Polypeptides inhibiting tau dephosphorylation activity and uses thereof

Abstract

This invention discloses a polypeptide that inhibits the desimilarization activity of tau protein and its applications, belonging to the field of biomedical technology. The polypeptide of this invention is tau. 438 The peptide is obtained through endogenous tau protein synthesis. The peptide provided by this invention can inhibit tau protein desimulation and increase tau mimicry, exhibiting high specificity and safely and effectively improving cognitive and memory function in AD model mice.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a polypeptide that inhibits the desimilation activity of tau protein and its application in disease treatment and drug preparation. Background Technology

[0002] Aging has become a major global public health challenge, and age-related neurodegenerative diseases are among the most prominent problems. Alzheimer's disease (AD), as the most common and serious neurodegenerative disease, is characterized by progressive memory decline and cognitive impairment, severely impacting patients' quality of life and imposing a heavy burden on families and society. According to incomplete statistics, there are currently over 50 million AD patients worldwide, and this number is projected to reach 152 million by 2050 as the aging process intensifies. However, because the etiology and pathogenesis of AD are not fully understood, effective prevention and treatment methods are still lacking. Therefore, identifying key molecular targets and developing targeted therapies have become urgent scientific problems to be solved.

[0003] As research into tau protein deepens, its clinical value as a biomarker for Alzheimer's disease (AD) has gradually gained recognition. Currently, phosphorylated subtypes such as p-tau181, p-tau217, and p-tau231 in plasma have been proven to serve as early diagnostic biomarkers for AD, with results highly consistent with positron emission tomography (PET) and cerebrospinal fluid (CSF) analysis. Furthermore, the analysis of Alz-tau biomarkers by measuring the ratio of high-molecular-weight tau to low-molecular-weight tau in platelets, as well as the detection of total tau and p-tau levels in saliva, provide effective means for early AD screening. Saliva testing, in particular, has broad application prospects due to its non-invasive nature. These tau-based monitoring technologies not only aid in the early diagnosis of AD but also serve as key indicators for assessing disease progression and treatment response, supporting the clinical management of AD. However, regrettably, current clinical applications of tau-related research are still primarily focused on the diagnostic field; breakthrough progress in the development of therapeutic drugs targeting tau has not yet been achieved, indicating a significant technological gap. Besides phosphorylation, tau protein undergoes various post-translational modifications, including acetylation, methylation, ubiquitination, and mimetic modification. These modifications directly regulate tau protein's function and aggregation tendency by altering its charge properties, hydrophobicity, and spatial conformation. Among these, mimetic modification, as an emerging post-translational modification, has attracted widespread attention in recent years for its role in neurodegenerative diseases. Similar to ubiquitination, this modification process involves a cascade reaction of E1 activators, E2 conjugates, and E3 ligases to covalently link the NEDD8 molecule to the target protein, thereby regulating the target protein's metabolism, localization, and function. Studies have shown that abnormal mimetic modification of tau protein may participate in the pathogenesis of Alzheimer's disease (AD) by affecting tau protein metabolism, regulating neuroinflammatory responses, and synaptic function. This provides new insights for developing therapeutic strategies centered on tau protein mimetic regulation. Summary of the Invention

[0004] This invention proposes a polypeptide that inhibits the desimilation activity of tau protein and its application.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: The first objective of this invention is to provide a polypeptide that inhibits the desimilarization activity of tau protein, said polypeptide being tau. 438 The polypeptide tau 438 The sequence includes at least LATLADEVSASLARQGL.

[0006] Furthermore, the polypeptide tau 438 It is obtained through the synthesis of endogenous tau protein; the polypeptide sequence of the endogenous tau protein includes at least LATLADEVSASLAKQGL.

[0007] Furthermore, the polypeptide tau 438 Its molecular weight is 1.93 kilodaltons, and it has good water solubility; its isoelectric point is higher than the plasma hydrogen ion concentration index, and it does not easily ionize into cations in the blood, resulting in low bioirritation.

[0008] The second objective of this invention is to provide the application of a polypeptide in the preparation of a drug that inhibits the desimilarization of tau protein, wherein the polypeptide is tau 438 The polypeptide tau 438 The sequence includes at least LATLADEVSASLARQGL.

