Beta2-microglobulin blocking peptides, pharmaceutical compositions thereof and uses thereof

Abstract

The present application belongs to the field of biological medicine, and relates to beta2-microglobulin blocking peptide, a pharmaceutical composition thereof and purposes. Specifically, the present application relates to an isolated polypeptide which is a polypeptide shown in SEQ ID NO: 3 or a truncated fragment of the polypeptide shown in SEQ ID NO: 3, wherein the truncated fragment comprises a polypeptide shown in SEQ ID NO: 8. The isolated polypeptide of the present application can effectively prevent and treat AD or cognitive impairment caused by AD, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to β2-microglobulin blocking peptide, its pharmaceutical composition and uses. Background Technology

[0002] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in humans. According to the World Alzheimer's Disease Report 2018, 50 million people worldwide had AD in 2018, and this number is projected to rise to 152 million by 2050. The main pathological features of the disease include amyloid deposits formed by the oligomerization of β-amyloid protein (Aβ) produced by the cleavage of amyloid precursor protein (APP) in the brain; neurofibrillary tangles (NFTs) formed by the aberrant phosphorylation of the microtubule-binding protein tau; neuronal loss; and excessive neuroinflammation. The main clinical manifestations of AD are memory decline and cognitive impairment, which worsens as the disease progresses, ultimately leading to the loss of all memory and the ability to live independently, resulting in death. Given the global aging population, the continuously rising incidence of AD, and the current lack of effective treatments, AD has become one of the most serious threats to human health.

[0003] Since the discovery of Alzheimer's disease (AD) in 1901, numerous studies have been conducted on its pathogenesis; however, no definitive conclusion has yet been reached. The "Aβ cascade hypothesis" is one of the mainstream theories regarding the pathogenesis of AD. This hypothesis posits that abnormal cleavage of APP (application enzymes) produces neurotoxic Aβ (alpha-β). 1-40 / 42 Aβ plays a central role in the pathogenesis of Alzheimer's disease (AD) and is a common pathway for AD induced by various factors. Aβ oligomers induce a series of neurotoxic reactions that can trigger neuroinflammation, leading to neuronal dysfunction and loss, ultimately resulting in dementia. Therefore, reducing Aβ production in the brain, promoting Aβ clearance, inhibiting Aβ aggregation, and reducing its neurotoxicity have become one of the main measures for treating AD.

[0004] β2-microglobulin (B2M) is an aging factor that has received increasing attention in recent years. B2M is a subunit of Major Histocompatibility Complex I (MHC-I), encoded by a gene on human chromosome 15, and contains 119 amino acids. Because B2M is not anchored to the cell membrane via a transmembrane domain, it can migrate from the MHC-I complex into the intercellular space. Normally, B2M exists as a soluble monomer, but it can aggregate and deposit under certain pathological conditions. These pathological factors include aging, long-term renal dysfunction, and inflammation. B2M amyloid deposition is mainly found in the bone and joint regions, ultimately leading to severe arthritis, fractures, and carpal tunnel syndrome. Furthermore, serum and plasma B2M levels are increased in many disease states. Of particular note is the significantly higher B2M levels in the plasma and cerebrospinal fluid of Alzheimer's disease (AD) patients compared to age-matched normal controls. Stereotactic injection of B2M into the brain inhibits neuronal regeneration and impairs cognitive function in mice, while B2M deficiency promotes neuronal regeneration and reverses age-related cognitive deficits. However, no studies have reported whether B2M has a direct or indirect impact on the development and progression of Alzheimer's disease. Summary of the Invention

[0005] Through in-depth research and creative work, the inventors discovered the role of B2M in the occurrence and development of AD. Surprisingly, they found that inhibiting B2M and Aβ... 1-42 The bound blocking peptide has the potential to be used as a drug for the prevention and treatment of AD, particularly cognitive impairment caused by AD. Therefore, the following invention is provided:

[0006] One aspect of the present invention relates to an isolated polypeptide, which is the polypeptide shown in SEQ ID NO:3 or a truncated fragment of the polypeptide shown in SEQ ID NO:3, wherein the truncated fragment comprises the polypeptide shown in SEQ ID NO:8.

[0007] In some embodiments of the present invention, the isolated polypeptide, wherein the truncated fragment does not contain the six histidine residues at the N-terminus of the polypeptide shown in SEQ ID NO:3.

[0008] In some embodiments of the present invention, the isolated polypeptide is a polypeptide represented by any of the sequences in SEQ ID NOs:7-8.

