Voltage-dependent anion channel protein VDAC1 inhibitory peptide and application thereof

Abstract

According to the voltage-dependent anion channel protein VDAC1 inhibitory peptide and the application thereof, active ingredients are a group of polypeptides extracted from leguminous plant seeds or gene expression, the polypeptides can specifically bind to the mitochondrial voltage-dependent anion channel protein 1 (VDAC1) of cells, the overexpression of VDAC1 in SH-SY5Y cells is inhibited, the mitochondrial damage of the SH-SY5Y cells is reduced, and the activity of the voltage-dependent anion channel protein VDAC1 inhibitory peptide is improved. Therefore, the normal physiological function of the SH-SY5Y cells can be maintained.

Description

Technical Field

[0001] The present invention belongs to the field of biology and new medicine technology, and specifically relates to a variety of polypeptides as voltage-dependent anion channel protein (VDAC1) inhibitors and their application in neurodegenerative diseases. Background Art

[0002] Mitochondrial dysfunction induces stress responses, such as those triggered by increased mitochondrial reactive oxygen species (mROS), which links the adaptability of the organelle to the condition of the entire organism. Voltage-dependent anion channel protein 1 (VDAC1) is a 31kDa membrane protein present on the outer membrane of mitochondria. It can form a hydrophilic channel on the membrane, regulate the entry and exit of anions, cations, ATP and other metabolites into and out of mitochondria, and play an important role in regulating cellular metabolism, maintaining intracellular calcium homeostasis, regulating cell apoptosis and necrosis. As a gatekeeper, VDAC1 plays an important role in maintaining mitochondrial function and cell viability by regulating its channel conductance and maintaining ΔΨm homeostasis. In addition, VDAC1 serves as a docking site for cellular kinases (such as hexokinase), allowing the enzyme to preferentially obtain ATP produced by oxidative phosphorylation. Studies have found that in many diseases such as T2D and Alzheimer's disease, VDAC1 is abnormally highly expressed, forming larger oligomeric channels on the outer membrane of mitochondria, promoting the release of mitochondrial DNA (mtDNA). VDAC1 oligomerization also affects mitochondrial calcium homeostasis and autophagy, further affecting cellular energy metabolism and cell fate.

[0003] It has been confirmed that VDAC1 undergoes expression changes during the occurrence and development of Alzheimer's disease (AD). VDAC1 expression can be detected in brain tissues of AD patients and transgenic mice, and gradually increases with the severity of the disease. VDAC1 interacts with APP, Aβ, and phosphorylated tau protein, which may block the mitochondrial pore and cause mitochondrial dysfunction. VDAC1 is involved in the toxic reaction process induced by Aβ. Pre-incubation of VDAC1 antibodies with different neural cells can resist Aβ-induced cell apoptosis. For example, downregulating VDAC1 expression in SH-SY5Y cells with small interfering RNA (siRNA) inhibited the cell penetration and toxicity of Aβ, indicating that VDAC1 is necessary for Aβ to enter cells and mediate cell apoptosis. Studies on APP-stable HEK293 cells found that downregulating VDAC1 expression with small interfering siRNA reduced the generation of Aβ, while overexpression of VDAC1 did not lead to increased Aβ levels.

[0004] It has been reported that peptides that specifically bind to VDAC1 protein have a variety of biological activities, such as regulating glucose and lipid metabolism, protecting pancreatic β cells from apoptosis, alleviating atherosclerosis, reducing lipopolysaccharide-induced inflammation, and promoting Ca2+. 2+ Influx and cell secretion, inhibiting the growth of colon cancer cells, etc. However, there is no report on these peptides interacting with VDAC1, inhibiting its expression and protecting mitochondrial damage in neurons. Summary of the invention

[0005] The present invention provides a class of peptides extracted from leguminous plant seeds including but not limited to soybeans and peas as VDAC1 inhibitors to avoid mitochondrial damage in nerve cells such as SH-SY5Y cells, thereby treating neurodegenerative diseases. This group of peptides can specifically target VDAC1, and by forming 7 hydrogen bond interactions between molecules, the binding free energy is as low as -9.256 kcal / mol, and can be used as VDAC1 inhibitors. In the Aβ-induced model, cellular ROS increased significantly, ATP decreased significantly, and mitochondrial membrane potential MMP decreased significantly. This group of peptides can significantly reverse the ROS, ATP and MMP levels under Aβ-induced acute conditions, thereby reducing the damage to nerve cell mitochondria induced by Aβ.