[0009] A third objective of this invention is to provide the application of a polypeptide in the preparation of a drug that reduces the content of phosphorylated tau protein (p-tau). The polypeptide is tau. 438 The polypeptide tau 438The sequence includes at least LATLADEVSASLARQGL.

[0010] The fourth objective of this invention is to provide the application of a polypeptide in the preparation of drugs for the prevention and treatment of neurodegenerative diseases and memory loss. The polypeptide is tau. 438 The polypeptide tau 438 The sequence includes at least LATLADEVSASLARQGL.

[0011] Furthermore, the neurodegenerative diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, amyotrophic lateral sclerosis, multiple systemic atrophy, spinocerebellar ataxia, and frontotemporal dementia.

[0012] The fifth object of this invention is to provide a polypeptide drug, said polypeptide drug comprising the polypeptide tau 438 The composition formed with pharmaceutical excipients, wherein the polypeptide is tau 438 The polypeptide tau 438 The sequence includes at least LATLADEVSASLARQGL; the polypeptide drug is an injectable dosage form for intravenous or intramuscular injection, the injectable dosage form including powder for injection and aqueous injection.

[0013] Furthermore, the method for selecting pharmaceutical excipients in the polypeptide drug: based on tau 438 The amino acid sequence was calculated or experimentally determined to have an isoelectric point of 4.28. A buffer system with a hydrogen ion concentration index deviating from the isoelectric point by 1–2 units was selected. A phosphate buffer with a hydrogen ion concentration index of 5.0 to 6.0 (100 mL formulation: 0.038 g sodium dihydrogen phosphate, 0.294 g disodium hydrogen phosphate; after dissolution, the hydrogen ion concentration index was adjusted to 5.5 ± 0.1) was preferred to ensure the solubility and stability of the peptide. Sodium chloride (0.9% concentration, the standard concentration for human isotonicity, 0.9 g per 100 mL formulation) was used as an osmotic pressure regulator to adjust the osmotic pressure of the formulation to the isotonic range (280–320 mmol / kg). Mannitol (4% concentration, i.e., 4 g per 100 mL formulation) could be used as a lyophilization protectant. The polypeptide tau of the present invention 438It is synthesized through the demimicry of endogenous tau protein functional fragments, and can specifically target the demimicry site of tau protein, safely, effectively and reversibly reducing the level of tau protein mimicry, reducing the generation of p-tau from the source, avoiding interference with the normal protein modification process. It is highly specific, has good safety, and definite curative effect. It is easily soluble in water, has a small molecular weight, and is easily hydrolyzed after exertion. The amino acids produced can nourish nerves. It is convenient to synthesize and formulate, and has a low cost. It has obvious and unique advantages over small molecule chemical inhibitors and monoclonal antibody drugs, making it an ideal drug for the prevention and treatment of AD, and has strong clinical translational value.

[0014] This invention relates to a polypeptide that inhibits the desimilarization activity of tau protein and its applications, the beneficial effects of which are: (1) The polypeptide provided by the present invention can significantly improve cognitive and memory dysfunction in AD model mice.

[0015] (2) The polypeptide administration method provided by the present invention is more flexible and is not limited to intravenous injection. It can be administered by intramuscular injection or other methods.

[0016] (3) The polypeptide provided by the present invention has a small molecular weight, is easily soluble in water, has an isoelectric point higher than the plasma hydrogen ion concentration index, does not ionize into cations in the blood, has low irritation, is not easily degraded by ubiquitination, and is easily degraded and cleared by proteases and peptidases in the body after exerting its effect. It is not easy to accumulate and has unique effects and economic advantages compared to small molecule inhibitors and monoclonal antibody drugs. It is a polypeptide drug that can be used for the prevention and treatment of AD in a truly meaningful sense.

[0017] (4) The polypeptide synthesis process provided by this invention is simple, easy to produce in large quantities, and can be made into lyophilized powder. It has great potential in the prevention and treatment of AD, and has good promotional and clinical translational value. It has a very good long-term prospect. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0019] Figure 1 This figure shows the verification results of tau protein mimicry modification in Example 2. The figure confirms through immunoprecipitation experiments that tau protein can undergo mimicry modification, providing the core theoretical basis for the invention idea of ​​"targeting tau protein mimicry for intervention".