[0009] HHHHHHHSDLSFSKDW SFYLLYYTEFTPTEK (SEQ ID NO:3)

[0010] SFYLLYYTEFTPTEK(SEQ ID NO:7)

[0011] SFYLLYYTE (SEQ ID NO:8)

[0012] In some embodiments of the present invention, the isolated polypeptide is a polypeptide represented by any of the sequences in SEQ ID NOs:11-15.

[0013] SFYLLYYTEFTPTE (SEQ ID NO:11)

[0014] SFYLLYYTEFTPT (SEQ ID NO:12)

[0015] SFYLLYYTEFTP(SEQ ID NO:13)

[0016] SFYLLYYTEFT (SEQ ID NO:14)

[0017] SFYLLYYTEF (SEQ ID NO:15)

[0018] Another aspect of the present invention relates to an isolated polynucleotide encoding an isolated polypeptide as described in any one of the present invention.

[0019] Another aspect of the invention relates to a recombinant expression vector comprising the isolated polynucleotides of the present invention.

[0020] Another aspect of the invention relates to a transformed cell comprising the recombinant expression vector of the invention.

[0021] Another aspect of the invention relates to a pharmaceutical composition comprising the isolated polypeptide described in any one of the invention.

[0022] In some embodiments of the present invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0023] Another aspect of the invention relates to the use of the isolated polypeptide described in any one of the invention in the preparation of a medicament for the treatment or prevention of Alzheimer's disease.

[0024] Another aspect of the present invention relates to the use of the isolated polypeptide described in any one of the present invention in the preparation of the following pharmaceutical products:

[0025] Inhibition of B2M-induced Aβ 1-42 Drugs that promote oligomerization, drugs that inhibit B2M-induced β-amyloid plaque formation, or drugs that inhibit Aβ 1-42 Neurotoxic drugs.

[0026] This invention is the first to discover that B2M expression is significantly elevated in the brain tissue of AD patients, and that the level of B2M expression in the brain is related to Aβ. 1-42 There is a significant positive correlation between the levels of B2M and Aβ. Further increasing the B2M content in the brain of an AD mouse model (5×FAD) via stereotactic injection can increase β-amyloid plaque deposition in the brain of 5×FAD mice. Furthermore, the inventors discovered that B2M and Aβ... 1-42 There is a direct interaction; using a truncated B2M amino acid sequence as a blocking peptide can inhibit B2M and Aβ. 1-42 Aggregation, inhibiting B2M and Aβ 1-42 Neurotoxicity: In vivo experiments showed that the blocking peptide could significantly inhibit the effect of B2M on Aβ in the brain. 1-42 The aggregation-promoting effect reduces β-amyloid plaque deposition in the brain. This finding provides a potential drug target and novel therapeutic approaches based on this target for the clinical treatment of AD.

[0027] The isolated polypeptide according to any one of the present invention is used for the treatment or prevention of Alzheimer's disease.

[0028] The isolated polypeptide according to any one of the present invention is used to inhibit B2M-induced Aβ. 1-42 Oligomerization, inhibition of B2M-induced β-amyloid plaque formation, or use to inhibit Aβ 1-42 Neurotoxicity.

[0029] Another aspect of the invention relates to a method for treating or preventing Alzheimer's disease, comprising the step of administering to a subject in need an effective amount of the isolated polypeptide described in any one of the invention.

[0030] Another aspect of the present invention relates to a method for inhibiting B2M-induced Aβ. 1-42 Oligopolymerization or inhibition of B2M-induced β-amyloid plaques or inhibition of Aβ 1-42 Methods for treating neurotoxicity include administering an effective amount of the isolated polypeptide described in any one of the present invention to a subject in need.

[0031] In some embodiments of the present invention, the amino acid sequence of the β2-microglobulin is shown in SEQ ID NO:9.

[0032] The amino acid sequence (N-terminus to C-terminus) of human B2M protein is as follows:

[0033] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:9)

[0034] In this invention, when referring to the amino acid sequence of β2-microglobulin (B2M), it includes the full-length β2-microglobulin and its fusion protein. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of β2-microglobulin without affecting its biological function. In one embodiment of the invention, β2-microglobulin is human β2-microglobulin. In one embodiment of the invention, the amino acid sequence of β2-microglobulin is as shown in SEQ ID NO:9.

[0035] In this invention, the term "host cell" refers to the cell into which the vector is introduced, including many cell types such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or animal cells such as human cells.

[0036] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a protein can be inserted. For example, vectors include: plasmids; phage particles; Cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression.

[0037] The term "disease and / or symptom" refers to a physical condition of the subject that is related to the disease and / or symptom described in this invention.

[0038] The term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives the pharmaceutical composition of the present invention to treat, prevent, reduce and / or alleviate the disease or condition described in the present invention.