[0006] The present invention provides a polypeptide that specifically binds to voltage-dependent anion channel protein 1 (VDAC1), wherein the polypeptide comprises at least one of amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0007] Furthermore, each amino acid sequence of the polypeptide contains 37 amino acids, among which there are 6 cysteines, which are located at site 3, site 7, site 15, site 20, site 22 and site 32, wherein the cysteine ​​at site 3 forms a pair of disulfide bonds with the cysteine ​​at site 20, the cysteine ​​at site 7 and the cysteine ​​at site 22, and the cysteine ​​at site 15 and the cysteine ​​at site 32, respectively, and the three pairs of disulfide bonds form a cysteine ​​knot domain.

[0008] Furthermore, the binding energy between the polypeptide and the VDAC1 protein ranges from -7.324 to -9.256 kcal / mol.

[0009] Furthermore, the polypeptide is extracted from leguminous plant seeds.

[0010] The present invention also proposes the use of the voltage-dependent anion channel protein VDAC1 inhibitory peptide in the preparation of a drug for reducing nerve cell damage.

[0011] Furthermore, the neural cell is a human neuroblastoma cell SH-SY5Y.

[0012] The present invention also provides a pharmaceutical preparation for reducing nerve cell damage, wherein the active ingredient of the pharmaceutical preparation includes the above-mentioned polypeptide binding to voltage-dependent anion channel protein 1 (VDAC1).

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The disulfide bond formed by the polypeptide itself forms a cysteine ​​knot domain, making the polypeptide compound resistant to degradation by digestive tract proteases, trypsin and endoproteinase Glu-C. At the same time, the pharmaceutical composition with this polypeptide as the main active ingredient can be taken orally to work without injection, thus alleviating the pain of the patient.

[0015] (2) Interaction simulation experiments showed that the binding energy range of the combined peptides and VDAC1 was -7.324 to -9.256 kcal / mol, both of which had low binding free energy and were mainly caused by multiple hydrogen bonds between molecules.

[0016] (3) The peptide drug combination was verified to interact with VDAC1 through co-ip and pull-down experiments.

[0017] (4) The peptide drug combination can significantly reduce the levels of ROS, ATP and MMP under Aβ-induced conditions by inhibiting the overexpression of VDAC1, thereby reducing the mitochondrial damage of neurons induced by Aβ, thereby treating neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Simulation of the interaction between peptide and VDAC1;

[0019] Figure 2 A in the figure is the silver staining result of the interaction between polypeptide AG and VDAC1. Figure 2 B in the figure is the result of western hybridization identification;

[0020] Figure 3 Pull down results of the interaction between peptide VG and VDAC1;

[0021] Figure 4 This is the pull-down result of the interaction between peptide VG and his-VDAC1;

[0022] Figure 5The results of the experiment in which the peptide protected SH-SY5Y cells (human neuroblastoma cells) from Aβ-induced mitochondrial damage by inhibiting VDAC1 expression, where A is the result of Western blot experiment, B is the result of mitochondrial ROS level detection in SH-SY5Y cells, C is the result of mitochondrial membrane potential detection in SH-SY5Y6 cells, and D is the result of ATP level detection in SH-SY5Y cells. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Example 1: Extraction of polypeptides from seeds of leguminous plants including but not limited to soybean and pea, extraction process, sequencing and identification of the results, and naming.

[0025] 200g of soybeans were germinated, ground and immersed in 1L of water, 50ml of white vinegar was added, stirred and dissociated for 6h, and filtered. The crude soybean peptide extract was obtained by centrifugation in a large refrigerated high-speed centrifuge at 8000r for 15min and freeze-dried for 72h. The crude extracted polypeptide was dissolved and filtered with a 0.22um filter element. The dissolved and filtered soybean polypeptide solution was separated and purified using preparative liquid chromatography technology. The purified polypeptide sample was analyzed using high performance liquid chromatography (HPLC). The purified polypeptide was VDAC1 inhibitory polypeptide, and the peak time was 33min-36min.