[0020] Figure 2 This is a diagram identifying the mimetic modification sites of the tau protein in Example 2; this diagram clearly identifies the specific sites on the tau protein where demimetic modification occurs, providing a basis for subsequent design of specific peptides (tau...). 438The targeting sequence provides precise molecular target support.

[0021] Figure 3 The figure shows the effect of the peptide on the tau protein mimicry level in Example 2. By comparing the peptide group with the target group, the figure intuitively demonstrates that the peptide of the present invention can significantly improve the tau protein mimicry level, and verifies the regulatory activity of the peptide on tau protein mimicry.

[0022] Figure 4 This figure shows the effect of the peptide on the phosphorylation level of tau protein in Example 2. By comparing the peptide group with the cell model group, this figure visually demonstrates that the peptide of the present invention can significantly reduce the phosphorylation level of tau protein, verifying the regulatory activity of the peptide on tau protein phosphorylation.

[0023] Figure 5 This figure shows the effect of the peptide on the escape latency in the water maze test of model mice in Example 3. The quantitative comparison of experimental data from each group clearly shows that, compared with the model group, the escape latency of the model group + peptide mice was significantly shortened after peptide intervention. This indicates that the peptide can effectively improve the spatial learning acquisition ability of model mice, providing direct and crucial behavioral experimental support for the cognitive-improving efficacy of the peptide and laying the foundation for its application in cognitive impairment intervention.

[0024] Figure 6 This figure shows the effect of the peptide on the number of target crossings in the water maze experiment of model mice in Example 3. The figure clearly presents the results through a systematic comparison of the data from each group. After peptide intervention, the number of target crossings in the model group + peptide mice was significantly increased compared to the model group, suggesting that the peptide can effectively enhance the model mice's ability to retain and reproduce spatial target locations. This further confirms its effect on improving the spatial cognitive function of mice, adding core experimental evidence to the efficacy value of the peptide.

[0025] Figure 7 This figure shows the effect of the peptide on the time spent in the target quadrant in the water maze experiment of model mice in Example 3. Quantitative analysis of the data from each group clearly shows that, compared with the model group, the model group plus peptide mice had a significantly longer time spent in the target quadrant. This indicates that the peptide can effectively enhance the selective exploration preference of model mice for spatial target regions, further supporting its effect on improving the spatial memory retrieval function of mice.

[0026] The above Figure 5 , Figure 6 and Figure 7The experimental results complement each other and form a closed loop, comprehensively verifying the cognitive-improving efficacy of peptides from three core dimensions: spatial learning acquisition, memory retention, and memory retrieval, and providing sufficient behavioral experimental support for their application value in the intervention of cognitive impairment-related diseases. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0028] The first objective of this invention is to provide a polypeptide (tau) that inhibits the desimilarization activity of tau protein. 438 ) 。 The polypeptide is a polypeptide sequence derived from endogenous tau protein, with the sequence LATLADEVSASLARQGL.

[0029] A second objective of this invention is to provide the application of a polypeptide in the preparation of a drug that inhibits the desimilation of tau protein.

[0030] A third objective of this invention is to provide an application of a polypeptide in the preparation of a drug that reduces the content of phosphorylated tau protein (p-tau).

[0031] A fourth objective of this invention is to provide the use of a polypeptide in the preparation of medicaments for the prevention and treatment of neurodegenerative diseases and memory loss.

[0032] The fifth object of this invention is to provide a polypeptide drug, which is a polypeptide used alone or a composition formed by a polypeptide and a pharmaceutical excipient. The specific formulation of the composition formed by the polypeptide and pharmaceutical excipient is as follows: active ingredient: polypeptide tau 438 The dosage is 0.2 g, requiring a purity ≥95% (detected by high performance liquid chromatography) and pyrogen-free; the buffer is 0.038 g of sodium dihydrogen phosphate (monohydrate) and 0.294 g of disodium hydrogen phosphate (dodecanoate), used to adjust the hydrogen ion concentration index of the formulation; the osmotic pressure regulator is 0.9 g of sodium chloride, combined with 4 g of mannitol, which has the dual function of lyophilization protectant and osmotic pressure regulator; the stabilizer is 0.05 g of L-methionine, which has an antioxidant effect; the solvent is water for injection, added to a total volume of 100 ml; all excipients are pharmaceutical grade and comply with the standards of the Chinese Pharmacopoeia. The injection is selected from powder injection and water injection, and can be used for intravenous or intramuscular injection.

