CN117186199A - Beta 2-microglobulin blocking peptide, pharmaceutical composition and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to a beta 2-microglobulin blocking peptide, a pharmaceutical composition and application thereof. In particular, the invention 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 the polypeptide shown in SEQ ID NO. 8. The isolated polypeptide can effectively prevent and treat AD or cognitive impairment caused by AD, and has good application prospect.

Description

Beta 2-microglobulin blocking peptide, pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a beta 2-microglobulin blocking peptide, a pharmaceutical composition and application thereof.

Background

Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases in humans, and according to the 2018 report of Alzheimer's disease in the world, 5000 tens of thousands of people worldwide have AD by 2050, a figure that will increase to 1.52 billion. The main pathological features of the disease include amyloid deposition formed by oligomerization of beta-amyloid (aβ) produced by cleavage of brain amyloid precursor protein (Amyloid precursor protein, APP), neurofibrillary tangles (Neurofibrillary tangles, NFTs) formed by aggregation after abnormal phosphorylation of intracellular microtubule-associated protein tau, neuronal loss and excessive neuroinflammation, and the like. The clinical manifestations of AD patients are mainly memory decline and cognitive impairment, and this decline worsens with the progression of the disease, eventually losing all memory and life self-ability until death. In view of the advent of global aging, together with the continued rise in the incidence of AD, and the current lack of effective therapeutic measures, AD is one of the diseases that severely threatens human health.

Since AD was found in 1901, there has been considerable research on its pathogenesis, however, no clear definition exists at present. The "Abeta cascade hypothesis" is one of the main theories of the pathogenesis of AD, which considers that Abeta produced by aberrant cleavage of APP is neurotoxic _1-40/42 Plays a central role in the pathogenesis of AD and is a common pathway for the induction of AD for various reasons. A series of neurotoxic reactions caused by Abeta oligomers can excite neuroinflammation, lead to nerve cell dysfunction and neuron loss, and finally cause dementia. Thus, reduction of aβ production in the brain, promotion of aβ clearance, inhibition of aβ aggregation, and reduction of neurotoxicity have become one of the main measures for the treatment of AD.

Beta 2-microglobulin (B2M) is a senescence-promoting factor which has been recently receiving a great deal of attention, and B2M is a constituent subunit of the major histocompatibility complex I (Major histocompatibility complex I, MHC-I), encoded by the human chromosome 15 gene, comprising 119 amino acids. Since B2M is not anchored to the cell membrane by a transmembrane domain, B2M can be released from the MHC-I complex into the cell gap. Normally, B2M proteins exist in soluble monomeric form, but under the influence of some pathological factors, B2M aggregates and deposits. These pathological factors include aging, long-term renal dysfunction, inflammation, and the like. B2M amyloid deposition is mainly present in the osteoarticular area and ultimately leads to severe arthritis, fractures and carpal tunnel syndrome. In addition, in many disease states, there is an increase in B2M in both serum and plasma, of particular concern is that both plasma and cerebrospinal fluid in AD patients are significantly higher in B2M than in normal cohort controls. Brain stereotactic injection of B2M inhibits neuronal regeneration and impairs cognitive function in mice, whereas B2M deficiency promotes neuronal regeneration and reverses age-related cognitive dysfunction. However, whether B2M has direct or indirect influence on the occurrence and development of AD diseases is not reported.

Disclosure of Invention

The inventors have conducted intensive studies and inventive efforts to find out the role of B2M in the development of AD, and have surprisingly found that B2M and Abeta are hindered _1-42 The bound blocking peptides have potential as drugs for the prevention and treatment of AD, particularly cognitive impairment caused by AD. The following invention is thus provided:

one aspect of the invention relates to an isolated polypeptide which is a polypeptide as set forth in SEQ ID NO. 3 or a truncated fragment of a polypeptide as set forth in SEQ ID NO. 3, wherein the truncated fragment comprises a polypeptide as set forth in SEQ ID NO. 8.

In some embodiments of the invention, the isolated polypeptide, wherein the truncated fragment does not comprise the 6 histidines at the N-terminus of the polypeptide shown in SEQ ID NO. 3.

In some embodiments of the invention, the isolated polypeptide is a polypeptide as set forth in any one of SEQ ID NOs 7-8.

