Application of a polypeptide in preparing a drug for treating Tau protein disease

Abstract

The present invention relates to the field of biotechnology and discloses the use of a polypeptide in the preparation of a drug for treating tauopathy. The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1. The polypeptide can inhibit tau protein aggregation, inhibit cellular oxidative stress induced by K18 PFFs, and improve neuronal synaptic dysfunction induced by K18 PFFs. The polypeptide can be used to prepare tau protein aggregation inhibitors and drugs for treating tauopathy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of a polypeptide in preparing a medicine for treating Tau protein disease. Background Art

[0002] Tauopathies are a type of neurodegenerative disease characterized by abnormal tau protein deposition in the brain (Kovacs GG (2017) Tauopathies. Handb Clin Neurol 145: 355-368. https: / / 10.1016 / B978-0-12-802395-2.00025-0). Under physiological conditions, tau protein exists in neurons and interacts with microtubules to maintain the normal morphology of neurons (Avila J, Lucas JJ, Perez M, Hernandez F (2004) Role of tauprotein in both physiological and pathological conditions. Physiol Rev 84 (2): 361-384. https: / / 10.1152 / physrev.00024.2003). However, under pathological conditions, Tau protein undergoes abnormal phosphorylation, acetylation and other modifications to increase its aggregation tendency, causing its normal function to be lost and forming toxic pathological aggregates, leading to the occurrence of Tau disease. According to the different morphology of the aggregates, etiology and other conditions, Tau disease is divided into different types, such as Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, etc. (Arakhamia T, Lee CE, Carlomagno Y, Duong DM, Kundinger SR, Wang K, Williams D, Deture M et al (2020) Posttranslational Modifications Mediate the Structural Diversity of Tauopathy Strains. Cell 180 (4): 633-644. https: / / 10.1016 / j.cell.2020.01.027). At present, there is no effective treatment for Tau disease.

[0003] Tau pathology in the blood and cerebrospinal fluid of patients with tauopathy can characterize the severity of the patient's clinical symptoms (Zhang Y, Wu KM, Yang L, Dong Q, Yu JT (2022) Tauopathies: new perspectives and challenges. Mol Neurodegener 17(1):28. https: / / 10.1186 / s13024-022-00533-z). Injecting abnormal Tau protein into the brains of healthy mice can induce disease in healthy mice (Iba M, Mcbride JD, Guo JL, Zhang B, Trojanowski JQ, Lee VM (2015) Tau pathology spread in PS19 tau transgenic mice following locus coeruleus (LC) injections of synthetic tau fibrils is determined by the LC's afferent and efferent connections. Acta Neuropathol 130 (3): 349-362. https: / / 10.1007 / s00401-015-1458-4). These research results suggest that clearing or inhibiting Tau protein aggregation in the brains of patients may be an effective method for treating tauopathies. Summary of the Invention

[0004] The present invention aims to provide a polypeptide that inhibits Tau protein aggregation, a method for preparing the same, and the use of the polypeptide in preparing a medicament for treating tauopathies. The polypeptide of the present invention can inhibit Tau aggregation and alleviate cell and neuronal damage induced by Tau repeat domain preformed fibrils (K18 PFFs), and has great potential for the treatment of tauopathies.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a polypeptide for inhibiting Tau protein aggregation, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a biomaterial related to the polypeptide, which is any one of the following A1) to A4):

[0008] A1) a nucleic acid molecule encoding the polypeptide, the sequence of which is preferably as shown in SEQ ID NO. 2;

[0009] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0010] A3) a recombinant expression vector containing the nucleic acid molecule described in A1), or a recombinant expression vector containing the expression cassette described in A2); the recombinant expression vector is preferably pET28c;

[0011] A4) a recombinant cell containing the nucleic acid molecule described in A1), or a recombinant cell containing the expression cassette described in A2), or a recombinant cell containing the recombinant expression vector described in A3); the recombinant cell preferably uses Escherichia coli BL21 as a host cell.

[0012] The present invention also provides a method for preparing the above polypeptide, which comprises the following steps: inoculating the above recombinant cells into a culture medium, and obtaining the polypeptide by culture, or obtaining the polypeptide by culture and inducing expression.

[0013] The present invention also provides the use of the above polypeptide or related biological materials in the preparation of Tau protein aggregation inhibitors.