[0039] The term "blocking peptide" refers to a polypeptide that can competitively bind to Aβ with the full-length B2M protein, thereby inhibiting the biological effect of B2M binding to Aβ that promotes Aβ oligomerization.

[0040] In this invention, unless otherwise specified, the concentration units μM represent μmol / L, mM represent mmol / L, and nM represent nmol / L.

[0041] In this invention, when referring to the amount of drug added to cells, unless otherwise specified, it generally refers to the final concentration of the drug after addition.

[0042] Aβ 1-42 The amino acid sequence is as follows:

[0043] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID NO:10)

[0044] Beneficial effects of the invention

[0045] The present invention achieves one or more of the following technical effects (1)-(4):

[0046] (1) The polypeptides of the present invention can effectively prevent and / or treat Alzheimer's disease.

[0047] (2) The polypeptide of the present invention can effectively inhibit B2M-induced Aβ. 1-42 Oligopoly.

[0048] (3) The polypeptide of the present invention can effectively inhibit the formation of B2M-induced β-amyloid plaques.

[0049] (4) The polypeptide of the present invention can effectively inhibit Aβ 1-42 Neurotoxicity. Attached Figure Description

[0050] Figures 1A to 1B The expression level of B2M in the brains of AD patients is significantly increased. Among them:

[0051] Figure 1A Immunoblot results of B2M expression in the cerebral cortex tissue of AD patients.

[0052] Figure 1B Image J Analysis Figure 1A B2M expression levels were measured in the control group (n=8 individuals) and the AD group (n=21 individuals). Data were statistically analyzed using the Student t-test. No significant difference was found in ns (P>0.05); *P<0.05; **P<0.01; ***P<0.001.

[0053] Figure 1C : via Aβ 1-42 ELISA was used to detect Aβ in the brain tissue of the aforementioned 29 individuals. 1-42 Levels, through Pearson correlation analysis, revealed Aβ levels in the human brain 1-42 There is a significant positive correlation between the level and the level of B2M expression in the human brain.

[0054] Figures 1D to 1F Increased B2M levels in the brains of 5×FAD mice promoted amyloid plaque deposition. 1 μl (1 μg / μl) of purified B2M protein or 1 μl of PBS (left and right controls per mouse) was injected into the hippocampus of 4-month-old 5×FAD mice via stereotactic injection. Two months later, mice were anesthetized with 5% chloral hydrate, and their hearts were perfused with phosphate-buffered saline. Brain tissue was harvested, fixed overnight in 4% paraformaldehyde, dehydrated with 25% and 30% sucrose solutions, embedded using OCT, frozen sections, and subjected to immunofluorescence staining. Cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI), and Aβ was labeled with antibody 6E10. Images were acquired using laser confocal fluorescence microscopy. Scale bar: 300 μm. Data represent mean ± standard error.

[0055] Figure 1D Immunofluorescence staining results of amyloid plaques in the brain tissue of 5×FAD mice. Each group consisted of n=6 mice. The dashed boxes indicate the extent of the hippocampal DG region, which also represents the brain tissue region where the plaques were statistically analyzed.

[0056] Figure 1E : Figure 1D Statistical results of the number of β-amyloid deposits in the DG region of the mid-hippocampus. Data were analyzed using paired t-tests. No significant difference was found in ns, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

[0057] Figure 1F : Figure 1D Statistical results of the area of ​​β-amyloid deposits in the DG region of the mid-hippocampus. Data were analyzed using paired t-tests. No significant difference was found in ns, P>0.05; *P<0.05; **P<0.01; ***P<0.001.

[0058] Figures 2A to 2B : In vitro purified B2M protein and purified Aβ 1-42 Results of co-immunoprecipitation (co-IP) experiments on protein-protein interactions. Among them:

[0059] Figure 2A Immunoprecipitation of Aβ with anti-Aβ antibodies 1-42 With B2M.

[0060] Figure 2B Immunoprecipitation of B2M and Aβ with anti-B2M antibodies. 1-42 .

[0061] Figure 2C In vivo B2M and Aβ comparisons using brain tissue from 5×FAD mice. 1-42 The results of the co-IP experiment on the interaction were obtained using an anti-Aβ antibody.

[0062] Figure 2D Using thiamine-T as an indicator for in vitro B2M incubation of Aβ 1-42 After promoting Aβ 1-42 Oligomeric results were continuously detected using a fluorescent microplate reader. At 37°C, different concentrations of full-length B2M protein were mixed with Aβ... 1-42 The mixture was co-incubated, with thiosulfate-T added to the experimental system. The changes in the thiosulfate-T fluorescence signal value were detected every 5 minutes using a fluorescence microplate reader under excitation wavelength λ = 448 nm and absorption wavelength λ = 488 nm. A graph showing the change in fluorescence signal value over time was plotted after the experiment. The data represent the average absolute fluorescence signal intensity.

