KR20170029672A - Pharmaceutical composition for treating Parkinson's disease containing cell-transducible PRAS40 fusion protein - Google Patents

Abstract

The Parkinsons disease is a neurodegenerative disease in which dopamine neurons are gradually removed from the substantia nigra of the brain. The inventors have clarified that a PRAS40 fusion protein can protect dopamine neurons from oxidation stress in SH-SY5Y neuroblastoma cells and Parkinsons disease animal models. The PRAS40 fusion protein has been confirmed to be able to smoothly move into SH-SY5Y cells and into the substantia nigra of the brain through a Western blot analysis. The PRAS40 fusion protein remarkably inhibits DNA fragment and production of active oxygen species induced by MPP^+, and the survival of SH-SY5Y cells is eventually induced. In addition, immunohistochemical analyzing data using TH antibody and cresyl violet coloration shows that the PRAS40 fusion protein remarkably protects dopamine cells in the substantia nigra with respect to oxidation stress such as MPTP. Accordingly, the PRAS40 fusion protein can be used in preventing and treating the Parkinsons disease.

Description

[0001] The present invention relates to a pharmaceutical composition for treating and preventing Parkinson's disease comprising a cell-permeable PRAS40 fusion protein,

The present invention relates to the protective action of dopamine neurons of the PRAS40 fusion protein, wherein the PRAS40 fusion protein covalently bound to the protein transport domain smoothly migrates into the brain tissue, significantly inhibits reactive oxygen species generation and DNA fragmentation, Suggesting that dopamine neurons in the substantia nigra are prominently protected. The PRAS40 fusion protein can be used for the prevention and treatment of Parkinson's disease.

Parkinson's disease is a representative neurodegenerative disorder with signs of stable tremor, stiffness, slow motion, and postural instability. Parkinson's disease is characterized by morphological features such as the specific death of dopaminergic brain cells in Substantia nigra . The causes of active cell tumors (ROS), oxidative stress, abnormal structure of proteins, and mitochondrial damage have been identified, but the exact cause has not yet been elucidated.

The most closely related mTOR pathway among the various pathways of the AKT signaling pathway regulates cell growth as apoptosis occurs. The PRAS40 protein is a component of mTORC1, which forms a complex with Raptor, Deptor, and mTOR. PI3K activated by the cell growth factor phosphorylates Akt, thereby activating mTORC1. The PRAS40 protein, which inhibits mTORC1, is phosphorylated by pAkt and is released from its binding with mTORC1 to activate mTORC1. This activated mTORC1 regulates cell growth, gene transcription and translation by regulating S6 kinase and 4EBP1, and is involved in protein stabilization and insulin signaling. This mTORC1 is closely related to cell survival. Recently, the phosphorylated PRAS40 protein has been known to have various functions as well as mTORC1 modulation. In particular, the PRAS40 protein has been shown to bind to 14-3-3 sigma protein and inhibit apoptosis. This PRAS40 inhibition of apoptosis has been shown to be protective effect in a stroke animal model, which is a representative disease of reactive oxygen species .

The present inventors used protein transduction domains (PTDs) to transport proteins into living cells. The present inventors efficiently transduced Tat-Catalase in various cells in the prior art. As a result of experiments using astrocytes, it was found that when Tat-catalase increased, radical scavenger activity was increased. In addition, it has been shown that various transduction fusion proteins function to protect against cell death on Invitro and Invivo.

The present inventors examined whether PRAS40 fusion protein effectively permeates cell membrane and cerebrovascular barrier to show neuroprotective effect in an animal model of Parkinson's disease and aims to provide a pharmaceutical composition for prevention and treatment of Parkinson's disease comprising PRAS40 fusion protein do.

The inventors of the present invention have found that Tat-PRAS40 fusion protein induces both MPP ⁺ and MPTP-induced dopaminergic cell death and Parkinson's disease disease model by allowing Tat to penetrate into cells or tissues by fusing Tat peptide, which is a protein transduction domain, Protection efficacy was demonstrated. As a result, it has been confirmed that the cell permeable Tat-PRAS40 fusion protein has potential as an effective protein therapeutic agent in various apoptotic diseases including Parkinson's disease.

The present inventors examined the protective effect of Tat-PRAS40 fusion protein in animal models of SH-SY5Y cells and Parkinson's disease. Parkinson's disease is a neurodegenerative disease in which dopaminergic neurons gradually disappear in the substantia nigra of the brain. The inventors have found that Tat-PRAS40 fusion proteins can protect dopamine neurons from oxidative stress in SH-SY5Y neuroblastoma cells and Parkinson's disease animal models. Tat-PRAS40 fusion protein was confirmed by Western blot analysis to transfer into SH-SY5Y cells and into the substantia nigra of the brain. Tat-PRAS40 fusion protein significantly inhibited the production of MPP ⁺ -induced reactive oxygen species and DNA fragmentation, resulting in the survival of SH-SY5Y cells. Neuroprotective effects are obtained by the Tat-PRAS40 fusion protein affecting the levels of apoptotic-mediated apoptotic mediator and anti-apoptotic mediator. Furthermore, immunohistochemistry data using TH antibody and cresyl violet staining indicate that the Tat-PRAS40 fusion protein significantly protects dopamine cells in the substantia nigra against oxidative stress such as MPTP. Thus, the Tat-PRAS40 fusion protein is available for the prophylactic and therapeutic use of Parkinson's disease.

The pharmaceutical composition containing the Tat-PRAS40 fusion protein as an active ingredient can be formulated in an oral or injection form by a conventional method in combination with a carrier that is conventionally acceptable in the pharmaceutical field. Oral compositions include, for example, tablets and gelatin capsules, which may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, , Magnesium stearate, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) and the tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone ), And may optionally contain a disintegrant (e.g., starch, agar, alginic acid or a sodium salt thereof) or a boiling mixture and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The injectable composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterilized and / or contains adjuvants such as preservatives, stabilizers, wetting or emulsifying solution accelerators, salts for controlling osmotic pressure and / or buffering agents. They may also contain other therapeutically valuable substances.

The pharmaceutical preparations thus prepared may be administered orally or parenterally, that is, intravenously, subcutaneously, intraperitoneally, or topically, as desired. The dose may be administered in a single dose of 0.01 to 100 mg / kg per day. The dosage level for a particular patient may vary depending on the patient's body weight, age, sex, health condition, time of administration, method of administration, excretion rate, severity of disease, and the like.

The definitions of the main terms used in the description of the present invention and the like are as follows.