[0026] The amino acid sequences of the six polypeptides extracted are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0027] Example 2: Simulation of the interaction between peptide and VDAC1

[0028] In this example, Autodock vina was used for molecular docking.

[0029] First, prepare the protein receptor and polypeptide ligand. The protein receptor of VDAC1 is downloaded from the PDB database in the pdb format (PDB: 2JK4), which is the receptor of VDAC1.

[0030] The ligands are the six polypeptides separated in Example 1.

[0031] SEQ ID NO.1: ASCNG VCSPF EMPPC GSSAC RCIPV GLVVG YCRHP SG, abbreviated as AG.

[0032] SEQ ID NO.2: VSCNG VCSPF EMPPC GSSAC RCIPY GLVVG NCRHP SG, abbreviated as VG.

[0033] SEQ ID NO.3: ASCNGVCSPFEMPPCGTSACRCIPVGLVIGYCRNPSG, abbreviated as PA1F.

[0034] SEQ ID NO.4: VSCNG VCSPF DIPPC GTPLC RCIPY GLFVG NCRHP YG, referred to as DG.

[0035] SEQ ID NO.5: ISCNG VCSPF DIPPC GTPLC RCIPA GLFVG KCRHP YG, referred to as IG.

[0036] SEQ ID NO.6: VDCSGACSPFEVPPCGSRDCRCIPIGLVVGFCIYPTG, aM1 for short.

[0037] First, the process of protein tertiary structure prediction and evaluation was carried out using online tools, especially for the six homologous peptides mentioned above.

[0038] 1. Use the Swiss-model website to predict the tertiary structure of the target sequence online, and download the generated PDB (Protein Data Bank) file, which contains the detailed information of the predicted protein tertiary structure. 2. Use the SAVES v6.0 online website to evaluate and score the quality of the modeled PDB protein structure. 3. After the model predicted in step 1 is qualified in the SAVES v6.0 evaluation in step 2, it needs to be evaluated as qualified by 2 different evaluation software (including PROCHECK, Molprobity, Errat and Verify_3D) before the small molecule protein of homology modeling can be used. After the prediction and the evaluation of multiple softwares are qualified, it is proved that in this embodiment, the homology modeling detection evaluation of this group of polypeptides is qualified.

[0039] Then, we used online tools to conduct molecular docking experiments on the receptor (VDAC1 protein) and the ligand (six peptides) to simulate the molecular interaction between the two. The specific steps are:

[0040] First, the receptor and each ligand were pre-processed using Autodock vina software to set the active pocket, that is, the area on the receptor that may bind to the ligand. 20 models were generated using the program, from which the model with the lowest binding energy was selected for docking the molecule.

[0041] The above test results show that the binding energy between VDAC1 protein and this group of polypeptides ranges from -7.324 to -9.256 kcal / mol, among which the binding energy between VDAC1 and AG (SEQ ID NO.1) is the lowest (-9.256 kcal / mol), followed by the binding energy with VG (SEQ ID NO.2) (-9.254 kcal / mol), which indicates that there is a strong interaction between VDAC1 and this group of polypeptides.

[0042] Finally, Pymol software was used for visualization analysis. Figure 1 As shown, this group of peptides can enter the α-helical region ion channel formed by the VDAC1 molecule, and may block the substance transport of VDAC1 in the cell by forming intermolecular hydrogen bonds.

[0043] Example 3: Interaction test between polypeptide AG (SEQ ID NO.1) and VDAC1

[0044] In this example, the polypeptide AG selected in Example 2 was used as the object, and the interaction between the polypeptide and VDAC1 was detected by co-immunoprecipitation (Co-IP), and then the protein interacting with the polypeptide antigen was identified by western hybridization.