[0063] Figure 2E :and Figure 2D The results of thiosulfate-T fluorescence detection of B2M protein at corresponding concentrations under the same experimental conditions. The data represent the average absolute fluorescence signal intensity.

[0064] Figure 3A : A schematic diagram of segments of a B2M amino acid sequence truncated into four non-overlapping peptide sequences.

[0065] Figure 3B Truncated B2M peptides inhibit B2M and promote Aβ. 1-42 Experimental results of oligomerized thiosulfinate-T. The truncated B2M amino acid sequence was used as a blocking peptide with Aβ. 1-42 Pre-incubate at 37°C for 3 hours, then add full-length B2M protein and continue incubation for Aβ. 1-42 The effect of the blocking peptide on B2M-promoted Aβ was analyzed by examining changes in the fluorescence signal intensity of thiosulfate-T. 1-42 Oligopolymerization inhibition effect. Data represent the average absolute fluorescence signal intensity.

[0066] Figure 3C Based on the above thiosulfate-T experiment, the effects of different concentrations of B2M-3 blocking peptide on B2M-promoting Aβ were detected. 1-42The inhibitory effect of oligomerization. The data represent the average absolute fluorescence signal intensity.

[0067] Figure 3D : Detection of B2M-3 blocking peptides that inhibit B2M-promoting Aβ using transmission electron microscopy (TEM) 1-42 Oligomerization on Aβ 1-42 The morphology of the formed fibers is affected. Scale bar, 1 μm.

[0068] Figure 3E To further narrow down the range of amino acid sequences that exert the inhibitory effect, the inventors further divided the B2M-3 blocking peptide into three segments of short peptides with non-overlapping sequences.

[0069] Figures 3F to 3H The thiamine-T experiment was performed on three further truncated peptides to analyze the effects of different concentrations of the peptide sequences on B2M-mediated Aβ promotion. 1-42 The inhibitory effect of oligomerization. Data represent the average absolute fluorescence signal intensity. Wherein:

[0070] Figure 3F Pre-incubation of Aβ with three different concentrations of the first-phase B2M-3 truncated peptide (B2M-3-1) 1-42 After 3 hours, the full-length B2M protein was added, and the changes in thiamine-T fluorescence signal were continuously detected using a fluorescence microplate reader.

[0071] Figure 3G Pre-incubation of Aβ with three different concentrations of the second-strand B2M-3 truncated peptide (B2M-3-2) 1-42 After 3 hours, the full-length B2M protein was added, and the changes in thiamine-T fluorescence signal were continuously detected using a fluorescence microplate reader.

[0072] Figure 3H Pre-incubation of Aβ with three different concentrations of the third-segment B2M-3 truncated peptide (B2M-3-3) 1-42 After 3 hours, the full-length B2M protein was added, and the changes in thiamine-T fluorescence signal were continuously detected using a fluorescence microplate reader.

[0073] Figures 4A to 4B B2M-3 blocking peptide promotes Aβ by B2M 1-42 In vitro electrophysiological experiments demonstrating the inhibitory effect of oligomerization on neurotoxicity. Aβ was pre-incubated at 37°C with a B2M-3 blocking peptide or a control no-significant peptide sequence (NS). 1-42 After 3 hours, the full-length B2M protein was added and incubated for another 12 hours to obtain two oligomerized Aβ proteins with different degrees of oligomerization. 1-42 Product. Based on oligomerization of Aβ 1-42Exhibiting neurotoxicity, the inventors incubated isolated brain slices of 2-month-old wild-type mice with the two different oligomerized Aβ products (referred to as oAβ), respectively. After 1.5 hours of incubation, a long-term potentiation (LTP) electrophysiological experiment was performed to detect the effect of oAβ on the synaptic plasticity of neural circuits in the isolated brain slices. Figure 4B Data were analyzed using one-way ANOVA. For each ns, no significant difference was observed; P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.001. Where:

[0074] Figure 4A Results of LTP recording analysis of isolated brain slices in the CA1 region. In the oAβ treatment group, n = 4 mice per group, and n = 8 brain slices were recorded per group. In the control group treated with peptides alone, n = 4 mice per group, and n = 7 brain slices were recorded per group.

[0075] Figure 4B : Figure 4A Statistical analysis of fEPSP amplitude in the last 10 minutes of LTP recording results. The n value is the same for each group. Figure 4A .