"Tat-PRAS40 fusion protein" refers to a covalent complex formed by genetic fusion or chemical bonding between a protein transport domain and a PRAS40 protein, and a protein transport domain and a cargo molecule (i.e., PRAS40 in the present invention) it means. As one specific example in the present specification and drawings, "Tat-PRAS40" refers to the binding of the HIV-Tat protein transport domain to the N-terminus of the PRAS40 protein among the PRAS40 fusion proteins.

In addition, the term "genetic fusion" means a link consisting of a linear, covalent bond formed through genetic expression of a DNA sequence encoding a protein.

The term "target cell" refers to a cell to which a cargo molecule is delivered by a transport domain. That is, the target cell is a cell, that is, a living organism or a cell or organ It is meant to include microorganisms that are found. In addition, the target cell means an extracellular cell, that is, a cultured animal cell, a human cell or a microorganism. Specifically, in the present specification, the target cell means a brain cell or a nerve cell.

As used herein, the term "protein transport domain" refers to a peptide or protein that is covalently bonded to a cargo molecule (target protein) peptide or protein and can introduce the peptide or protein into cells without requiring additional receptor, carrier, energy, For example, HIV-Tat peptide.

As used herein, the term "target protein" means a molecule that is covalently bound to a protein transport domain and introduced into cells to exhibit activity. It is synonymous with "cargo molecule".

Also, in the present specification, the term "introduction", "transport", "penetration", "transport", "transfer", "permeation", "pass" Respectively.

The present invention relates to a pharmaceutical composition for the prevention and treatment of Parkinson's disease, which comprises a PRAS40 fusion protein in which a protein transport domain is covalently bonded to one or more of N-terminal and C-terminal of human PRAS40 protein.

The present invention also relates to a pharmaceutical composition for the prevention and treatment of Parkinson's disease comprising the PRAS40 fusion protein, wherein the protein transport domain is an HIV-Tat peptide.

In addition, the present invention relates to a pharmaceutical composition for preventing and treating Parkinson's disease containing the PRAS40 fusion protein, wherein the fusion protein is SEQ ID NO: 11.

The PRAS40 fusion protein of the present invention was permeabilized intracellularly in a time-dependent, dose-dependent manner into SH-SY5Y cells, and further maintained intracellular PRAS40 fusion protein levels for a considerable period of time.

In addition, the PRAS40 fusion protein of the present invention significantly inhibited the production of reactive oxygen species, which is a response to MPP ⁺ .

The PRAS40 fusion protein of the present invention can protect cells from oxidative stress induced by MPP ⁺ .

The PRAS40 fusion protein of the present invention can effectively cross the cerebral vascular barrier and protects dopamine neurons against MPTP toxicity in MPTP-induced Parkinson's disease mouse models. MPTP-induced dopaminergic neurons It is predicted that it will be possible to reduce motor dysfunction by inhibiting the death.

FIG. 1 is a schematic diagram (A) of a vector for producing a PRAS40 protein and a Tat-PRAS40 fusion protein, and (B) a result of overexpression and purification of these vectors in E. coli and SDS-PAGE and Western blotting, respectively.
Fig. 2 shows the result of cell penetration of Tat-PRAS40 fusion protein by immunofluorescence staining.
FIG. 3 is a graph showing the result of DCF-DA staining of the Tat-PRAS40 fusion protein, which confirmed the inhibitory effect on the production of reactive oxygen species.
FIG. 4 is a fluorescence microscope photograph showing the results of DCF-DA staining of the Tat-PRAS40 fusion protein confirming the production of reactive oxygen species.
Figure 5 shows the results of TUNEL analysis confirming the inhibitory effect on Tat-PRAS40 fusion protein DNA fragmentation.
Figure 6 shows the protective effect of the Tat-PRAS40 fusion protein on the apoptosis markers caspase-3, Bax and Bcl2.
Fig. 7 shows the binding mechanism of Tat-PRAS40 fusion protein and 14-3-3 sigma protein.
FIG. 8 shows the effect of Tat-PRAS40 fusion protein penetrating into tissues and protecting cells and tissues in an animal model of Parkinson's disease by immunofluorescence staining.
FIG. 9 shows the tissue protective effect of the Tat-PRAS40 fusion protein in a Parkinson's disease animal model by CV staining and TH immunochemical staining.
FIG. 10 shows the protective mechanism of the Tat-PRAS40 fusion protein in an animal model of Parkinson's disease by immunochemical staining and tissue Western blotting.
FIG. 11 shows the protective mechanism of the Tat-PRAS40 fusion protein in an animal model of Parkinson's disease by immunofluorescence staining.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited by the descriptions of the embodiments. Particularly, as a result of each experimental example, data on the Tat-PRAS40 fusion protein among the various fusion proteins prepared in the specific examples were described, but other fusion proteins were also similar to the results obtained using the Tat-PRAS40 fusion protein as a sample And the results are shown.

material

Restriction enzymes and T4 DNA ligase were purchased from promega (USA) and Tat-oligonucleotides were synthesized from Gibco BRL (USA). IPTG was purchased from Duchefa (Netherland). The pET15b vector and the BL21 (DE3) plasmid were purchased from Novagen (USA), and the Ni ^{2 +} -nitrile trichlorosaccharide sepharose superfluous column was purchased from Qiagen (Germary). Anti-histidine antibody, anti-PRAS40 antibody and pPRAS40 and anti-14-3-3-sigma antibody were purchased from Santa Cruz (CA, USA) and anti-pAkt primary antibody, Bax rabbit antibody and Bcl-2 rabbit antibody Cell Signaling Technology (Denvers, MA, USA). Other primary antibodies and secondary anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (USA). Tat-peptide synthesis was custom-made on PEPTRON (Deajeon, Korea). All of the other reagents were made using the express product. Other chemicals and reagents not specified were purchased at the highest level in Sigma-Aldrich (St. Louis, Mo., USA).

Experimental Method

Tat- PRAS40 Of the fusion protein  Expression and purification

PRAS40 protein and Tat-PRAS40 fusion protein expression vector were constructed. The sense primer sequence is 5'-CTCGAGCGCAGCCCC-3 '(SEQ ID NO: 6) and comprises the Xho I restriction site and the antisense primer sequence is 5'-GGATCCTCAATATTTCCGCTTCAG-3' (SEQ ID NO: 7) and contains the Bam HI restriction site. Polymerase chain reaction (PCR) was performed using PRAS40 primers and cDNA template, and PCR products were inserted into the TA cloning vector. The TA vector containing the PRAS40 cDNA was ligated to two pET-15b vectors with PRAS40 cDNA digested with Xho I and Bam HI and digested . One vector contains the Tat sequence and six consecutive histidine sequences as Tat-PRAS40 expression vectors. The other vector contains only six consecutive histidine sequences as the PRAS40 expression vector.