[0014] The present invention also provides the use of the above-mentioned polypeptide or related biomaterials in the preparation of a drug for treating tauopathy, which includes Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, chronic traumatic encephalopathy, etc.

[0015] Advantages and beneficial effects of the present invention: The polypeptide of the present invention can inhibit Tau protein aggregation, inhibit K18 PFFs-induced cellular oxidative stress, and improve K18 PFFs-induced neuronal synaptic dysfunction. The present invention provides a new drug for the treatment of Tauopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the peptide inhibition of Tau aggregation in Example 1 are shown. K18 monomers were incubated in the presence or absence of the peptide, and their aggregation kinetics were monitored using thioflavin T (ThT). The results showed that the peptide significantly inhibited K18 aggregation ( Figure 1 A), n = 3 independent replicates. K18 PFFs were added to Tau RD-YFP HEK293 cells in the presence or absence of peptide to observe Tau aggregation. The results showed that K18 PFFs successfully promoted the formation of Tau inclusion bodies, which was inhibited by the peptide ( Figure 1 B, C), n = 5 independent replicates, scale bar: 20 μm. ANOVA, **P < 0.01, ****P < 0.0001.

[0017] Figure 2The results of Example 2 show that the polypeptide inhibits K18 PFFs-induced cellular oxidative stress. K18 PFFs were added to SH-SY5Y cells in the presence or absence of the polypeptide, and oxidative stress indicators were detected. The results showed that the polypeptide significantly reduced the intracellular reactive oxygen species (ROS) induced by K18 PFFs. Figure 2 A, B), malondialdehyde ( Figure 2 C) and hydrogen peroxide ( Figure 2 D) levels, indicating that the peptide significantly inhibited K18 PFFs-induced oxidative stress. n = 5 independent replicates. Scale bar: 50 μm. ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0018] Figure 3 This figure shows the results of Example 3 demonstrating that the peptide improves K18 PFFs-induced synaptic dysfunction in neurons. K18 PFFs were added to neurons in the presence or absence of the peptide. DiI staining revealed that the peptide significantly improved the K18 PFFs-induced decrease in dendritic spine density. n = 8 independent replicates. Scale bar: 10 μm. ANOVA, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0019] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0020] [Example 1] Peptide inhibits Tau aggregation

[0021] 1. Preparation of recombinant polypeptide protein

[0022] The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and its coding sequence is shown in SEQ ID NO.2. The NotⅠ and SalⅠ double enzyme digestion method is used to obtain a polypeptide coding sequence with sticky ends and a pET28c expression vector. The two are ligated with T4 DNA ligase to insert the polypeptide coding sequence into the pET28c expression vector to obtain a recombinant plasmid. Subsequently, the recombinant plasmid is transformed into Escherichia coli BL21 to obtain protein expression bacteria. An appropriate amount of protein expression bacteria is added to LB liquid culture medium and placed in a shaker, shaken at 37°C and 160 rpm for 6 hours, then the shaker temperature is lowered to 20°C. After the bacterial solution is adapted to 20°C for 1 hour, isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to a final concentration of 1 mM, and the bacterial solution is shaken overnight (cultured for 14 hours). The next day, the bacteria are collected by centrifugation at 10,000 rpm for 10 minutes, and the resulting precipitate is placed in a solvent (20 mM Tris-HCl mixture, pH = 8), and then sonicated at a power of 450 W for 20 minutes. The precipitate was collected by centrifugation at 10,000 rpm for 15 minutes. The precipitate was washed repeatedly with a washing buffer (20 mM Tris-HCl, 1 mM EDTA, 2 M urea, 1 M sodium chloride, 1% Triton X-100, pH = 8.0) and centrifuged at 10,000 rpm for 15 minutes to collect the precipitate. This was repeated three times. The resulting precipitate was dissolved in a lysis buffer (20 mM Tris-HCl, 5 mM DTT, 8 M urea, pH = 8.0) and allowed to stand overnight at 4°C. On the third day, the precipitate was discarded by centrifugation at 10,000 rpm for 15 minutes. A diluent (20 mM Tris-HCl, 0.15 M sodium chloride, pH = 8.0) was slowly added dropwise to the supernatant to gradually renature the protein. Dilution was stopped when the solution became slightly turbid. The resulting solution was placed in a dialysis bag and dialyzed against double-distilled water at 4°C for 24 hours. On the last day, the concentration and purity of the soluble protein were measured, and the protein solution was frozen at -80°C and then freeze-dried in a freeze dryer to obtain the target protein dry powder.