[0076] Figures 4C to 4D Intracerebral injection of B2M-3 blocking peptide into the brains of 5×FAD mice significantly reduced the number and area of ​​β-amyloid plaques in the brains of AD mice. Figure 4C and 4D Data were statistically analyzed using a paired t-test. For each ns, no significant difference was observed (P>0.05); *P<0.05; **P<0.01; ***P<0.001). Where:

[0077] Figure 4C Immunofluorescence staining results of β-amyloid plaques deposited in the hippocampal DG region of 5×FAD mice injected with B2M-3 blocking peptide or meaningless peptide. Cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI), and Aβ was labeled with antibody 6E10. Scale bar, 300 μm. n = 6 mice per group.

[0078] Figure 4D : Figure 4C Statistical analysis results of the number of β-amyloid plaques in the DG region of the mid-hippocampus.

[0079] Figure 4E : Figure 4C Statistical analysis results of the area of ​​β-amyloid plaques in the DG region of the mid-hippocampus. Detailed Implementation

[0080] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0081] In the examples below, the 5×FAD mice used are transgenic mouse models of AD disease, which exhibit AD-specific β-amyloid plaque pathological features in the brain; the 5×FADA mice were purchased from Jackson Laboratory (Ellsworth, ME, USA), serial number 34840-JAX.

[0082] In the examples below, all ThT experiments (thioflavin-T detection) used the same buffer, specifically: 50 mM sodium phosphate buffer (pH 7.4), 50 mM NaCl, 10 μM ThT (a dye), and 0.01% sodium azide.

[0083] Example 1: B2M expression is increased in the brain tissue of Alzheimer's disease patients, and Aβ expression is also increased in the brain. 1-42 Content and B2M expression levels showed a significant positive correlation.

[0084] Brain tissue from AD patients and brain tissue from age-matched non-AD-like dementia controls were collected (obtained from the National Human Brain Tissue Resource Bank (Zhejiang University) and the Neurodegenerative Research Center of the University of Science and Technology of China; informed consent was obtained before the brain tissue samples were stored). The tissues were ground, lysed with RIPA protein lysis buffer, and total protein was extracted. BCA concentration was measured and samples were prepared, followed by immunoblotting detection.

[0085] The results are as follows Figures 1A to 1B As shown in the results, B2M protein levels were significantly elevated in the cerebral cortex of AD patients compared to the control group. Simultaneously, the expression level of presynaptic vesicle protein (synaptophysin) in the brain tissue of AD patients was significantly lower than that in the control group.

[0086] Aβ 1-42 ELISA testing is mainly used to detect Aβ in the brain. 1-42 The method can measure the content of Aβ per unit weight of brain tissue. 1-42 Quantitative detection was performed. For the human brain tissue used in the above immunoblotting experiments, Aβ was used... 1-42 ELISA kit (ThermoFisher Scientific, catalog number KHB3441) for measuring Aβ 1-42 Content. Total protein concentration was determined by BCA method.

[0087] Aβ in the human brain 1-42Pearson correlation analysis was performed between the content and relative B2M expression level. Results are as follows: Figure 1C As shown. The results indicate that Aβ in human brain tissue 1-42 content a Relative B2M expression level in the brain b There is a significant positive correlation between them.

[0088] A: Based on the standards provided in the kit, a standard curve was constructed after serial dilution. The samples were diluted and tested together with the standards. After the ELISA reaction, the OD value of each sample was measured using a microplate reader. Then, based on the standard curve, the Aβ value of each sample was calculated. The standard curve for this experiment was y = 154.21x + 5.4233, R0. 2 =0.9932, y is the Aβ content, x is the OD value, the control sample was diluted 40 times before detection, and the AD human brain sample was diluted 200 times before detection.

[0089] b: The relative expression levels here are based on Figure 1A The image is from [source]. Figure 1B The statistical results were obtained. The average value of B2M expression level (protein band gray value) in the brain of the control group was used as the baseline value. Each sample was compared with the baseline value, and the resulting value was the fold change in relative expression level.

[0090] Example 2: Increasing B2M content in the brain can increase β-amyloid plaque deposition in the brain.

[0091] A cannula was implanted in the hippocampus of 3-month-old 5×FAD mice using stereotaxic techniques. Every 7 days, mice were injected with 1 μl (1 μg / μl) of purified B2M protein or PBS through the cannula for two consecutive months. After injection, mice were anesthetized with 5% chloral hydrate, and their hearts were perfused with phosphate-buffered saline. Brain tissue was harvested, fixed overnight in 4% paraformaldehyde, dehydrated with 25% and 30% sucrose solutions, embedded using OCT, frozen sections, and subjected to immunofluorescence staining. Cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI), and Aβ was labeled with antibody 6E10 (Biolegend, catalog number 803016). Images were acquired using a laser confocal fluorescence microscope.