E. coli BL21 (DE3) cells were transformed with each vector construct to produce PRAS40 and Tat-PRAS40 fusion proteins. The transformed bacteria were cultured in 100 ml of LB medium at 37 ° C. until the optical density of 600 nm became 0.5, and then 0.5 mM IPTG (isopropyl-β-D-thiogalactoside) was added thereto, followed by induction at 37 ° C. for 4 hours . Cells were harvested and lysed and lysed by centrifugation at 4 ° C in binding buffer (10 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The supernatant was added to a sepharose sepharose super flow column with 2.0 ml of Ni ^{2 +} -nitrile. The column was washed with 10 times of binding buffer and 7 times of wash buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The PRAS40 and Tat-PRAS40 fusion proteins were eluted with elution buffer (250 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The salts contained in the purified protein were removed by PD-10 desalting column chromatography (Amersham, Braunschweig, Germany). Protein concentration was determined by Bradford method using bovine serum albumin as a standard.

Tat- PRAS40 Of the fusion protein SH - SY5Y  Neuroblastoma cell permeation

SH-SY5Y human neuroblastoma cells were cultured in DMEM (Dulbecco ' s Modified Eagle ' s Medium) medium supplemented with heat-activated activated rabbit serum (10% FBS) and antibiotics (100 mu g / ml streptomycin, 100 U / % Air and 5% CO ₂ And cultured under a humid environment.

To observe intracellular permeation of Tat-PRAS40 fusion protein, cultured cells were grown in 6-well plate for 4 to 6 hours, replaced with 1 ml of fresh medium without FBS and mixed with various concentrations of Tat-PRAS40 fusion Proteins were treated in media. After one hour, the cells were treated with trypsin-EDTA (Gibco Grand Island, NY, USA), washed thoroughly with PBS (phosphate-buffered saline), and the amount and activity of the fusion protein permeated into the cells were analyzed and measured by Western blot .

Fluorescence microscopy analysis

SH-SY5Y human neuroblastoma cells were cultured on cover slips and treated with 3 uM Tat-PRAS40 fusion protein. Then, the cells were incubated at 37 ° C for 2 hours, washed twice with PBS, and fixed with 4% paraformaldehyde for 5 minutes at room temperature. After making the cells permeable, they were blocked with 3% bovine serum albumin, PBS containing 0.1% Triton X-100 (PBS-BT) for 40 minutes, and washed with PBS-BT. The primary antibody (His-probe, Santa Cruze Biotechnology) was diluted 1: 2000 and over-night cultured at 4 ° C. The secondary antibody (Alexa flour 488, Invitrogen) was diluted 1: 15000 and incubated in the dark for one hour at room temperature. Nuclei were stained with 1 μg / ml DAPI (Roche) for 2 minutes. Fluorescence distribution was analyzed with a confocal laser scanning system (Bio-Rad MRC-1024ES).

MTT  analysis

The biological activity of the Tat-PRAS40 fusion protein can be measured by determining the number of viable cells after treating the cells with hydrogen peroxide. SH-SY5Y human neuroblastoma cells were plated in 96-well plates at 80%, and the Tat-PRAS40 fusion protein was first treated at a concentration of 0.5 ~ 3 uM. And treated with 0.8 mM of hydrogen peroxide and cultured for 10 hours. Cell viability was evaluated by color development using MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-dipheyltetrazolium bromide}. Absorbance was measured at 570 nm using an ELISA microplate reader (Labsystems Multiskan MCC / 340) and the cell viability was expressed as a percentage of control cells that did not receive the fusion protein.

Intracellular Active oxygen species  Measure

The level of reactive oxygen species in the cells was measured using DCF-DA (2 ', 7'-dichlorodihydrofluorescein diacetate). DCF-DA converts to DCF in cells by reactive oxygen species and emits fluorescence. SH-SY5Y human neuroblastoma cells were compared with those of Tat-PRAS40 fusion protein treated and untreated. Tat-PRAS40 fusion protein was treated for 1 hour and then hydrogen peroxide was treated at 0.8 mM for 2 hours. Then, the cells were washed twice with PBS and DCF-DA was treated at a concentration of 20 μM for 1 hour. Fluorescence images were obtained using a fluorescence microscope (Nikon eclipse 80i, Japan).

Western Blat  analysis

For Western blot analysis, the proteins in the cell lysate were separated by 12% SDS polyacrylamide gel, and the proteins in the gels were electrochromatically transferred to a nitrocellulose membrane (Amersham, UK). The membranes were blocked with 5% skim milk powder in TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 hour and reacted with primary and secondary antibodies. Secondary antibody conjugated with rabbit anti histidine antibody, beta -actin antibody, caspase-3 antibody, truncated caspase-3 antibody, Bax antibody, Bcl-2 antibody (1: 10,000 dilution) and horseradish peroxidase (1: 10,000 dilution). The conjugated antibody complexes were detected using a chemiluminescence detection kit according to manufacturer's instructions (Amersham, Piscataway, NJ, USA).

Confocal  Microscopic observation

To detect Tat-PRAS40 fusion protein and PRAS40 protein in SH-SY5Y cells, cells were seeded on glass cover slip and treated with Tat-PRAS40 fusion protein and PRAS40 protein at 3 μM concentration for 3 hours, respectively. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 5 minutes at room temperature, and incubated with anti-histidine primary antibody and Alexa Fluor 488 conjugated secondary antibody (Invitrogen, Carlsbad, Calif., USA). The nuclei were stained with 1 [mu] g / ml of 4'6-diamidino-2-phenylindole (Roche Applied Science, Basel, Switzerland) for 5 minutes. Fluorescence was analyzed with an Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan).

TUNEL  (Terminal deoxynucleotidyl transferase -mediated dUTP  nick-end-labeling) analysis

SH-SY5Y cells on glass cover slip were incubated with 2mM each of Tat-PRAS40 fusion protein and PRAS40 protein for 3 hours at 37 ° C and exposed to MPP ⁺ (4.0mM) for 14 hours and 30 minutes. TUNEL staining was performed using a cell death kits (Roche Applied Science, Basel, Switzerland). Fluorescence microscopy photographs were taken with an Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).

Experimental animal

Male, 6 weeks old, C57BL / 6 mice were purchased from Hallym University Laboratory Animals Center. The mice were bred at a constant temperature (23 ° C) and at a constant relative humidity (60%) for 12 hours. Water and feed were freely accessible. All laboratory procedures and controls related to animals have been followed by the National Veterinary Research and Quarantine Service of Korea, which is guided by the Committee on Animal Protection and Utilization. Parkinson 's disease model was constructed as previously studied. Mice were injected with MPTP at a dose of 20 mg / kg every two hours. In order to confirm the protective effect of Tat-PRAS40 fusion protein on Parkinson's disease, 2.0 mg / kg of protein was subcutaneously injected 12 hours before MPTP treatment. Mice were divided into five groups as follows: 1) untreated control group, 2) MPTP treated group, 3) MPTP ⁺ Tat-PRAS40 fusion protein treated group, 4) MPTP ⁺ PEP-1 Peptide treatment group, and 5) MPTP ⁺ control PRAS40 protein treatment group. Mice were sacrificed one week after the last injection.