[0045] 1. Cell culture: SH-SY5Y cells (human neuroblastoma cells) were inoculated in a 10 cm culture dish and cultured in a high glucose medium (40 mM) for 12 h;

[0046] 2. Peptide treatment: select the polypeptide AG (such as SEQ ID NO.1) in the above Example 1, add it to the high-glucose medium, and continue incubation for 12 hours to allow the polypeptide to interact with the cells;

[0047] 3. Fix the cells, remove the culture medium, wash the cells three times with PBS to remove unbound peptides and other impurities, and then add 4% paraformaldehyde to fix the cells for 15 minutes to maintain cell morphology and prepare for subsequent antibody incubation.

[0048] 4. Antibody incubation and elution: Use peptide-specific antibodies (aglycin antibodies) for incubation to allow the antibodies to bind to peptides that may be bound to the cells, and then go through an elution step to remove unbound antibodies.

[0049] 5. Protein separation and visualization: The eluted samples were separated by SDS-PAGA (sodium dodecyl sulfate polyacrylamide gel electrophoresis) to separate the proteins according to their molecular weight. Finally, the protein bands were visualized by silver staining to observe the proteins that the polypeptides may bind to.

[0050] In the above experiment, three groups of experiments were set up, including a blank group (SH-SY5Y cells), a control group (SH-SY5Y cells, polypeptide AG) and an experimental group (SH-SY5Y cells, polypeptide AG and polypeptide-specific antibody). Figure 2 As shown in the silver staining results of A in the figure, the anti-Aglcin antibody pulled down a specific band at around 35Kd, and the protein was identified as VDAC1 by mass spectrometry, indicating that the polypeptide AG may interact with VDAC1.

[0051] Next, in order to verify whether the band pulled down by the anti-Aglcin antibody contains VDAC1 protein, a co-immunoprecipitation (Co-IP) experiment was performed using VDAC1 protein antibody for further confirmation.

[0052] First, the cells in step 4 are treated and lysed, and the cells that have been incubated and fixed with the polypeptide AG are lysed to release the proteins in the cells; then, the antibody is incubated, and the lysed protein mixture is incubated with the VDAC1 protein-specific antibody, and the VDAC1 protein antibody can bind to the protein that it specifically binds to; then, immunoprecipitation is performed, and a bead support (such as Protein A / G beads) that can bind to the antibody is used to fix and precipitate the antibody-protein complex. In this process, the protein that binds to the VDAC1 protein-specific antibody (including VDAC1 in the protein pulled down by the aglcin antibody) will be precipitated; then, the precipitate is washed to remove non-specifically bound proteins, and the protein that binds to the VDAC1 protein-specific antibody is eluted; finally, detection and analysis are performed, and the eluted protein is separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and the presence of VDAC1 protein is detected by Western blot.

[0053] The results are as follows Figure 2 As shown in B, it is proved that the protein that interacts with polypeptide AG in this example is VDAC1 protein.

[0054] Example 4: Peptide (SEQ ID NO.2) and VDAC1 Pull-down Test

[0055] In this example, a pull-down experiment was used to detect the interaction between the polypeptide and VDAC1, and then western hybridization was used to identify the protein that interacted with the polypeptide antigen.

[0056] 1. Synthesize SEQ ID NO.2 with his tag, referred to as his-VG.

[0057] 2. Collect 4 large dishes of SH-SY5Y cell (human neuroblastoma cell) suspension, then resuspend it in mitochondrial isolation buffer and supplement with protease inhibitors. Then transfer the cell suspension to a 5ml glass homogenizer on ice and homogenize about 20 times. Centrifuge the obtained homogenate at 600g for 10 minutes and collect the supernatant. Centrifuge the supernatant at 12,000g for 10 minutes, discard the supernatant, and the precipitate obtained is the cell precipitate, which is the crude mitochondria. The crude mitochondria were lysed with IP lysis buffer, the sample was sonicated 4 times, 4s each time, and centrifuged at 12000g for 25min to obtain the supernatant mitochondrial protein.