[0092] The results are as follows Figures 1D to 1F As shown, compared with PBS injection, B2M protein injection significantly increased the deposition of β-amyloid plaques in the brain.

[0093] The results showed that the level of B2M protein in the brain of AD mice was elevated, and that increasing the amount of B2M in the brain could further increase the deposition of β-amyloid plaques in the brain of AD mice.

[0094] Example 3: B2M and Aβ 1 -42 There is an interaction between them

[0095] Under in vitro conditions, purified B2M protein was mixed with purified Aβ. 1-42 Protein co-incubation was performed using an anti-Aβ antibody (BioLegend, 6E10) or an anti-B2M antibody (abcam, #75853) for immunoprecipitation. Then, Western blot analysis was used to determine whether there was a direct interaction between the two in vitro.

[0096] Experimental results are as follows Figures 2A to 2B As shown in the figure. The results indicate that both anti-Aβ antibody (6E10) and anti-B2M antibody can be used for immunoprecipitation to remove Aβ. 1-42 It precipitated along with the two purified proteins, B2M.

[0097] Furthermore, cerebral cortex tissue was taken from 12-month-old 5×FAD mice, and after tissue lysis, immunoprecipitation was performed using an anti-Aβ antibody (6E10). Then, immunoblotting analysis was performed to determine whether there was a direct interaction between the two in vivo.

[0098] Experimental results are as follows Figure 2C As shown in the figure. The results indicate that immunoprecipitation using an anti-Aβ antibody (6E10) can remove Aβ from the body. 1-42 It settled down along with B2M.

[0099] The results showed that, under both in vivo and in vitro conditions, B2M and Aβ 1-42 There are interactions between them.

[0100] Example 4: B2M and Aβ 1 -4 2 The interaction between them can promote Aβ 1 -42 of Oligomerization

[0101] Thioflavin-T is a fluorescent dye that binds to β-sheet-rich proteins. After binding, the fluorescence intensity of thioflavin-T increases, making it an effective indicator for detecting fibroblast formation. In the presence of thioflavin-T, purified B2M is combined with Aβ... 1-42 The two proteins were co-incubated at 37°C, and fluorescence values ​​were read every 5 minutes using a fluorescence microplate reader (excitation light λ = 448 nm, absorption light λ = 488 nm) for 12-14 hours. After the experiment, a curve was plotted based on the change in fluorescence values ​​over time to analyze the Aβ levels within the reaction system. 1-42 The formation of fibrils.

[0102] Experimental results are as follows Figure 2D As shown. The results indicate that B2M protein incubation with Aβ 1-42 Afterwards, it can promote Aβ1-42 The formation of fibrils exhibits a certain concentration dependence.

[0103] In addition, considering that the B2M protein itself also has a certain oligomerization ability, the inventors conducted a thiosulfate-T experiment on the purified B2M protein alone under the same experimental conditions.

[0104] Experimental results are as follows Figure 2E As shown in the figure. The results indicate that, under the same experimental conditions, the B2M protein alone does not oligomerize, does not produce fibrils, and does not interfere with [the experiment / work]. Figure 2D The experimental results.

[0105] The results showed that B2M and Aβ 1-42 The interaction between them can promote Aβ 1-42 Oligo-aggregation.

[0106] Example 5: The truncated B2M peptide can act as a blocking peptide, preventing the interaction between B2M and Aβ. 1-4 2 Binding, inhibiting B2M-induced Aβ1- 42 widow Polymerization

[0107] To clarify B2M and Aβ 1-42 The amino acid regions where interactions occur, such as Figure 3A As shown, the inventors truncated the full-length human B2M sequence into four small peptides with non-overlapping sequences, named B2M-1, B2M-2, B2M-3, and B2M-4 respectively (detailed sequences are shown in Table 1 below). The peptides in Table 1 were synthesized.

[0108] Table 1

[0109]

[0110] Using B2M truncated peptides with Aβ 1-42 Pre-incubation allows small peptides that bind effectively to inhibit the binding of the full-length B2M protein to Aβ. 1-42 The binding of B2M to Bβ inhibits its induced Aβ activity. 1-42 Oligomerization. The inventors reacted 10 μM of four truncated peptides and a meaningless control peptide with 10 μM of Aβ. 1-42 Pre-incubate at 37°C for 3 hours, then add 1 μM full-length B2M protein for thiamine-T detection.

[0111] Experimental results are as follows Figure 3B As shown, the results indicate that only the third B2M-3 truncated peptide can effectively block the interaction between B2M and Aβ. 1-42 Binding, inhibiting B2M-induced Aβ 1-42 Oligopoly.