Immunohistochemistry

Immunostaining was performed as in the previous study. Tissue sections were blocked for 30 min at room temperature in PBS containing 3% bovine serum albumin, blocked to detect histidine antibodies or dopaminergic neurons to detect Tat-PRAS40 fusion protein and PRAS40 protein, and tyrosine hydroxylase TH) antibody. Biotin - conjugated goat anti - rabbit antibody was used as a secondary antibody. Cresyl violet staining was performed to stain Nissl bodies, which are granules found in living neurons and considered as rough endoplasmic reticulum. The sections were visualized with DAB (3,3'-diaminobenzidine) in 0.1 M Tris buffer and placed on gelatin-coated slides. The images were taken and analyzed with an Olympus DP72 digital camera and DP2-BSW microscope digital camera software. The drawing was made using Adobe Photoshop 7.0 (San Jose, CA, USA).

Statistical analysis

Data were expressed as mean ± standard deviation from each independent experiment. The differences between the mean values were analyzed using one - way ANOVA. Newman-Keuls post hoc analyzes were employed when differences were observed in the ANOVA test ( p <0.01).

Results 1: Tat- PRAS40 Of the fusion protein  Purification and cell permeation

It was over-expressed in a cell-permeable Tat-PRAS40 with the Tat vector and PRAS40 gene of a type of including a HIV-Tat peptide coding sequence of the protein transport domain recombination to produce the fusion protein (A in Fig. 1), E. coli Ni ^{2 +} - Purification was carried out using a nitriloacetic acid sepharose affinity column and PD-10 column chromatography, and confirmed by SDS-PAGE and Western blotting (FIG. 1B).

In order to confirm whether Tat-PRAS40 fusion protein smoothly penetrates into cells, it was treated with SH-SY5Y human neuroblastoma cells having the property of dopaminergic neurons to stain green fluorescence with Tat-PRAS40 fusion protein specific antibody, And confirmed by observable confocal microscopy. As a result, the intracellular permeation of the Tat-PRAS40 fusion protein was effected effectively, whereas the negative control and the control PRAS40 protein without Tat did not permeate (Fig. 2).

Result 2: MPP ⁺ Lt; RTI ID = 0.0 &gt; Tat- PRAS40 Of the fusion protein  Inhibitory effect

In the model of Parkinson's disease, the cell death is induced by treating MPP ^+, which is a cell death inducer, and the MTT assay that can identify the living cell specifically can inhibit the cell death of Tat-PRAS40 fusion protein Respectively. When SH-SY5Y cells were treated with MPP ⁺ alone, the number of viable cells decreased to about 55%, but when the Tat-PRAS40 fusion protein was treated, the cell viability increased to about 77%. On the other hand, it was confirmed that there was no change in cell viability when PRAS40 protein having no cell penetration ability was treated. In addition, Tat-PRAS40 fusion protein was phosphorylated by activated pAkt after treatment with Akt inhibitor and mTOR inhibitor (Rapamycin), and it was confirmed that it exhibited cell killing effect independent of mTOR (FIG. 3). In order to confirm that Tat-PRAS40 fusion protein effectively inhibited reactive oxygen species induced by MPP ⁺ treatment, H ₂ DCFDA (2 ', 7'-dichlorofluorescin diacetate) (2 ', 7'-dichloro-fluorescein) by hydrolysis and hydrolysis of impermeable H ₂ DCF (2', 7'-dichlorofluorescin) by reaction with DCF-DA staining method Respectively.

In the MPP ⁺ untreated cells, reactive oxygen species were generated to show green fluorescence, whereas the pretreatment of the Tat-PRAS40 fusion protein inhibited the production of reactive oxygen species in the cells (FIG. 4). In order to confirm DNA fragmentation induced by active oxygen species, TUNEL staining was performed by attaching fluorescent pigment to the cut DNA fragment to confirm DNA fragmentation. Similar to the DCF-DA staining method, it was confirmed that the increased DNA fragment by treating MPP ⁺ singly was very effectively inhibited when the Tat-PRAS40 fusion protein was treated (FIG. 5).

The expression of caspase-3 and Bax, Bcl2, which are markers of apoptosis induced by MPP ⁺ , was confirmed by treatment with Tat-PRAS40 fusion protein. Confirmed that the truncated caspase-3 and Bax induced by MPP ⁺ decreased in a dose-dependent manner as Tat-PRAS40 fusion protein was treated, and Bcl2 increased. Conversely, there was no change in the treatment with the control PRAS40 protein without Tat. In addition, it was confirmed that when the Akt inhibitor and the mTOR inhibitor rapamycin were treated, the activated Akt phosphorylated the PRAS40, thereby exhibiting an apoptosis-inhibiting effect independent of mTOR (FIG. 6).

The protective effect against apoptosis was confirmed by immunoprecipitation method to determine whether PRAS40 phosphorylated is due to binding to 14-3-3 sigma protein. As a result, , Indicating that phosphorylated PRAS40 binds to the 14-3-3 sigma protein (Fig. 7).

Result 3: Cell permeability  Tat- PRAS40  Protective Effects of Fusion Proteins in Animal Model of Parkinson's Disease

Cell permeability to Parkinson's disease To examine the protective effect of the Tat-PRAS40 fusion protein, C57BL / 6 male mice were injected with 2 mg / kg of Tat-PRAS40 fusion protein and PRAS40 protein, respectively, and induced Parkinson's disease MPTP was injected intraperitoneally to produce an animal model of Parkinson 's disease.

It was confirmed that the fusion protein penetrated the blood-brain barrier (SN) region where dopaminergic neurons were present. Tissue immunofluorescence staining revealed that the control PRAS40 protein did not infiltrate, but when Tat-PRAS40 fusion protein was treated, it penetrated into brain tissue effectively. In addition, it was confirmed that the Tat-PRAS40 fusion protein infiltrating into brain tissue showed a protective effect against Parkinson's disease (Fig. 8). In addition, CV (Cresyl Violet) staining for staining only living cells and TH (Tyrosine Hydroxylase) staining for a dopaminergic neuron were confirmed by immunohistochemistry (Fig. 9). In animals treated with Tat-PRAS40 fusion protein for 4 days after induction of Parkinson's disease, protective effect against dopaminergic neuron apoptosis was shown, whereas in the animal treated with PRAS40 control protein, there was no protection against apoptosis I did. In addition, immunohistochemistry and tissue Western blotting confirmed that the same results as in the cell test were obtained in the tissues. As a result, it was indirectly confirmed that apoptosis was suppressed by the phosphorylated PRAS40 in tissues (FIG. 10). It was confirmed more precisely by immunohistochemical staining method with excellent sensitivity. The phosphorylated PRAS40 was identified at the same position as the 14-3-3 sigma protein, so that the Tat-PRAS40 fusion protein was expressed in the animal model Parkinson's disease (Fig. 11).