HHHHHHHSDLSFSKDWSFYLLYYTEFTPTEK(SEQ ID NO:3)

SFYLLYYTEFTPTEK(SEQ ID NO:7)

SFYLLYYTE(SEQ ID NO:8)

In some embodiments of the invention, the isolated polypeptide is a polypeptide as set forth in any one of SEQ ID NOs 11-15.

SFYLLYYTEFTPTE(SEQ ID NO:11)

SFYLLYYTEFTPT(SEQ ID NO:12)

SFYLLYYTEFTP(SEQ ID NO:13)

SFYLLYYTEFT(SEQ ID NO:14)

SFYLLYYTEF(SEQ ID NO:15)

Another aspect of the invention relates to an isolated polynucleotide encoding an isolated polypeptide according to any one of the invention.

Yet another aspect of the invention relates to a recombinant expression vector comprising an isolated polynucleotide of the invention.

A further aspect of the invention relates to a transformed cell comprising a recombinant expression vector of the invention.

A further aspect of the invention relates to a pharmaceutical composition comprising an isolated polypeptide according to any one of the invention.

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

A further aspect of the invention relates to the use of an isolated polypeptide according to any one of the invention for the manufacture of a medicament for the treatment or prevention of alzheimer's disease.

A further aspect of the invention relates to the use of an isolated polypeptide according to any of the invention for the preparation of a medicament as follows:

inhibition of B2M-induced Abeta _1-42 Oligomerizing agents, agents inhibiting B2M-induced formation of beta-amyloid plaques or inhibiting aβ _1-42 Is a neurotoxic drug.

The invention discovers that the expression of B2M in brain tissue of AD patients is obviously increased for the first time, and the expression level of B2M in brain and Abeta _1-42 There is a significant positive correlation between levels, and further increasing B2M levels in AD mouse model (5 x FAD) brains by brain stereotactic injection can increase β -amyloid plaque deposition in 5 x FAD mice. Further, the present inventors found that B2M and Abeta _1-42 Direct interactions exist and B2M and Abeta can be inhibited by using truncated B2M amino acid sequence as blocking peptide _1-42 Aggregation, inhibition of B2M and Abeta _1-42 Neurotoxicity, in vivo experiments show that the blocking peptide can obviously inhibit B2M to Abeta in brain _1-42 Promoting aggregation, and reducing beta-amyloid plaque deposition in brain. The discovery provides a potential drug target point and a new therapeutic mode based on the target point for clinical treatment of AD.

An isolated polypeptide according to any one of the present invention for use in the treatment or prevention of alzheimer's disease.

An isolated polypeptide according to any one of the present invention for use in inhibiting B2M-induced aβ _1-42 Oligomerization and inhibition of B2M-induced betaAmyloid plaque formation or for inhibition of aβ _1-42 Is a neurotoxicity of (a).

A further aspect of the invention relates to a method for the treatment or prevention of alzheimer's disease comprising the step of administering to a subject in need thereof an effective amount of an isolated polypeptide according to any of the invention.

In yet another aspect, the invention relates to a method of inhibiting B2M-induced Abeta _1-42 Oligomerization or inhibition of B2M-induced beta-amyloid plaques or inhibition of Abeta _1-42 Comprising the step of administering to a subject in need thereof an effective amount of an isolated polypeptide of any one of the present invention.

In some embodiments of the invention, the amino acid sequence of the beta 2-microglobulin is shown in SEQ ID NO 9.

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

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:9)

in the present invention, when referring to the amino acid sequence of β2-microglobulin (B2M), it includes the full length of β2-microglobulin, and also includes fusion proteins thereof. However, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced in the amino acid sequence of the β2-microglobulin without affecting its biological function. In one embodiment of the invention, the beta 2-microglobulin is a human beta 2-microglobulin. In one embodiment of the invention, the amino acid sequence of the beta 2-microglobulin is shown in SEQ ID NO 9.

In the present invention, the term "host cell" refers to a cell into which a vector is introduced, and includes many cell types such as prokaryotic cells such as E.coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells, or human cells.

In the present invention, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide that inhibits a protein can be inserted. For example, the carrier comprises: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40). One vector may contain a variety of elements that control expression.

The term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., receiving a pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate a disease or disorder described herein.