[0058] 3. Dissolve 1 mg of his-VG in 200ul dd H2O, incubate his-VG and Ni-NTA His-Tag purified agarose beads (a substance used to purify recombinant proteins containing polyhistidine (6xHis) sequences) (200ul) together for 2 hours, then add the mitochondria purified from SH-SY5Y cells obtained in step 2, and continue incubating overnight. In this step, a control group was set up, which included the his-tag agarose beads incubated with the mitochondria purified from SH-SY5Y cells in step 2, but without the addition of his-VG in step 1.

[0059] 4. After incubation, wash three times with TBST, then elute with imidazole, and prepare samples for western hybridization identification using VDAC1 protein antibody.

[0060] like Figure 3 The western results showed that his-VG could interact with VDAC1.

[0061] Example 5: Peptide (SEQ ID NO.2) and his-VDAC1 pull down test

[0062] In this example, a pull-down assay was used to detect the interaction between endogenous peptides and his-VDAC1, and then western hybridization was used to identify proteins that interacted with the peptide antigen.

[0063] 1. Construct PET28a-his-VDAC1 plasmid.

[0064] 1.1 Obtain the coding sequence of the target gene VDAC1, perform PCR amplification with the designed primers and template DNA (cDNA of VDAC1) to obtain the target gene fragment, and use a PCR product purification kit to recover the amplified product containing the target gene.

[0065] 1.2 Connection with the vector: amplify PET28a as an expression vector in Escherichia coli, extract plasmid DNA, and use the same restriction site as in the primer in step 1.1 to treat the PET28a vector with restriction enzymes to produce the same sticky ends as the target gene fragment. Use DNA ligase to connect the target gene fragment purified in step 1.1 to the PET28a vector after restriction enzyme treatment to form a recombinant plasmid.

[0066] 1.3 Transformation and screening: The ligated recombinant plasmid was transformed into BL21 cells, and the transformed recipient cells were cultured in a medium containing kanamycin to screen out positive clones that integrated the target gene.

[0067] 1.4 Identification and purification: The positive clones in the above steps were selected for colony PCR, and the clones identified as positive by colony PCR were sequenced to verify the target gene, and then expressed in the recipient cells of step 1.3. After protein purification, his-VDAC1 protein (obtained by laboratory purification) was obtained.

[0068] 2. Construct pCMV-C-EGFP-VG plasmid.

[0069] 2.1 Using the VG sequence (SEQ ID NO.2) of Example 1 as the target gene, design and synthesize upstream and downstream primers; obtain the target gene fragment by PCR amplification, and purify the amplified target gene fragment using a PCR product purification kit;

[0070] 2.2 pCMV-C-EGFP containing an EGFP (enhanced green fluorescent protein) tag was used as a eukaryotic expression vector, which was amplified in Escherichia coli, and the plasmid DNA was extracted. The plasmid DNA was treated with enzymes using the same restriction sites as the target gene primers. The purified target gene fragment was connected to the enzyme-treated vector using DNA ligase to form a recombinant plasmid, and then the recombinant plasmid was endogenously expressed in 293t cells to express the polypeptide VG in 239t cells. After the cells were lysed, the cell protein supernatant was obtained by centrifugation.

[0071] 3. Pull Down test.

[0072] 3.1 Prepare IP (immunoprecipitation) samples. Incubate the purified His-VDAC1 protein obtained in step 1.4 with Ni-NTAHis-Tag purified agarose beads (200ul) for 2 hours to allow the His-VDAC1 protein to bind to the agarose beads; then add the protein supernatant of 239t cells transfected with the recombinant plasmid obtained in step 2.2, and continue to incubate overnight to allow the EGFP-VG polypeptide in the cells to interact with the his-VDAC1 protein bound to the agarose beads. Wash the agarose beads with washing buffer PBST to remove unbound proteins and impurities.

[0073] 3.2 The interaction between His-VDAC1 protein and EGFP-VG was detected by Western blot (immunoblotting) method, i.e., using VDAC1 antibody and GFP antibody. The bound protein sample obtained by immunoprecipitation in step 3.1 was subjected to SDS-PAGE electrophoresis to separate the proteins. The separated proteins were incubated with VDAC1 antibody and GFP antibody respectively.