[0023] 2. Preparation of K18 Monomer Protein

[0024] First, the pRK172-His-K18 expression plasmid was constructed. Specifically, a truncated version of the human Tau coding sequence (SEQ ID NO. 3) containing only the microtubule-binding domain (K18) was fused to an N-terminal His tag and inserted into the Kpn1 site of the pRK172 bacterial expression vector. The plasmid was then transformed into Escherichia coli BL21(DE3). Expression of pRK172-His-K18 was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 6-8 hours. The induced bacteria were sonicated in a buffer containing 20 mM Tris (pH 8.0), 500 mM NaCl, and 5 mM imidazole. After the solution cleared, it was centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant collected after centrifugation was applied to a column pre-equilibrated with Ni-NTA His·Bind Resin (MERCK). The column was washed with 10 column volumes of a buffer containing 20 mM Tris (pH = 8.0), 500 mM NaCl, and 15 mM imidazole; the protein was eluted in the same buffer containing 100 mM imidazole; the purified protein was dialyzed against double-distilled water for 48 hours, lyophilized in a freeze dryer, and stored in a -80°C refrigerator until use.

[0025] 3. K18 PFFs Preparation and ThT Assay

[0026] The lyophilized peptide and K18 monomer were dissolved in PBS and centrifuged at 15,000 rpm for 20 minutes to remove insoluble components. Three solutions were prepared: PBS, K18 solution (final concentration 1 mg / mL), and K18 (final concentration 1 mg / mL) + peptide (final concentration 0.05 mg / mL). DTT (final concentration 2 mM) and low molecular weight heparin (final concentration 12.5 μM) were added to each solution. The solutions were then incubated on a shaker at 37°C and 1000 rpm for 13 hours. During the incubation period, the aggregation of K18 in the presence or absence of peptide was assessed using a thioflavin T (ThT) assay. Specifically, a 10 μL aliquot was removed from the incubation mixture, and PBS and ThT were added to a total volume of 100 μL, with a final ThT concentration of 2 mM. After incubation at room temperature for 10 minutes, fluorescence of the mixture was recorded using a SpectraMax microplate reader at an excitation wavelength of 450 nm and an emission wavelength of 510 nm. Repeat the above operation at different time points, and the results are shown in Figure 1 A, indicating that the peptide significantly inhibited K18 aggregation. The remaining solution in the K18 solution group was K18 PFFs, which were used for subsequent experiments.

[0027] 4. Cell Immunofluorescence

[0028] HEK293 cell lines stably expressing the Tau repeat domain (Tau RD-YFP HEK293 cells) produce Tau inclusions when exposed to exogenous K18 PFFs. Tau aggregate morphology can be observed under a fluorescence microscope and is used as a cell model for detecting Tau aggregation (Sanders DW, Kaufman SK, Devos SL, Sharma AM, Mirbaha H, Li A, Barker SJ, Foley AC et al (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82(6): 1271-1288. https: / / 10.1016 / j.neuron.2014.04.047).

[0029] Tau RD-YFP HEK293 cells were plated in a six-well plate. When the cells adhered and the cell density reached about 50%, peptides (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL) were added, or equal amounts of PBS and K18 PFFs were added. Only PBS was added as the control group, and the cells were cultured for 36 hours. The culture medium in the six-well plate was removed, PBS was slowly added along the wall of the well, the six-well plate was gently shaken to wash the cells, and the PBS was removed. Then 4% paraformaldehyde containing 1% Triton X-100 was added to the well for fixation and permeabilization for 30 minutes. Blocked with 3% bovine serum albumin (BSA) for 30 minutes, stained with DAPI for 5 minutes, and images were taken using a fluorescence microscope. The results are shown in Figure 1 B, C, K18 PFFs successfully promoted the formation of Tau inclusion bodies, and this promotion was inhibited by the peptide.

[0030] [Example 2] Peptide inhibits K18 PFFs-induced cellular oxidative stress

[0031] SH-SY5Y cells were plated into the dish. When the cells adhered and the density reached about 50%, peptides (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL) were added, or equal amounts of PBS and K18 PFFs were added. The control group was treated with PBS alone. After the cells were cultured for 36 hours, the culture medium was removed and PBS was slowly added along the wall of the well. The dish was gently shaken to wash the cells and the PBS was removed. 1 mL of PBS was added to the dish again, and the cells in the dish were resuspended and placed in a centrifuge tube. Centrifuged at 3700 rpm for 5 minutes at room temperature, the supernatant was removed, and the cell pellet was obtained for subsequent operations.