[0112] Furthermore, the inventors used different concentrations (5 μM, 10 μM, 20 μM) of B2M-3 truncated peptide and 10 μM of Aβ 1-42 Pre-incubate at 37°C for 3 hours, then add 1 μM full-length B2M and perform thiosulfate-T detection again.

[0113] Experimental results are as follows Figure 3C As shown in the figure. The results indicate that the B2M-3 peptide can effectively inhibit B2M-induced Aβ. 1-42 It oligomerizes and exhibits a certain concentration dependence.

[0114] Aβ 1-42 Oligomerization forms fibrous oligomers, which then deposit to form amyloid plaques. 10 μM of Aβ... 1-42 The peptide was pre-incubated with 10 μM B2M-3 small peptide at 37°C for 3 hours, and then co-incubated with purified 1 μM B2M protein at 37°C for 72 hours. The sample was then spotted onto a carbon-coated grid and stained with 1% uranyl acetate. Images were acquired using a Hitachi HT-7800 transmission electron microscope (Hitachi High Technology, Japan). Experimental results are as follows: Figure 3D As shown in the figure. The results showed that, compared with the control group, pre-incubation with the B2M-3 small peptide could effectively inhibit the Aβ-induced response of the full-length B2M protein. 1-42 It forms fibrous oligomers.

[0115] like Figure 3E As shown, based on B2M-3, the inventors further shortened the amino acid sequence to narrow the effective binding range. The detailed truncated sequence of B2M-3 is shown in Table 2 below.

[0116] Table 2

[0117] 6 B2M-3-1 HSDLSFSKDW 7 B2M-3-2 SFYLLYYTEFTPTEK 8 B2M-3-3 SFYLLYYTE

[0118] After synthesizing the small peptides, using the same experimental method described above, the inventors synthesized three truncated small peptide sequences (B2M-3-1, B2M-3-2, and B2M-3-3) of B2M-3 at different concentrations (1 μM, 5 μM, and 10 μM) and reacted them with 10 μM of Aβ. 1-42 Pre-incubate, then add 1 μM of full-length B2M protein for thiamine-T detection. Experimental results are as follows: Figures 3F to 3H As shown in the figure. The results indicate that both B2M-3-2 and B2M-3-3 small peptide sequences can effectively inhibit B2M-induced Aβ. 1-42 Oligomerization occurs, and it exhibits a certain concentration dependence.

[0119] The results showed that B2M and Aβ 1-42The interacting amino acid regions are mainly located within the third truncated region. Using B2M-3 peptide, B2M-3-2, or B2M-3-3 as blocking peptides can effectively inhibit B2M-induced Aβ. 1-42 Oligomerization and effective inhibition of B2M-induced β-amyloid plaque formation.

[0120] Example 6: B2M-3 blocking peptide inhibits B2M-induced Aβ 1-42 widow Aggregation, thereby inhibiting Aβ 1-42 Neurotoxicity

[0121] Oligomeric Aβ 1-42 It has strong neurotoxicity, and B2M promotes Aβ. 1-42 Oligomerization further enhances its neurotoxicity. Based on the B2M-3 blocking peptide, B2M-induced Aβ can be inhibited. 1-42 Oligomerization is a concern, therefore it is necessary to investigate whether B2M-3 blocking peptides can inhibit B2M-induced oligomerization of Aβ. 1-42 Neurotoxicity.

[0122] 10 μM B2M-3 peptide or a meaningless control peptide was used with 10 μM Aβ. 1-42 Pre-incubate at 37°C for 3 hours, then add 1 μM of full-length B2M protein and continue incubation for 12 hours to obtain two types of Aβ. 1-42 Incubation products with varying degrees of oligomerization.

[0123] Two-month-old wild-type (WT) mice were anesthetized, and brain tissue was rapidly extracted and cooled in ice-cold, oxygenated artificial cerebrospinal fluid (ACSF). The tissue was then transferred to a microtome for coronal sectioning, with each section being 400 μm thick. The brain sections were incubated in oxygen-saturated ACSF at 32°C for 1 hour, followed by incubation at room temperature for another hour. Oligomeric Aβ... 1-42 Brain slices were diluted to 200 nM with ACSF and incubated at room temperature for 1.5 hours. The slices were then transferred to a recording chamber, with recording electrodes placed in the CA1 region of the Schaffer collateral-commissural pathway and stimulation electrodes placed in the CA3 region. The stimulation intensity was 30% of the maximum field excitatory postsynaptic potential (fEPSP). After 20 minutes of baseline fEPSP recording, high-frequency stimulation (HFS) induced long-term potentiation (LTP) (two stimuli, each containing 100 pulses, with a 30-second interval between each stimuli), and recording continued for 60 minutes.