[0074] like Figure 4 The results in A show that GFP-VG can be overexpressed in 293t cells. Figure 4 The results in B (WB) indicate that VG can interact with VDAC1.

[0075] Embodiment 6:

[0076] In the Aβ (β-amyloid protein)-induced SH-SY5Y cell (human neuroblastoma cell) mitochondrial damage model, this group of peptides can reverse the ROS (reactive oxygen species), ATP (adenosine triphosphate) and MMP (mitochondrial membrane potential) levels under Aβ-induced conditions by inhibiting the overexpression of VDAC1, thereby protecting mitochondrial damage.

[0077] The specific steps are as follows:

[0078] 1. Cell culture and inoculation: cultured SH-SY5Y cells were inoculated into 6-well plates and cultured adherently.

[0079] 2. Group treatment: The cells obtained in step 1 were divided into 3 groups. The first group was used as the control group and cultured with normal culture medium without any treatment. The second experimental group was treated with 10umol / L Aβ (β-amyloid protein) to induce mitochondrial damage. The third experimental group was first treated with Aβ and then added with polypeptide VG and incubated for 72 hours.

[0080] 3. Collect cells and prepare protein samples: collect cells, lyse them with NP40 to release the proteins in the cells, and then prepare protein samples.

[0081] 4. Protein quantification and detection: The protein samples were quantified using the BCA kit to ensure that the protein content in each sample was the same. Afterwards, the VDAC1 oligomerization was detected using western detection technology.

[0082] 5. Detection indicators: (1) ROS (reactive oxygen species) level: After incubating the three groups of cells in step 2 with the fluorescent probe DCFH-DA, the ROS level was detected using a fluorescent microplate reader; (2) ATP (adenosine triphosphate) level: according to the method of the ATP detection kit (Biyuntian), it was detected using a fluorescent microplate reader; (3) Mitochondrial membrane potential (MMP): After incubating the three groups of cells in step 2 above with the JC-1 probe for 30 minutes, it was detected using a fluorescent microplate reader.

[0083] Figure 5 From the results of western blot in A, we can see that the protein band of the third experimental group, i.e. the peptide group, decreased, indicating that the addition of peptide reduced the expression of VDAC1. Figure 5 B in the figure shows that the ROS level in the Aβ (β-amyloid protein)-treated group increased significantly, while the ROS level in the peptide-added group decreased significantly. Figure 5 C in the figure shows that the Aβ (β-amyloid protein) treatment group significantly reduced the mitochondrial membrane potential, while the peptide addition group was able to restore the mitochondrial membrane potential. Figure 5 D in the figure shows that the ATP level of SH-SY5Y cells treated with Aβ (β-amyloid protein) was significantly reduced, while the ATP level was significantly increased in the peptide addition group. These results indicate that peptide VG reduces the mitochondrial damage of SH-SY5Y cells treated with Aβ (β-amyloid protein) by inhibiting the overexpression of VDAC1.

Claims

1. Application of a voltage-dependent anion channel protein VDAC1 inhibitory peptide in the preparation of a drug for reducing nerve cell damage, characterized in that: The polypeptide can specifically bind to the VDAC1 protein, and the polypeptide comprises at least one of the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

2. The use according to claim 1, characterized in that: Each amino acid sequence of the polypeptide contains 37 amino acids, among which there are 6 cysteines, which are located at position 3, position 7, position 15, position 20, position 22 and position 32. The cysteine ​​at position 3 forms a pair of disulfide bonds with the cysteine ​​at position 20, the cysteine ​​at position 7 forms a pair of disulfide bonds with the cysteine ​​at position 22, and the cysteine ​​at position 15 forms a pair of disulfide bonds with the cysteine ​​at position 32, and the three pairs of disulfide bonds form a cysteine ​​knot domain.

3. The use according to claim 1, characterized in that: The binding energy of the polypeptide to the VDAC1 protein ranges from -7.324 to -9.256 kcal / mol.

4. The use according to claim 1, characterized in that: The polypeptide is extracted from seeds of leguminous plants.

5. The use according to claim 1, characterized in that: The neural cells are human neuroblastoma cells SH-SY5Y.

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