[0032] Intracellular reactive oxygen species (ROS) levels were measured using DCFH-DA (Nanjing Construction Bioengineering Institute, E004-1-1). A malondialdehyde (MDA) detection kit (Nanjing Construction Bioengineering Institute, A003-1-2) was used to assess lipid peroxidation in cells. A hydrogen peroxide detection kit (Nanjing Construction Bioengineering Institute, A064-1-1) was used to measure the concentration of hydrogen peroxide in cells. All assays were performed according to the manufacturer's instructions. All results were normalized to the corresponding total protein content.

[0033] See the results Figure 2 The peptide significantly reduced the intracellular reactive oxygen species induced by K18 PFF ( Figure 2 A, B), malondialdehyde ( Figure 2 C) and hydrogen peroxide ( Figure 2 D) levels, indicating that the peptide significantly inhibited K18 PFF-induced oxidative stress.

[0034] [Example 3] Peptide improves K18 PFFs-induced neuronal synaptic dysfunction

[0035] 1. Neuron Isolation and Culture: 18-day-old gestational female C57BL / 6 mice were sacrificed and, after disinfection with alcohol, mouse embryos were removed from their uteri. After washing the embryos with 75% ethanol and sterile PBS, the brains were removed from the head using a dissecting microscope and transferred to a fresh culture dish containing PBS. The meninges were stripped away, and the cerebral cortex was removed. The cortical tissue was carefully minced with sterile scissors and added to DMEM (DMEM-HS-FS) supplemented with horse serum (final concentration 5%) and fetal bovine serum (final concentration 5%). The cells were gently pipetted with a sterile Pasteur pipette until no large clumps of tissue were visible. The cells were centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 3 mL of DMEM-HS-FS, pipetted again, and centrifuged to obtain a neuronal cell suspension. Neurons were plated onto six-well plates lined with round glass slides, with the ratio of one pup to one full six-well plate.

[0036] 2. DiI staining: After 7 days of neuronal culture, add polypeptide (final concentration 0.25 μg / mL) and K18 PFFs (final concentration 5 μg / mL), or add equal amounts of PBS and K18 PFFs. Only PBS was added as the control group, and the neurons were cultured for another 7 days. Discard the culture medium, add PBS to wash the neurons, discard the PBS, add 4% paraformaldehyde to fix for 30 minutes, rinse with PBS and discard the PBS, leaving only a small amount of liquid on the circular glass slide. Carefully add DiI crystals (Invitrogen, D282) to the circular glass slide and incubate at room temperature for 30 minutes. Then, wash the DiI crystals with PBS. Observe the circular glass slide under a confocal microscope (Lecia). In order to measure the density of neuronal dendritic spines, 8-10 neurons were randomly selected from each group, and the number of dendritic spines on the 50 μm long secondary dendrites of each neuron was counted, and then the dendritic spine density was calculated. See the results. Figure 3 The peptide significantly improved the decrease in dendritic spine density induced by K18 PFFs.

[0037] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A polypeptide for inhibiting Tau protein aggregation, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A biomaterial related to the polypeptide according to claim 1, characterized in that: Any of the following A1) to A4): A1) a nucleic acid molecule encoding the polypeptide; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant expression vector containing the nucleic acid molecule described in A1), or a recombinant expression vector containing the expression cassette described in A2); A4) A recombinant cell containing the nucleic acid molecule of A1), or a recombinant cell containing the expression cassette of A2), or a recombinant cell containing the recombinant expression vector of A3).

3. The biomaterial according to claim 2, wherein: The sequence of the nucleic acid molecule encoding the polypeptide is shown in SEQ ID NO.

2.

4. The biomaterial according to claim 2, wherein: The recombinant expression vector is pET28c.

5. The biomaterial according to claim 2, characterized in that: The recombinant cells use Escherichia coli BL21 as host cells.

6. The method for preparing the polypeptide according to claim 1, characterized in that: The method comprises the following steps: inoculating the recombinant cell according to any one of claims 2 to 5 into a culture medium, and obtaining the polypeptide by culture, or obtaining the polypeptide by culture and inducing expression.

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