[0033] The sixth objective of this invention is to provide a method for synthesizing polypeptides.

[0034] Example 1 A polypeptide that inhibits the desimilarization activity of tau protein (tau 438 The synthesis of ) includes the following steps: 1.1. Based on the first amino acid at the C-terminus of the peptide (i.e., C-terminal amino acid L), 0.5 mmol of fluorenemethyloxycarbonyl-L-leucine-Wang's resin was added to a solid-phase reactor. 10-15 mL of dichloromethane was added to the reactor, and the resin was soaked and swollen for 30 minutes before being dried. The resin was washed three times with N,N-dimethylformamide, each time for 1 minute. Subsequently, a 20% (v / v) piperidine-N,N-dimethylformamide solution was added, and the reaction was carried out at room temperature for 5 minutes. After drying, the same concentration of piperidine-N,N-dimethylformamide solution was added again, and the reaction was carried out at room temperature for 10 minutes to remove the fluorenemethyloxycarbonyl protecting group. After the reaction, the reactor was dried, and the resin was washed three times with N,N-dimethylformamide, each time for 1 minute, to obtain the activated resin-initial amino acid complex. 1.2. Following the amino acid sequence of the polypeptide (from C-terminus to N-terminus: LGQRALSASVEDALTAL), condensation and fluorene methyloxycarbonyl removal reactions were carried out alternately in a reaction volume of 1.5 mmol to condense all remaining amino acids of the polypeptide onto the resin. After the last amino acid was condensed, a 20% (v / v) piperidine-N,N-dimethylformamide solution was added, and the reaction was carried out for 5 minutes. Then, a 20% (v / v) piperidine-N,N-dimethylformamide solution was added again, and the reaction was carried out for 10 minutes. The mixture was washed once with N,N-dimethylformamide during the reaction. After the reaction was completed, the mixture was dried and washed three times with N,N-dimethylformamide. 1.3 Add 2 mmol of FITC and N-methylmorpholine to the reactor and react under conventional condensation conditions for 5-10 minutes. Use ninhydrin to check whether the reaction is complete. After the reaction is complete, wash three times alternately with N,N-dimethylformamide and dichloromethane, and then wash with methanol to shrink and obtain dry polypeptide-resin. 1.4 Place the peptide-resin obtained in step 1.3 into a round-bottom flask, and slowly add the prepared lysis buffer at 0 degrees Celsius (the volume ratio of the reagent formula used in the lysis buffer is trifluoroacetic acid: benzyl sulfide: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2). Stir slowly and react in an ice bath for 0.5 hours, then react at room temperature for 2 hours. Filter to obtain the lysis buffer, slowly add the lysis buffer to anhydrous ice-cold ether and stir. Filter to separate the crude peptide, wash 3 times with ice-cold ether to obtain the crude peptide. 1.5. The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC): A C18 column (5 μm, 4.6 × 250 mm) was used. Mobile phase A was 0.1% trifluoroacetic acid-water solution, and mobile phase B was 0.1% trifluoroacetic acid-acetonitrile solution. Gradient elution was performed (the proportion of phase B gradually increased from 20% to 80%, elution time 30 min). The detection wavelength was 220 nm. The eluent corresponding to the target peak at a retention time of approximately 15 min was collected, concentrated by rotary evaporation, and then freeze-dried (freeze-drying conditions: -50℃, vacuum ≤10 Pa, freeze-drying time 24 hours) to obtain a pure peptide with a purity ≥95%. The molecular weight of the pure peptide was determined to be 1.93 kDaltons, consistent with the theoretical molecular weight, i.e., peptide tau438, and its amino acid sequence was LATLADEVSASLARQGL.