From these results, it was confirmed that the Tat-PRAS40 fusion protein effectively penetrates into cells and tissues and protects against dopaminergic neuronal apoptosis and Parkinson's disease caused by MPP ⁺ and MPTP. Thus, the present inventors have found that Tat-PRAS40 fusion protein Suggesting the possibility of a therapeutic agent for Parkinson's disease.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for treating Parkinson's disease          containing cell-transducible PRAS40 fusion protein <130> hallym-PRAS40-PD <160> 33 <170> Kopatentin 1.71 <210> 1 <211> 29 <212> DNA <213> Human immunodeficiency virus type 1 <400> 1 taggaagaag cggagacagc gacgaagac 29 <210> 2 <211> 31 <212> DNA <213> Human immunodeficiency virus type 1 <400> 2 tcgagtcttc gtcgctgtct ccgcttcttc c 31 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ctcgagatgg cagaaccgca gcccccgtcc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctcgagatga aggtagaggt gctgcctgcc 30 <210> 5 <211> 9 <212> PRT <213> Human immunodeficiency virus type 1 <400> 5 Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctcgagcgca gcccc 15 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggatcctcaa tatttccgct tcag 24 <210> 8 <211> 771 <212> DNA <213> Homo sapiens <400> 8 atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg tgggggccgc tgagcgcttc 60 cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg ccccgccgcc accaccccgc 120 ccgggcccct gtgcctatgc tgcccatggt cgaggagccc tggcggaggc agcgcgccgt 180 tgcctccacg acatcgcact ggcccacagg gctgccactg ctgctcggcc tcctgcgccc 240 ccaccagcac cacagccacc cagtcccaca cccagcccac cccggcctac cctggccaga 300 gaggacaacg aggaggacga ggatgagccc acagagacag agacctccgg ggagcagctg 360 ggcattagtg ataatggagg gctctttgtg atggatgagg acgccaccct ccaggacctt 420 ccccccttct gtgagtcaga ccccgagagt acagatgatg gcagcctgag cgaggagacc 480 cccgccggcc cccccacctg ctcagtgccc ccagcctcag ccctacccac acagcagtac 540 gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca aggagaagag gacagaggcg 600 cggtcatcag atgaggagaa tgggccgccc tcttcgcccg acctggaccg catcgcggcg 660 agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc aggtcttcgg ggacctgcca 720 cggccgcggc ttaacaccag cgacttccag aagctgaagc ggaaatattg a 771 <210> 9 <211> 256 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 <210> 10 <211> 858 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding Tat-PRAS40 fusion protein <400> 10 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggcgtcg gggcgccccg aggagctgtg ggaggccgtg 120 gtgggggccg ctgagcgctt ccgggcccgg actggcacgg agctggtgct gctgaccgcg 180 gccccgccgc caccaccccg cccgggcccc tgtgcctatg ctgcccatgg tcgaggagcc 240 ctggcggagg cagcgcgccg ttgcctccac gacatcgcac tggcccacag ggctgccact 300 gctgctcggc ctcctgcgcc cccaccagca ccacagccac ccagtcccac acccagccca 360 ccccggccta ccctggccag agaggacaac gaggaggacg aggatgagcc cacagagaca 420 gagacctccg gggagcagct gggcattagt gataatggag ggctctttgt gatggatgag 480 gacgccaccc tccaggacct tccccccttc tgtgagtcag accccgagag tacagatgat 540 ggcagcctga gcgaggagac ccccgccggc ccccccacct gctcagtgcc cccagcctca 600 gccctaccca cacagcagta cgccaagtcc ctgcctgtgt ctgtgcccgt ctggggcttc 660 aaggagaaga ggacagaggc gcggtcatca gatgaggaga atgggccgcc ctcttcgccc 720 gacctggacc gcatcgcggc gagcatgcgc gcgctggtgc tgcgagaggc cgaggacacc 780 caggtcttcg gggacctgcc acggccgcgg cttaacacca gcgacttcca gaagctgaag 840 cggaaatatt gaggatcc 858 <210> 11 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Tat-PRAS40 fusion protein <400> 11 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr             260 265 <210> 12 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding PRAS40-Tat fusion protein <400> 12 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggcgtc 60 ggggcgcccc gaggagctgt gggaggccgt ggtgggggcc gctgagcgct tccgggcccg 120 gactggcacg gagctggtgc tgctgaccgc ggccccgccg ccaccacccc gcccgggccc 180 ctgtgcctat gctgcccatg gtcgaggagc cctggcggag gcagcgcgcc gttgcctcca 240 cgacatcgca ctggcccaca gggctgccac tgctgctcgg cctcctgcgc ccccaccagc 300 accacccca cccagtccca cgaggaggac gaggatgagc ccacagagac agagacctcc ggggagcagc tgggcattag 420 tgataatgga gggctctttg tgatggatga ggacgccacc ctccaggacc ttcccccctt 480 ctgtgagtca gaccccgaga gtacagatga tggcagcctg agcgaggaga cccccgccgg 540 cccccccacc tgctcagtgc ccccagcctc agccctaccc acacagcagt acgccaagtc 600 cctgcctgtg tctgtgcccg tctggggctt caaggagaag aggacagagg cgcggtcatc 660 agatgaggag aatgggccgc cctcttcgcc cgacctggac cgcatcgcgg cgagcatgcg 720 cgcgctggtg ctgcgagagg ccgaggacac ccaggtcttc ggggacctgc cacggccgcg 780 gcttaacacc agcgacttcc agaagctgaa gcggaaatat ggatcctagg aagaagcgga 840 gacagcgacg aagatag 857 <210> 13 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-Tat fusion protein <400> 13 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Arg Lys Lys Arg Arg Gln Arg Arg Arg             260 265 <210> 14 <211> 886 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Tat-PRAS40-Tat fusion protein <400> 14 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatag gaagaagcgg 60 agacagcgac gaagactcga gatggcgtcg gggcgccccg aggagctgtg ggaggccgtg 120 gtgggggccg ctgagcgctt ccgggcccgg actggcacgg agctggtgct gctgaccgcg 180 gccccgccgc caccaccccg cccgggcccc tgtgcctatg ctgcccatgg tcgaggagcc 240 ctggcggagg cagcgcgccg ttgcctccac gacatcgcac tggcccacag ggctgccact 300 gctgctcggc ctcctgcgcc cccaccagca ccacagccac ccagtcccac acccagccca 360 ccccggccta ccctggccag agaggacaac gaggaggacg aggatgagcc cacagagaca 420 gagacctccg gggagcagct gggcattagt gataatggag ggctctttgt gatggatgag 480 gacgccaccc tccaggacct tccccccttc tgtgagtcag accccgagag tacagatgat 540 ggcagcctga gcgaggagac ccccgccggc ccccccacct gctcagtgcc cccagcctca 600 gccctaccca cacagcagta cgccaagtcc ctgcctgtgt ctgtgcccgt ctggggcttc 660 aaggagaaga ggacagaggc gcggtcatca gatgaggaga atgggccgcc ctcttcgccc 720 gacctggacc gcatcgcggc gagcatgcgc gcgctggtgc tgcgagaggc cgaggacacc 780 caggtcttcg gggacctgcc acggccgcgg cttaacacca gcgacttcca gaagctgaag 840 cggaaatatg gatcctagga agaagcggag acagcgacga agatag 886 <210> 15 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> Tat-PRAS40-Tat fusion protein <400> 15 Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr Gly Ser Arg Lys Lys             260 265 270 Arg Arg Gln Arg Arg Arg         275 <210> 16 <211> 895 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Pep-PRAS40 fusion protein <400> 16 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggcgtcgggg cgccccgagg agctgtggga ggccgtggtg ggggccgctg agcgcttccg 180 ggcccggact ggcacggagc tggtgctgct gaccgcggcc ccgccgccac caccccgccc 240 gggcccctgt gcctatgctg cccatggtcg aggagccctg gcggaggcag cgcgccgttg 300 cctccacgac atcgcactgg cccacagggc tgccactgct gctcggcctc ctgcgccccc 360 accagcacca cagccaccca gtcccacacc cagcccaccc cggcctaccc tggccagaga 420 ggacaacgag gaggacgagg atgagcccac agagacagag acctccgggg agcagctggg 480 cattagtgat aatggagggc tctttgtgat ggatgaggac gccaccctcc aggaccttcc 540 ccccttctgt gagtcagacc ccgagagtac agatgatggc agcctgagcg aggagacccc 600 cgccggcccc cccacctgct cagtgccccc agcctcagcc ctacccacac agcagtacgc 660 caagtccctg cctgtgtctg tgcccgtctg gggcttcaag gagaagagga cagaggcgcg 720 gtcatcagat gaggagaatg ggccgccctc ttcgcccgac ctggaccgca tcgcggcgag 780 catgcgcgcg ctggtgctgc gagaggccga ggacacccag gtcttcgggg acctgccacg 840 gccgcggctt aacaccagcg acttccagaa gctgaagcgg aaatattgag gatcc 895 <210> 17 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> Pep-PRAS40 fusion protein <400> 17 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Ser Gly Arg Pro Glu Glu Leu              20 25 30 Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly          35 40 45 Thr Glu Leu Val Leu Leu Thr Ala Pro Pro Pro Pro Arg Pro      50 55 60 Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Leu Ala Glu Ala  65 70 75 80 Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr                  85 90 95 Ala Ala Arg Pro Pro Ala Pro Pro Ala Pro Gln Pro Pro Ser Pro             100 105 110 Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu         115 120 125 Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly     130 135 140 Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu 145 150 155 160 Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp                 165 170 175 Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val             180 185 190 Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro         195 200 205 Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg     210 215 220 Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Pro Asp Leu Asp Arg 225 230 235 240 Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr                 245 250 255 Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe             260 265 270 Gln Lys Leu Lys Arg Lys Tyr         275 <210> 18 <211> 894 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding PRAS40-Pep fusion protein <400> 18 catcatcatc atcatcacag cagcggcctg gtgccggcgg cagccactcg agatggcgtc 60 ggggcgcccc gaggagctgt gggaggccgt ggtgggggcc gctgagcgct tccgggcccg 120 gactggcacg gagctggtgc tgctgaccgc ggccccgccg ccaccacccc gcccgggccc 180 ctgtgcctat gctgcccatg gtcgaggagc cctggcggag gcagcgcgcc gttgcctcca 240 cgacatcgca ctggcccaca gggctgccac tgctgctcgg cctcctgcgc ccccaccagc 300 accacccca cccagtccca cgaggaggac gaggatgagc ccacagagac agagacctcc ggggagcagc tgggcattag 420 tgataatgga gggctctttg tgatggatga ggacgccacc ctccaggacc ttcccccctt 480 ctgtgagtca gaccccgaga gtacagatga tggcagcctg agcgaggaga cccccgccgg 540 cccccccacc tgctcagtgc ccccagcctc agccctaccc acacagcagt acgccaagtc 600 cctgcctgtg tctgtgcccg tctggggctt caaggagaag aggacagagg cgcggtcatc 660 agatgaggag aatgggccgc cctcttcgcc cgacctggac cgcatcgcgg cgagcatgcg 720 cgcgctggtg ctgcgagagg ccgaggacac ccaggtcttc ggggacctgc cacggccgcg 780 gcttaacacc agcgacttcc agaagctgaa gcggaaatat ggatcctaaa agaaacctgg 840 tgggaaacct ggtggaccga atggtctcag ccgaaaaaaa aacgtaaagt gtag 894 <210> 19 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-Pep fusion protein <400> 19 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Gly Ser Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln             260 265 270 Pro Lys Lys Lys Arg Lys Val         275 <210> 20 <211> 960 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding Pep1-PRAS40-Pep1 fusion protein <400> 20 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccataa aagaaacctg 60 gtgggaaacc tggtggaccg aatggtctca gccgaaaaaa aaacgtaaag tgctcgagat 120 ggcgtcgggg cgccccgagg agctgtggga ggccgtggtg ggggccgctg agcgcttccg 180 ggcccggact ggcacggagc tggtgctgct gaccgcggcc ccgccgccac caccccgccc 240 gggcccctgt gcctatgctg cccatggtcg aggagccctg gcggaggcag cgcgccgttg 300 cctccacgac atcgcactgg cccacagggc tgccactgct gctcggcctc ctgcgccccc 360 accagcacca cagccaccca gtcccacacc cagcccaccc cggcctaccc tggccagaga 420 ggacaacgag gaggacgagg atgagcccac agagacagag acctccgggg agcagctggg 480 cattagtgat aatggagggc tctttgtgat ggatgaggac gccaccctcc aggaccttcc 540 ccccttctgt gagtcagacc ccgagagtac agatgatggc agcctgagcg aggagacccc 600 cgccggcccc cccacctgct cagtgccccc agcctcagcc ctacccacac agcagtacgc 660 caagtccctg cctgtgtctg tgcccgtctg gggcttcaag gagaagagga cagaggcgcg 720 gtcatcagat gaggagaatg ggccgccctc ttcgcccgac ctggaccgca tcgcggcgag 780 catgcgcgcg ctggtgctgc gagaggccga ggacacccag gtcttcgggg acctgccacg 840 gccgcggctt aacaccagcg acttccagaa gctgaagcgg aaatatggat cctaaaagaa 900 acctggtggg aaacctggtg gaccgaatgg tctcagccga aaaaaaaacg taaagtgtag 960                                                                          960 <210> 21 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> Pep1-PRAS40-Pep1 fusion protein <400> 21 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val Leu Glu Met Ala Ser Gly Arg Pro Glu Glu Leu              20 25 30 Trp Glu Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly          35 40 45 Thr Glu Leu Val Leu Leu Thr Ala Pro Pro Pro Pro Arg Pro      50 55 60 Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Leu Ala Glu Ala  65 70 75 80 Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr                  85 90 95 Ala Ala Arg Pro Pro Ala Pro Pro Ala Pro Gln Pro Pro Ser Pro             100 105 110 Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu         115 120 125 Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly     130 135 140 Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu 145 150 155 160 Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp                 165 170 175 Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val             180 185 190 Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro         195 200 205 Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg     210 215 220 Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Pro Asp Leu Asp Arg 225 230 235 240 Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr                 245 250 255 Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe             260 265 270 Gln Lys Leu Lys Arg Lys Tyr Gly Ser Lys Glu Thr Trp Trp Glu Thr         275 280 285 Trp Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val     290 295 300 <210> 22 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9lys-PRAS40 fusion protein <400> 22 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatattg aggatcc 857 <210> 23 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 9Lys-PRAS40 fusion protein <400> 23 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr             260 265 <210> 24 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding PRAS40-9Lys fusion protein <400> 24 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccactc gagatggcgt 60 cggggcgccc cgaggagctg tgggaggccg tggtgggggc cgctgagcgc ttccgggccc 120 ggactggcac ggagctggtg ctgctgaccg cggccccgcc gccaccaccc cgcccgggcc 180 cctgtgccta tgctgcccat ggtcgaggag ccctggcgga ggcagcgcgc cgttgcctcc 240 acgacatcgc actggcccac agggctgcca ctgctgctcg gcctcctgcg cccccaccag 300 caccacagcc acccagtccc acacccagcc caccccggcc taccctggcc agagaggaca 360 acgaggagga cgaggatgag cccacagaga cagagacctc cggggagcag ctgggcatta 420 gtgataatgg agggctcttt gtgatggatg aggacgccac cctccaggac cttcccccct 480 tctgtgagtc agaccccgag agtacagatg atggcagcct gagcgaggag acccccgccg 540 gcccccccac ctgctcagtg cccccagcct cagccctacc cacacagcag tacgccaagt 600 ccctgcctgt gtctgtgccc gtctggggct tcaaggagaa gaggacagag gcgcggtcat 660 cagatgagga gaatgggccg ccctcttcgc ccgacctgga ccgcatcgcg gcgagcatgc 720 gcgcgctggt gctgcgagag gccgaggaca cccaggtctt cggggacctg ccacggccgc 780 ggcttaacac cagcgacttc cagaagctga agcggaaata tggatccaaa aaaaaaaaaa 840 aaaaaaaaaa aaaatag 857 <210> 25 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-9Lys fusion protein <400> 25 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Gly Ser Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys             260 265 <210> 26 <211> 884 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9Lys-PRAS40-9Lys fusion protein <400> 26 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatatgg atccaaaaaa aaaaaaaaaa aaaaaaaaaa atag 884 <210> 27 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> 9Lys-PRAS40-9Lys fusion protein <400> 27 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Ser Ser Gly Leu Val Pro Arg   1 5 10 15 Gly Ser His Leu Glu Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu              20 25 30 Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu          35 40 45 Leu Val Leu Leu Thr Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro      50 55 60 Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Ala Gul Ala Ala Arg  65 70 75 80 Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala                  85 90 95 Arg Pro Pro Ala Pro Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro             100 105 110 Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu         115 120 125 Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser     130 135 140 Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp 145 150 155 160 Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser                 165 170 175 Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro             180 185 190 Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser         195 200 205 Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser     210 215 220 Asp Glu Glu Asn Gly Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala 225 230 235 240 Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val                 245 250 255 Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys             260 265 270 Leu Lys Arg Lys Tyr Gly Ser Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys         275 280 285 <210> 28 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9Arg-PRAS40 fusion protein <400> 28 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatattg aggatcc 857 <210> 29 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> 9Arg-PRAS40 fusion protein <400> 29 Arg Arg Arg Arg Arg Arg Arg Arg Leu Glu Met Ala Ser Gly Arg   1 5 10 15 Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala Glu Arg Phe Arg              20 25 30 Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr Ala Ala Pro Pro          35 40 45 Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala His Gly Arg Gly Ala      50 55 60 Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp Ile Ala Leu Ala His  65 70 75 80 Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro Pro Ala Pro Gln                  85 90 95 Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu             100 105 110 Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly         115 120 125 Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu Phe Val Met Asp Glu     130 135 140 Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu 145 150 155 160 Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro                 165 170 175 Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala             180 185 190 Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly Phe Lys Glu Lys Arg         195 200 205 Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly Pro Ser Ser Ser Pro     210 215 220 Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala Leu Val Leu Arg Glu 225 230 235 240 Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn                 245 250 255 Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr             260 265 <210> 30 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding PRAS40-9 Arg fusion protein <400> 