The term "blocking peptide" refers to a polypeptide that is capable of competitively binding aβ with a full length B2M protein, thereby inhibiting the biological effect of B2M binding aβ that promotes oligomerization of aβ.

In the present invention, if not specified, the concentration unit/. Mu.M represents/. Mu.mol/L, mM represents mmol/L, and nM represents nmol/L.

In the present invention, when the amount of drug to be added to cells is mentioned, it is generally referred to as the final concentration of the drug after the drug is added unless otherwise specified.

Aβ _1-42 The amino acid sequence of (2) is as follows:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID NO:10)

advantageous effects of the invention

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

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

(2) The polypeptide of the invention canCan effectively inhibit B2M induced Abeta _1-42 Oligomerization.

(3) The polypeptide of the invention can effectively inhibit the formation of B2M-induced beta-amyloid plaques.

(4) The polypeptide of the invention can effectively inhibit Abeta _1-42 Is a neurotoxicity of (a).

Drawings

Fig. 1A to 1B: the B2M expression level in the brain of AD patients was significantly increased. Wherein:

fig. 1A: the B2M expression immunoblotting detection result in the cerebral cortex tissue of the AD patient.

Fig. 1B: image J analyzed B2M expression levels in fig. 1A, control group n=8 human brain tissues, AD group n=21 human brain tissues. The data were statistically analyzed using student t test. ns, no significant difference, P >0.05; * P <0.05; * P <0.01; * P <0.001.

Fig. 1C: through Abeta _1-42 ELISA detected Abeta in the 29 human brain tissues _1-42 Level, abeta in human brain was found by Pearson correlation analysis _1-42 There is a significant positive correlation between levels and B2M expression levels in the human brain.

FIGS. 1D-1F increasing B2M levels in the brain of 5 XFAD mice promotes amyloid plaque deposition in the brain. Mice were anesthetized with 5% chloral hydrate after 2 months by brain stereotactic injection into the hippocampus of 4 month old 5×fad mice with 1 μl (1 μg/μl) of purified B2M protein or 1 μl PBS (control per mouse brain), heart perfused with phosphate buffer, brain tissue was taken, fixed overnight in 4% paraformaldehyde, dehydrated with 25% and 30% sucrose solution, brain tissue embedded with OCT, frozen sections were post immunofluorescent staining, dye 4',6-diamidino-2-phenylindole (DAPI) labeled nuclei, antibody 6E10 labeled aβ, and images were acquired by laser confocal fluorescence microscopy. Scale, 300 μm. Data represent mean ± standard error. Wherein:

fig. 1D: immunofluorescent staining of 5×fad mice brain tissue amyloid plaques. N=6 mice per group. Wherein the dashed box indicates the hippocampal DG area range, and also represents the brain tissue area where the statistical plaque is located.

Fig. 1E: FIG. 1D shows the statistics of the Number of β -amyloid plaques (Number of Abeta depositions) in the hippocampus DG region. The data were statistically analyzed using a Paired t test. ns, no significant difference, P >0.05; * P <0.05; * P <0.01; * P <0.001.

Fig. 1F: FIG. 1D shows statistics of the Area of β -amyloid plaques (Area of Abeta depositions) in the hippocampus DG region. The data were statistically analyzed using a Paired t test. ns, no significant difference, P >0.05; * P <0.05; * P <0.01; * P <0.001.

Fig. 2A to 2B: in vitro purified B2M protein and purified aβ _1-42 Co-immunoprecipitation (co-IP) experiments of interactions between proteins. Wherein:

fig. 2A: immunoprecipitation of Abeta with antibodies against Abeta _1-42 And B2M.

Fig. 2B: immunoprecipitation of B2M and Abeta with anti-B2M antibodies _1-42 。

Fig. 2C: in vivo B2M and Abeta using 5 XFAD mouse brain tissue _1-42 As a result of co-IP experiment of the interaction, the antibody used was an anti-Abeta antibody.

Fig. 2D: incubation of Abeta with B2M in vitro with thioflavin-T indicator _1-42 Post-promotion of Aβ _1-42 The result of continuous detection by the oligomerized fluorescent enzyme label instrument. At 37 ℃, different concentrations of full-length B2M protein and Abeta _1-42 Co-incubating, adding thioflavin-T into the experimental system, and detecting the change of a thioflavin-T fluorescent signal value every 5min under the conditions of excitation light wavelength lambda=448 nm and absorption light wavelength lambda=488 nm by using a fluorescence microplate reader. And (5) drawing a graph of the change of the fluorescence signal value with time after the test is finished. Data represent the average of absolute fluorescence signal intensities.