[0035] The polypeptides obtained by this invention can be prepared into intramuscular injection dosage forms such as powder injections and aqueous injections according to conventional existing standards commonly used by those skilled in the art.

[0036] Example 2 2.1 Mimetic modification of tau protein and verification of mutation sites Experimental Groups: The positive control (Input group) consisted of HEK293T cells co-transfected with wild-type human FLAG-tau plasmid and HA-NEDD8. This served as a loading control to verify the presence of Flag-tau protein and the HA-NEDD8 modified substrate in the sample, confirming the initial protein expression level and providing a reference for subsequent immunoprecipitation bands.

[0037] Negative control (IgG group): Total protein from HEK293T cells transfected with plasmid was collected and immunoprecipitated using irrelevant isotype IgG (non-anti-Flag antibody). This excluded non-specific antibody binding, background adsorption of Protein A / G magnetic beads, or non-specific protein interactions, confirming the specificity of the bands.

[0038] Target group (anti-Flag IP group): Total protein from HEK293T cells transfected with an equal amount was used for immunoprecipitation with anti-Flag antibody to enrich Flag-tau and its binding proteins. The interaction between Flag-tau protein and NEDD8 and the NEDDylation modification level of tau protein were specifically detected.

[0039] The specific experimental method is as follows: Positive control group: HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected with Flag-tau WT and HA-NEDD8 cells using standard transfection methods. Forty-eight hours after transfection, the culture medium was discarded, cells were washed with pre-cooled PBS, and immunoprecipitation lysis buffer containing protease inhibitors and phosphatase inhibitors was added. Lysis was performed on ice for 30 minutes, followed by centrifugation at 12,000 rpm for 15 minutes. The supernatant was collected as the total protein lysis buffer. Protein concentration was determined using the BCA method. 30 μg of total protein was directly loaded onto the sample without further immunoprecipitation.

[0040] Negative control group: HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected into Flag-tau WT and HA-NEDD8 cells using standard transfection methods. Forty-eight hours after transfection, the culture medium was discarded, cells were washed with pre-chilled PBS, and immunoprecipitation lysis buffer containing protease and phosphatase inhibitors was added. The cells were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was used as the total protein lysis buffer. Protein concentration was determined using the BCA method. 500 μg of the total protein lysis buffer was added to anti-IgG antibody and incubated overnight at 4°C with slow rotation. Protein A / G magnetic beads were then added and incubated at room temperature for 1 hour to enrich the immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specific binding proteins. 2× loading buffer was added to the immune complexes, mixed well, and boiled at 95°C for 10 minutes to denature the proteins. The supernatant was collected after centrifugation as the loading solution.

[0041] Objective group: HEK293T cells were cultured in conventional medium at 37℃ in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected into Flag-tau WT and HA-NEDD8 cells using standard transfection methods. Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed with pre-chilled PBS. Immunoprecipitation lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was collected as the total protein lysis buffer. Protein concentration was determined using the BCA method. 500 μg of total protein was added, and anti-Flag antibody was added. The mixture was incubated overnight at 4℃ with slow rotation. Subsequently, Protein A / G magnetic beads were added, and the mixture was incubated at room temperature for 1 hour to enrich the immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specifically bound proteins. 2× loading buffer was added to the immune complexes, and the mixture was boiled at 95℃ for 10 minutes to denature the proteins. The supernatant was collected after centrifugation as the loading solution.

[0042] The sample solutions from each group were analyzed using Western blotting, and the results are as follows: Figure 1 As shown.

[0043] 2.2 Verification of tau protein mutation sites Experimental groups: Wild-type tau cells marked with Flag and a series of lysine sites mutated to arginine sites were used as the research subjects, as detailed below: Wild-type tau group: HEK293T cells were transfected with the Flag-tau wild-type expression plasmid as a baseline control.

[0044] Lysine site mutation group: transfected with Flag-tau A438G, Flag-tau A395G, Flag-tau A390G, Flag-tau A385G, Flag-tau A383G, Flag-tau A375G, and Flag-tau A370G.

[0045] Control group: HEK293T cells transfected with empty vector (without tau sequence) to exclude vector background and non-specific signals.