30 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccactc gagatggcgt 60 cggggcgccc cgaggagctg tgggaggccg tggtgggggc cgctgagcgc ttccgggccc 120 ggactggcac ggagctggtg ctgctgaccg cggccccgcc gccaccaccc cgcccgggcc 180 cctgtgccta tgctgcccat ggtcgaggag ccctggcgga ggcagcgcgc cgttgcctcc 240 acgacatcgc actggcccac agggctgcca ctgctgctcg gcctcctgcg cccccaccag 300 caccacagcc acccagtccc acacccagcc caccccggcc taccctggcc agagaggaca 360 acgaggagga cgaggatgag cccacagaga cagagacctc cggggagcag ctgggcatta 420 gtgataatgg agggctcttt gtgatggatg aggacgccac cctccaggac cttcccccct 480 tctgtgagtc agaccccgag agtacagatg atggcagcct gagcgaggag acccccgccg 540 gcccccccac ctgctcagtg cccccagcct cagccctacc cacacagcag tacgccaagt 600 ccctgcctgt gtctgtgccc gtctggggct tcaaggagaa gaggacagag gcgcggtcat 660 cagatgagga gaatgggccg ccctcttcgc ccgacctgga ccgcatcgcg gcgagcatgc 720 gcgcgctggt gctgcgagag gccgaggaca cccaggtctt cggggacctg ccacggccgc 780 ggcttaacac cagcgacttc cagaagctga agcggaaata tggatccaga agaagaagaa 840 gaagaagaag aagatag 857 <210> 31 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> PRAS40-9 Arg fusion protein <400> 31 Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu Ala Val Val Gly Ala   1 5 10 15 Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu Leu Val Leu Leu Thr              20 25 30 Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro Cys Ala Tyr Ala Ala          35 40 45 His Gly Arg Gly Ala Leu Ala Glu Ala Ala Arg Arg Cys Leu His Asp      50 55 60 Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala Arg Pro Ala Pro  65 70 75 80 Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro Ser Pro Pro Arg Pro                  85 90 95 Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu Asp Glu Pro Thr Glu             100 105 110 Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser Asp Asn Gly Gly Leu         115 120 125 Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp Leu Pro Pro Phe Cys     130 135 140 Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser Leu Ser Glu Glu Thr 145 150 155 160 Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro Ala Ser Ala Leu Pro                 165 170 175 Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser Val Pro Val Trp Gly             180 185 190 Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser Asp Glu Glu Asn Gly         195 200 205 Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala Ala Ser Met Arg Ala     210 215 220 Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val Phe Gly Asp Leu Pro 225 230 235 240 Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys Leu Lys Arg Lys Tyr                 245 250 255 Gly Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg             260 265 <210> 32 <211> 884 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide coding 9Arg-PRAS40-9Arg fusion protein <400> 32 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccaaga agaagaagaa 60 gaagaagaag aagactcgag atggcgtcgg ggcgccccga ggagctgtgg gaggccgtgg 120 tgggggccgc tgagcgcttc cgggcccgga ctggcacgga gctggtgctg ctgaccgcgg 180 ccccgccgcc accaccccgc ccgggcccct gtgcctatgc tgcccatggt cgaggagccc 240 tggcggaggc agcgcgccgt tgcctccacg acatcgcact ggcccacagg gctgccactg 300 ctgctcggcc tcctgcgccc ccaccagcac cacagccacc cagtcccaca cccagcccac 360 cccggcctac cctggccaga gaggacaacg aggaggacga ggatgagccc acagagacag 420 agacctccgg ggagcagctg ggcattagtg ataatggagg gctctttgtg atggatgagg 480 acgccaccct ccaggacctt ccccccttct gtgagtcaga ccccgagagt acagatgatg 540 gcagcctgag cgaggagacc cccgccggcc cccccacctg ctcagtgccc ccagcctcag 600 ccctacccac acagcagtac gccaagtccc tgcctgtgtc tgtgcccgtc tggggcttca 660 aggagaagag gacagaggcg cggtcatcag atgaggagaa tgggccgccc tcttcgcccg 720 acctggaccg catcgcggcg agcatgcgcg cgctggtgct gcgagaggcc gaggacaccc 780 aggtcttcgg ggacctgcca cggccgcggc ttaacaccag cgacttccag aagctgaagc 840 ggaaatatgg atccagaaga agaagaagaa gaagaagaag atag 884 <210> 33 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> 9Arg-PRAS40-9Arg fusion protein <400> 33 Arg Arg Arg Arg Arg Arg Arg Arg Ser Ser Gly Leu Val Pro Arg   1 5 10 15 Gly Ser His Leu Glu Met Ala Ser Gly Arg Pro Glu Glu Leu Trp Glu              20 25 30 Ala Val Val Gly Ala Ala Glu Arg Phe Arg Ala Arg Thr Gly Thr Glu          35 40 45 Leu Val Leu Leu Thr Ala Ala Pro Pro Pro Pro Pro Arg Pro Gly Pro      50 55 60 Cys Ala Tyr Ala Ala His Gly Arg Gly Ala Ala Gul Ala Ala Arg  65 70 75 80 Arg Cys Leu His Asp Ile Ala Leu Ala His Arg Ala Ala Thr Ala Ala                  85 90 95 Arg Pro Pro Ala Pro Pro Pro Ala Pro Gln Pro Pro Ser Pro Thr Pro             100 105 110 Ser Pro Pro Arg Pro Thr Leu Ala Arg Glu Asp Asn Glu Glu Asp Glu         115 120 125 Asp Glu Pro Thr Glu Thr Glu Thr Ser Gly Glu Gln Leu Gly Ile Ser     130 135 140 Asp Asn Gly Gly Leu Phe Val Met Asp Glu Asp Ala Thr Leu Gln Asp 145 150 155 160 Leu Pro Pro Phe Cys Glu Ser Asp Pro Glu Ser Thr Asp Asp Gly Ser                 165 170 175 Leu Ser Glu Glu Thr Pro Ala Gly Pro Pro Thr Cys Ser Val Pro Pro             180 185 190 Ala Ser Ala Leu Pro Thr Gln Gln Tyr Ala Lys Ser Leu Pro Val Ser         195 200 205 Val Pro Val Trp Gly Phe Lys Glu Lys Arg Thr Glu Ala Arg Ser Ser     210 215 220 Asp Glu Glu Asn Gly Pro Pro Ser Ser Pro Asp Leu Asp Arg Ile Ala 225 230 235 240 Ala Ser Met Arg Ala Leu Val Leu Arg Glu Ala Glu Asp Thr Gln Val                 245 250 255 Phe Gly Asp Leu Pro Arg Pro Arg Leu Asn Thr Ser Asp Phe Gln Lys             260 265 270 Leu Lys Arg Lys Tyr Gly Ser Arg Arg Arg Arg Arg Arg Arg Arg Arg         275 280 285

Claims (3)

A pharmaceutical composition for the prevention and treatment of Parkinson's disease comprising a PRAS40 fusion protein in which a protein transport domain is covalently bonded to one or more of the N-terminal and C-terminal of human PRAS40 protein.
The method according to claim 1,
Wherein the protein transport domain is an HIV-Tat peptide. &Lt; RTI ID = 0.0 &gt; 25. &lt; / RTI &gt;
The method according to claim 1,
Wherein said fusion protein is SEQ ID NO. 11. 11. A pharmaceutical composition for preventing and treating Parkinson's disease comprising the PRAS40 fusion protein.

Images