Fig. 2E: thioflavin-T fluorescence detection results for B2M protein at corresponding concentrations under the same experimental conditions as in fig. 2D. Data represent the average of absolute fluorescence signal intensities.

Fig. 3A: segment schematic of a short peptide with a B2M amino acid full-length sequence truncated to 4 non-overlapping sequences.

Fig. 3B: truncated B2M small peptides inhibit B2M promotionAβ _1-42 Experimental results of oligomerized thioflavin-T. Truncated B2M amino acid sequence as blocking peptide and Abeta _1-42 Preincubation at 37℃for 3 hours followed by further addition of full length B2M protein and continued incubation of Abeta _1-42 Analysis of blocking peptides to promote Aβ to B2M by analysis of changes in thioflavin-T fluorescence signal intensity _1-42 Oligomerization inhibition effect. Data represent the average of absolute fluorescence signal intensities.

Fig. 3C: detection of B2M-3 blocking peptides at different concentrations for promotion of Abeta by B2M based on the thioflavin-T assay described above _1-42 Inhibition of oligomerization. Data represent the average of absolute fluorescence signal intensities.

Fig. 3D: detection of B2M-3 blocking peptides by Transmission Electron Microscopy (TEM) technique to promote Aβ by blocking B2M _1-42 Oligomerization of Abeta _1-42 Morphological effects of the formed filaments. Scale, 1 μm.

Fig. 3E: to further narrow down the amino acid sequence range that exerts the repressing effect, the present inventors further represented the B2M-3 blocking peptide as a segment schematic of 3 short peptides with no overlapping sequences.

Fig. 3F to 3H: the thioflavin-T experiments described above were performed on 3 further truncated small peptides, and small peptide sequences of different concentrations were analyzed to promote Aβ for B2M _1-42 Inhibition of oligomerization. Data represent the average of absolute fluorescence signal intensities. Wherein:

fig. 3F: three different concentrations of the first B2M-3 truncated small peptide (B2M-3-1) preincubate Abeta _1-42 And 3 hours, then adding full-length B2M protein, and continuously detecting the change condition of the thioflavin-T fluorescent signal based on a fluorescent enzyme-labeled instrument.

Fig. 3G: three different concentrations of the second-stage B2M-3 short-cut small peptide (B2M-3-2) preincubate Abeta _1-42 And 3 hours, then adding full-length B2M protein, and continuously detecting the change condition of the thioflavin-T fluorescent signal based on a fluorescent enzyme-labeled instrument.

Fig. 3H: three different concentrations of the third-stage B2M-3 short-cut small peptide (B2M-3-3) preincubated Abeta _1-42 And 3 hours, then adding full-length B2M protein, and continuously detecting the change condition of the thioflavin-T fluorescent signal based on a fluorescent enzyme-labeled instrument.

Fig. 4A to 4B: B2M-3 blocking peptides promote Aβ to B2M _1-42 In vitro electrophysiological experimental results of the inhibitory effect of neurotoxicity produced by oligomerization. Preincubation of Abeta with B2M-3 blocking peptide or control nonsensical peptide sequence (NS) at 37 ℃ _1-42 3 hours, then adding full-length B2M protein and further incubating for 12 hours to obtain two kinds of oligomerization Abeta with different oligomerization degrees _1-42 The product is obtained. Based on oligomerised Abeta _1-42 With neurotoxicity, the inventor incubates the two kinds of Abeta products (abbreviated as oAbeta) with different oligomerization states respectively for 2 months of in vitro brain slices of wild mice, incubates for 1.5 hours, and then performs electrophysiological long-time-interval enhancement (LTP) experiments to detect the influence of the oAbeta on synaptic plasticity of the in vitro brain slice nerve loops. Wherein, FIG. 4B data was statistically analyzed using one-way ANOVA. ns, no significant difference, P>0.05；*P<0.05；**P<0.01；***P<0.001；****P<0.001. Wherein:

fig. 4A: the analysis results were recorded in the isolated brain slice CA1 region LTP. oaβ -treated groups were n=4 mice per group, and brain numbers n=8 were recorded per group. Control group treated with the single peptide had n=4 mice per group, and brain number n=7 was recorded per group.