[0046] Specific experimental methods: Wild-type tau group: HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected into Flag-tau WT and HA-NEDD8 cells using standard transfection methods. Forty-eight hours after transfection, the culture medium was discarded, cells were washed with pre-chilled PBS, and immunoprecipitation lysis buffer containing protease and phosphatase inhibitors was added. Lysis was performed on ice for 30 minutes, followed by centrifugation at 12,000 rpm for 15 minutes. The supernatant was used as the total protein lysis buffer, and protein concentration was determined using the BCA method. 500 μg of the total protein lysis buffer was added to anti-Flag antibody and incubated overnight at 4°C with slow rotation. Protein A / G magnetic beads were then added, and incubation was performed at room temperature for 1 hour to enrich the immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specific binding proteins. 2× loading buffer was added to the immune complexes, mixed well, and boiled at 95°C for 10 minutes to denature the proteins. The supernatant was collected after centrifugation as the loading solution.

[0047] Lysine site mutation group: HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected with Flag-tau A438G, Flag-tau A395G, Flag-tau A390G, Flag-tau A385G, Flag-tau A383G, Flag-tau A375G, Flag-tau A370G, and HA-NEDD8 according to conventional transfection methods. Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed with pre-cooled PBS. Immunoprecipitation lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected as the total protein lysis buffer. Protein concentration was determined by the BCA method. Take 500 μg of total protein lysis buffer, add anti-Flag antibody, and incubate overnight at 4°C with slow rotation. Then add Protein A / G magnetic beads and incubate at room temperature for 1 hour to enrich the immune complexes. Wash the magnetic beads three times with pre-cooled PBS to remove non-specific binding proteins. Add 2× loading buffer to the immune complexes, mix well, and boil at 95°C for 10 minutes to denature the proteins. Centrifuge and collect the supernatant as the loading solution.

[0048] Control group: HEK293T cells transfected with an empty vector (without the tau sequence) were used to eliminate vector background and non-specific signals. HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids Flag-WT and HA-NEDD8 were co-transfected using conventional transfection methods. Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed with pre-chilled PBS. Immunoprecipitation lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected as the total protein lysis buffer. Protein concentration was determined by the BCA method. 500 μg of the total protein lysis buffer was taken, anti-Flag antibody was added, and the cells were incubated overnight at 4°C with slow rotation. Subsequently, Protein A / G magnetic beads were added, and the cells were incubated at room temperature for 1 hour to enrich immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specific binding proteins. Add 2× loading buffer to the immune complex, mix well, boil at 95°C for 10 minutes to denature the protein, centrifuge, and take the supernatant as the loading solution.

[0049] The sample solutions from each group were analyzed using Western blotting, and the results are as follows: Figure 2 As shown.

[0050] 2.3, Tau peptide 438 Effects on tau protein mimicry levels Experimental Groups: Negative control group (IgG): HEK293T cells were transfected with Flag-tau protein, and immunoprecipitation (IP) was performed using IgG (mouse IgG) instead of anti-Flag antibody. Purpose: To eliminate background interference such as non-specific antibody binding and non-specific adsorption of protein A / G beads, serving as a negative control for immunoprecipitation.

[0051] Target group (IP Flag): HEK293T cells were transfected with a Flag-tau labeled target protein. After cell lysis, immunoprecipitation was performed using an anti-Flag antibody to enrich Flag-tau and its interacting proteins. Purpose: To detect the presence of the target protein tau and its binding proteins (such as NEDD8) and to verify protein-protein interactions.

[0052] Objective + Peptide Group (IP Flag): HEK293T cells were transfected with Flag-tau, and then the peptide tau obtained in Example 1 was added to a final concentration of 20 μmol. 438 Immunoprecipitation was performed using anti-Flag antibodies.

[0053] The specific experimental method is as follows: Negative control group: HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected into Flag-tau WT and HA-NEDD8 cells using standard transfection methods. Forty-eight hours after transfection, the culture medium was discarded, cells were washed with pre-chilled PBS, and immunoprecipitation lysis buffer containing protease and phosphatase inhibitors was added. The cells were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was used as the total protein lysis buffer. Protein concentration was determined using the BCA method. 500 μg of the total protein lysis buffer was added to anti-IgG antibody and incubated overnight at 4°C with slow rotation. Protein A / G magnetic beads were then added and incubated at room temperature for 1 hour to enrich the immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specific binding proteins. 2× loading buffer was added to the immune complexes, mixed well, and boiled at 95°C for 10 minutes to denature the proteins. The supernatant was collected after centrifugation as the loading solution.

