoptosis was further increased by the influx of intracellular Ca ²⁺ , which was suppressed by BAPTA-AM. These results suggest that the influx of intracellular Ca ²⁺ may not only inhibit the physical binding of CIB1 and ASK1, but may also affect the activation of ASK1 and further ASK1 mediated various cell death.

Example 6 Effect of Calcium on Inhibitory Effect of CIB1 on 6-OHDA-induced ASK1 Activation and Apoptotic Apoptosis in Middle Brain Dopaminergic Neurons

In order to further solidify the physiological significance of ASK1 activation and ASK1-mediated apoptosis-induced apoptosis by Ca ²⁺ -dependent CIB1, the present inventors conducted a similar experiment shown in FIG. The model was carried out. In order to investigate the possibility that intracellular Ca ²⁺ influx regulates 6-OHDA-induced ASK1 activity and apoptosis apoptosis by ASK1, the expression of intracellular CIB1 protein in cultured midbrain dopaminergic neurons. CIB1 siRNA (SEQ ID NO: 5) or control (control siRNA; SEQ ID NO: 6) After transfection and drug treatment with the combination shown in FIG. 6A, the activity of ASK1 was quantified by the in vitro kinase assay method by the same method used in FIG. 2A.

In the group injected with control siRNA, ASK1 activated by 6-OHDA was further activated by the influx of intracellular Ca ²⁺ , and this phenomenon was completely suppressed by BAPTA-AM. The degree of activation of ASK1 according to the CIB1 siRNA was activated by 6-OHDA, but there was no change in the influx of intracellular Ca ²⁺ and BAPTA-AM treatment to inhibit it (see FIG. 6A). After treatment with cultured midbrain dopaminergic neurons in the combination shown in Figure 6b, and then stained with TH (tyrosine hydroxylase) antibody, a marker antibody of dopaminergic neurons to quantify cell death of dopaminergic neurons. Only cells positive for TH antibody were counted. Compared with the control group, apoptosis was further increased in cells injected with CIB1 siRNA, but there was no significant effect on apoptosis when BAPTA-AM treatment was performed to inhibit the influx of intracellular Ca ²⁺ . In the control group, 6-OHDA-induced apoptosis was significantly increased by the influx of intracellular Ca ²⁺ , and this phenomenon was shown to be inhibited by BAPTA-AM (FIG. 6B). As a result, the present inventors showed that CIB1 protein showed activity of ASK1 and apoptosis suppression and cytoprotective effect through ASK1 were reproduced not only in cell line model but also in midbrain dopaminergic neuron culture model. It is further strengthening its potential as gene therapy for the treatment of diseases of the immune system or inflammation.

1 is a diagram showing the binding between CIB1 and ASK1 protein.

2 is a diagram showing the effect of CIB1 on ASK1 activation by H2O2 or TNF-α.

3 is a diagram showing a mechanism for regulating ASK1 activation of CIB1.

4 shows the effect of CIB1 protein on ASK1 mediated apoptosis.

5 is a view showing the effect on the inhibition of ASK1 activation and ASK1 mediated apoptosis by CIB1 according to intracellular Ca ²⁺ influx.

6 is a diagram confirming the change in the properties of CIB1 according to intracellular Ca ²⁺ influx in the midbrain dopaminergic neuronal culture model.

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Human CIB protein and CIB gene according preventing stress-induced          cell death <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> CIB1 <400> 1 Met Gly Gly Ser Gly Ser Arg Leu Ser Lys Glu Leu Leu Ala Glu Tyr   1 5 10 15 Gln Asp Leu Thr Phe Leu Thr Lys Gln Glu Ile Leu Leu Ala His Arg              20 25 30 Arg Phe Cys Glu Leu Leu Pro Gln Glu Gln Arg Ser Val Glu Ser Ser          35 40 45 Leu Arg Ala Gln Val Pro Phe Glu Gln Ile Leu Ser Leu Pro Glu Leu      50 55 60 Lys Ala Asn Pro Phe Lys Glu Arg Ile Cys Arg Val Phe Ser Thr Ser  65 70 75 80 Pro Ala Lys Asp Ser Leu Ser Phe Glu Asp Phe Leu Asp Leu Leu Ser                  85 90 95 Val Phe Ser Asp Thr Ala Thr Pro Asp Ile Lys Ser His Tyr Ala Phe             100 105 110 Arg Ile Phe Asp Phe Asp Asp Asp Gly Thr Leu Asn Arg Glu Asp Leu         115 120 125 Ser Arg Leu Val Asn Cys Leu Thr Gly Glu Gly Glu Asp Thr Arg Leu     130 135 140 Ser Ala Ser Glu Met Lys Gln Leu Ile Asp Asn Ile Leu Glu Glu Ser 145 150 155 160 Asp Ile Asp Arg Asp Gly Thr Ile Asn Leu Ser Glu Phe Gln His Val                 165 170 175 Ile Ser Arg Ser Pro Asp Phe Ala Ser Ser Phe Lys Ile Val Leu             180 185 190 <210> 2 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> CIB1 <400> 2 atggggggct cgggcagtcg cctgtccaag gagctgctgg ccgagtacca ggacttgacg 60 ttcctgacga agcaggagat cctcctagcc cacaggcggt tttgtgagct gcttccccag 120 gagcagcgga gcgtggagtc gtcacttcgg gcacaagtgc ccttcgagca gattctcagc 180 cttccagagc tcaaggccaa ccccttcaag gagcgaatct gcagggtctt ctccacatcc 240 ccagccaaag acagccttag ctttgaggac ttcctggatc tcctcagtgt gttcagtgac 300 acagccacgc cagacatcaa gtcccattat gccttccgca tctttgactt tgatgatgac 360 ggaaccttga acagagaaga cctgagccgg ctggtgaact gcctcacggg agagggcgag 420 gacacacggc ttagtgcgtc tgagatgaag cagctcatcg acaacatcct ggaggagtct 480 gacattgaca gggatggaac catcaacctc tctgagttcc agcacgtcat ctcccgttct 540 ccagactttg ccagctcctt taagattgtc ctgtga 576 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CIB1 siRNA1 <400> 3 aaagacagcc ttagctttga g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> GFP siRNA, control <400> 4 ggctacgtcc aggagcgcac c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CIB1 siRNA <400> 5 aagagtcact gcatacccga g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFP siRNA <400> 6 ggctacgtcc aggagcgcac c 21  

Claims (11)

A pharmaceutical composition for treating or preventing cancer using the siRNA of SEQ ID NO: 3 as an active ingredient. delete delete delete delete delete delete delete delete The method of claim 1, wherein the cancer is breast cancer, liver cancer, lung cancer, gastric cancer, cerebral myeloma, head and neck cancer, thymic tumor, mesothelioma, esophageal cancer, colon cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, Pharmaceutical composition for the treatment or prophylaxis of cancer, characterized in that the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer. delete

Images