[0124] Experimental results are as follows Figures 4A to 4B As shown. The results indicate that, compared to brain slices incubated with simple small peptides, pre-incubation with meaningless peptides produced oligomeric Aβ. 1-42It significantly impairs the LTP of the Schaffercollateral-commissural pathway in the CA3-CA1 region of the hippocampus in WT mice, while Aβ produced by pre-incubation with B2M-3 blocking peptides... 1-42 Incubation products with varying degrees of oligomerization did not significantly damage the LTP in WT mice.

[0125] In summary, pre-incubation of Aβ with B2M-3 blocking peptides... 1-42 It can inhibit B2M from promoting Aβ 1-42 Neurotoxicity resulting from oligomerization.

[0126] Example 7: B2M-3 blocking peptide can inhibit the formation of B2M-induced β-amyloid plaques in the mouse brain.

[0127] The experiments in Examples 5-6 demonstrate that, under in vitro conditions, the B2M-3 blocking peptide can inhibit B2M-induced Aβ. 1-42 Oligomerization, and consequently, reduction of oligomerization Aβ 1-42 Neurotoxicity. To further verify the inhibitory effect of B2M-3 blocking peptide on B2M-induced β-amyloid plaque formation in vivo, the inventors implanted a cannula in the hippocampus of B2M-humanized 5×FAD mice using stereotaxic techniques. Subsequently, 1 μl (5 μg / μl) of a meaningless peptide or B2M-3 blocking peptide was injected into the left and right hippocampuses of the mice weekly through the cannula for two consecutive months. After the injection, the mice were anesthetized with 5% chloral hydrate, and the heart was perfused with phosphate buffer. Brain tissue was harvested, fixed overnight in 4% paraformaldehyde, dehydrated with 25% and 30% sucrose solutions, embedded in OCT, frozen sections, and subjected to immunofluorescence staining. The cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI) dye, and Aβ was labeled with antibody 6E10 (Biolegend, catalog number 803016). 1-42 Images were acquired using a laser confocal fluorescence microscope.

[0128] Experimental results are as follows Figures 4C to 4E As shown in the figure. The results showed that, compared with the injection of meaningless peptides, the injection of B2M-3 blocking peptides significantly reduced the deposition of β-amyloid plaques in the brain.

[0129] In summary, the B2M-3 blocking peptide effectively inhibited B2M-induced β-amyloid plaque deposition in the brains of 5×FAD mice.

[0130] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University <120> β2-microglobulin blocking peptide, its pharmaceutical composition and uses <130> IDC220074 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> B2M-1 <400> 1 His His His His His Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr 1 5 10 15 Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys 20 25 30 <210> 2 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> B2M-2 <400> 2 His His His His His Tyr Val Ser Gly Phe His Pro Ser Asp Ile 1 5 10 15 Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu 20 25 30 <210> 3 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> B2M-3 <400> 3 His His His His His His His Ser Asp Leu Ser Phe Ser Lys Asp Trp 1 5 10 15 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys 20 25 30 <210> 4 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> B2M-4 <400> 4 His His His His His His Asp Glu Tyr Ala Cys Arg Val Asn His Val 1 5 10 15 Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met 20 25 30 <210> 5 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Non-sense <400> 5 His His His His His His Gln Phe Lys Ser Ile Lys Asn Pro Gln His 1 5 10 15 Glu Val Gly Ala Ile Asn Arg Cys Arg Asn Pro Leu Thr Tyr Ser 20 25 30 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> B2M-3-1 <400> 6 His Ser Asp Leu Ser Phe Ser Lys Asp Trp 1 5 10 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> B2M-3-2 <400> 7 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys 1 5 10 15 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> B2M-3-3 <400> 8 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu 1 5 <210> 9 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> human B2M <400> 9 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp Met 115 <210> 10 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> human Aβ1-42 <400> 10 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment of B2M <400> 11 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment of B2M <400> 12 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment of B2M <400> 13 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro 1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment of B2M <400> 14 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> truncated fragment of B2M <400> 15 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe 1 5 10

Claims

1. The isolated polypeptide, which is the polypeptide shown in SEQ ID NO:

3.

2. An isolated polynucleotide encoding the isolated polypeptide of claim 1.

3. A recombinant expression vector comprising the isolated polynucleotide of claim 2.

4. A transformed cell comprising the recombinant expression vector of claim 3.

5. A pharmaceutical composition for treating Alzheimer's disease, comprising the isolated polypeptide of claim 1.

6. The pharmaceutical composition according to claim 5, further comprising one or more pharmaceutically acceptable excipients.

7. Use of the polypeptide shown in any one of SEQ ID NO: 3 and SEQ ID NO: 7-8 in the preparation of a medicament for treating Alzheimer's disease.

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