[0054] Objective group: HEK293T cells were cultured in conventional medium at 37℃ in a 5% CO2 incubator. When the cell confluence reached approximately 80%, plasmids were co-transfected into Flag-tau WT and HA-NEDD8 cells using standard transfection methods. Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed with pre-chilled PBS. Immunoprecipitation lysis buffer containing protease inhibitors and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was collected as the total protein lysis buffer. Protein concentration was determined using the BCA method. 500 μg of total protein was added, and anti-Flag antibody was added. The mixture was incubated overnight at 4℃ with slow rotation. Subsequently, Protein A / G magnetic beads were added, and the mixture was incubated at room temperature for 1 hour to enrich the immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specifically bound proteins. 2× loading buffer was added to the immune complexes, and the mixture was boiled at 95℃ for 10 minutes to denature the proteins. The supernatant was collected after centrifugation as the loading solution.

[0055] Objective + Peptide group: HEK293T cells were cultured in conventional medium at 37°C in a 5% CO2 incubator. When the cell confluence reached approximately 80%, Flag-tau WT and HA-NEDD8 were co-transfected using conventional transfection methods. 24 hours after transfection, the peptide tau obtained in Example 1 was added to a final concentration of 20 μmol. 438 Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed with pre-chilled PBS. Immunoprecipitation lysis buffer containing protease and phosphatase inhibitors was added, and the cells were lysed on ice for 30 minutes. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was collected as the total protein lysis buffer. Protein concentration was determined using the BCA method. 500 μg of total protein was taken, anti-Flag antibody was added, and the mixture was incubated overnight at 4°C with slow rotation. Subsequently, Protein A / G magnetic beads were added, and the mixture was incubated at room temperature for 1 hour to enrich the immune complexes. The magnetic beads were washed three times with pre-chilled PBS to remove non-specifically bound proteins. 2× loading buffer was added to the immune complexes, mixed well, and boiled at 95°C for 10 minutes to denature the proteins. After centrifugation, the supernatant was collected as the loading buffer. The sample solutions from each group were analyzed using Western blotting, and the results are as follows: Figure 3 As shown.

[0056] 2.4, Tau peptide 438 Effect on tau protein phosphorylation level This experiment used 293T cells as a research model. The Flag-tau expression plasmid was transfected into the cells to construct a tau-overexpressing cell model. After transfection, the cells were divided into two groups: a model cell group (transfected only with the tau plasmid, without the addition of peptides) and a model cell + peptide group (transfected with the tau plasmid and then supplemented with 20 μmol of the peptide tau obtained in Example 1). 438Cellular proteins were collected 24 hours later. After processing, total cellular protein was extracted, and Western blotting was used to detect the total tau and phosphorylated tau levels at two key phosphorylation sites (serine 199 and serine 404). GAPDH was used as an internal control protein to correct the sample loading amount. The results are shown below. Figure 4 As shown.

[0057] The results are as follows Figures 1-4 As shown, in this embodiment, the key lysine residue K438 for demimicry of human tau protein was successfully identified using immunoprecipitation. This site is the core site for demimicry modification of tau protein. The present invention's polypeptide tau 438 The assay can block demimicry at this site, inhibit tau protein demimicry activity, significantly increase tau protein mimicry levels, and reduce the expression of phosphorylated tau. These results clarify the key target of tau protein demimicry modification, providing direct experimental evidence for the mechanism of action of the peptide in this invention to target and inhibit tau protein demimicry, and confirming that regulating this mimicry site can intervene in the core pathological process of Alzheimer's disease (AD).

[0058] In this embodiment, both the immunoprecipitation experiment and the Western blot detection are experimental methods that are routinely performed in the field, and their specific steps will not be described in detail here.

[0059] Example 3 This embodiment uses the Morris water maze test to verify the learning and memory abilities of mice, with a total of 3 groups: Control group (WT mice): 11 wild-type mice, without disease model construction, were given 100 μL of phosphate-buffered saline via intraperitoneal injection for 3 consecutive months.

[0060] Model group (htau mice): 10 htau mice were given 100 μL of phosphate-buffered saline via intraperitoneal injection for 3 consecutive months.

[0061] Model group + peptide group (htau + peptide, drug-treated group): 13 htau model mice were given intraperitoneal injection of 5 mg / kg peptide (100 μL) for 3 consecutive months.

[0062] The Morris water maze experiment was used to validate the spatial learning and memory abilities of mice. The experiment was conducted in a circular pool with a radius of 1.5 meters and a height of 60 centimeters. The pool was divided into four quadrants, labeled NE, NW, SW, SE, or quadrants 1, 2, 3, and 4. Different shaped markers were affixed to the upper walls of each quadrant to aid the mice in spatial orientation. To create a relatively independent experimental environment, blue curtains were suspended around the pool, with corresponding shaped markers affixed to different positions on the curtains. During the experiment, a camera mounted above the pool tracked the mice's movements. Day 1 was the adaptation phase. Mice were gently placed in the pool, starting in quadrant 1, facing the pool wall, for 120 seconds of free exploration to adapt to the pool and its surroundings. No platform was placed underwater during this period. After exploration, the mice were removed, dried, and then returned to their cages. Days 2-6 were the learning phase. An underwater hiding platform was placed in the center of quadrant 3, with the top of the platform 1 centimeter above the water surface. During the experiment, mice were placed into the pool facing the pool wall from different quadrants. A successful escape was defined as the mouse finding a platform within 120 seconds and remaining there for 2 seconds. The escape latency (the time required to find the platform) was recorded. If a mouse failed to find the platform within the time limit, it was guided to the platform and remained there for 20 seconds; the latency was then recorded as 120 seconds. This learning was conducted four times daily, and the average of the four escape latency periods was calculated as the daily escape latency. On day 7, the testing phase began. The platform was removed, and mice were placed into the water facing the pool wall from quadrant 1. Their behavioral trajectory was recorded over 120 seconds, and the number of times they crossed the platform area was counted. Results are as follows: Figure 5 , Figure 6 and Figure 7 As shown, the peptides of this invention significantly improve spatial learning and memory in AD mice in the water maze.

[0063] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A polypeptide that inhibits the desimilarization activity of tau protein, characterized in that: The polypeptide is tau 438 The polypeptide tau 438 The sequence includes at least LATLADEVSASLARQGL.

2. The polypeptide that inhibits the desimilarization activity of tau protein according to claim 1, characterized in that: The polypeptide tau 438 It is obtained through the synthesis of endogenous tau protein; the polypeptide sequence of the endogenous tau protein includes at least LATLADEVSASLAKQGL.

3. The use of a polypeptide that inhibits tau protein desimilarization activity according to any one of claims 1-2 in the preparation of a drug that inhibits tau protein desimilarization.

4. The use of a polypeptide that inhibits the desimilarization activity of tau protein according to any one of claims 1-2 in the preparation of a drug that reduces the content of phosphorylated tau protein.

5. The use of a polypeptide according to claims 1-2 that inhibits the demimicry of tau protein in the preparation of drugs for the prevention and treatment of neurodegenerative diseases and memory loss; wherein the polypeptide is tau 438 The polypeptide tau 438 The sequence includes at least LATLADEVSASLARQGL.

6. The application according to claim 5, characterized in that: The neurodegenerative diseases mentioned are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Lewy body dementia, amyotrophic lateral sclerosis, multiple systemic atrophy, spinocerebellar ataxia, and frontotemporal dementia.

7. A polypeptide drug, characterized in that: Including the peptide tau 438 Composition formed with pharmaceutical excipients; said pharmaceutical excipients are pharmaceutically acceptable excipients for injection; said polypeptide tau 438 The polypeptide tau as described in any one of claims 1–2 438 .

8. A polypeptide drug according to claim 7, characterized in that: The polypeptide drug is an injectable dosage form for intravenous or intramuscular injection, and the injectable dosage form includes powder injection and aqueous injection.

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