Fig. 4B: LTP record in fig. 4A results last 10min fEPSP amplitude statistical analysis. Each set of n values is identical to fig. 4A.

Fig. 4C to 4D: the quantity of beta-amyloid plaque deposition and plaque deposition area in the brain of AD mice are significantly reduced after the 5X FAD mice are injected with the B2M-3 blocking peptide. Wherein, fig. 4C and 4D data were statistically analyzed using a Paired t test. ns, no significant difference, P >0.05; * P <0.05; * P <0.01; * P <0.001. Wherein:

fig. 4C: immunofluorescent staining of β -amyloid plaques deposited in hippocampal DG region in brain of 5×fad mice injected with B2M-3 blocking peptide or nonsensical peptide. Dye 4',6-diamidino-2-phenylindole (DAPI) labeled nuclei and antibody 6E10 labeled aβ. Scale, 300 μm. N=6 mice per group.

Fig. 4D: FIG. 4C shows the results of statistical analysis of the number of β -amyloid plaques in the hippocampus DG region.

Fig. 4E: FIG. 4C shows the results of statistical analysis of the area of β -amyloid plaques in the hippocampus DG region.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In the following examples, 5×fad mice are transgenic model mice for AD disease, which exhibit AD-specific β -amyloid pathological characteristics in the brain; 5×FADA mice were purchased from Jackson Laboratory (Ellsworth, ME, USA), numbered 34840-JAX.

In the following examples, the buffers used for all ThT experiments (thioflavin-T assay) were identical, and the specific formulation was: 50mM sodium phosphate buffer (pH 7.4), 50mM NaCl, 10. Mu.M ThT (a dye) and 0.01% sodium azide.

_1-42 Example 1: the expression level of B2M in brain tissue of Alzheimer's disease patient is increased, and the content of Abeta in brain is equal to that of There is a significant positive correlation in B2M expression levels

Respectively taking brain cortex tissue of AD patients and brain tissue of the same-age non-AD-like dementia control human (obtained from national brain tissue resource library (Zhejiang) and the university of Chinese science and technology neurodegenerative research center, and obtaining the known consent before the brain tissue sample is put in storage), extracting total protein after tissue grinding and RIPA protein lysate cleavage, preparing a sample by BCA concentration measurement, and then performing immunoblotting detection.

The results are shown in fig. 1A to 1B. The results show that B2M protein is significantly elevated in the cerebral cortex of AD patients relative to the control group. The expression level of the presynaptic vesicle protein (syntophysin) detected at the same time in brain tissues of AD patients is obviously lower than that of a control group.

Aβ _1-42 ELISA detection is mainly used for detecting Abeta in brain _1-42 Content of the method canTo A beta in brain tissue per unit weight _1-42 And (5) carrying out quantitative detection. For human brain tissue used in the immunoblotting experiments, abeta is used _1-42 ELISA kit (Thermo Fisher Scientific Co., ltd., product No. KHB 3441) for measuring Abeta _1-42 The content is as follows. The BCA method determines total protein concentration.

Aβ in human brain _1-42 And carrying out Pearson correlation analysis between the content and the relative B2M expression quantity. The results are shown in FIG. 1C. The results show that Abeta in human brain tissue _1-42 Content of ^a Relative B2M expression level in brain ^b There is a significant positive correlation between them.

a: and (3) based on a standard substance provided by the kit, performing gradient dilution and then making a standard curve. And (3) diluting the samples, detecting the diluted samples together with a standard substance, detecting the OD value of each sample by using an enzyme-labeled instrument after ELISA reaction is finished, and then calculating the Abeta value of each sample based on a standard curve. The standard curve of the experiment is y=154.21x+5.4233, R ² =0.9932, y is aβ content, x is OD, 40-fold dilution before control sample detection, 200-fold dilution before AD human brain sample detection.

b: the relative expression results are based on the gel diagram of fig. 1A, and the data are derived from the statistical results of fig. 1B. The average value of the B2M expression quantity (protein band gray value) in the brain of the control group is taken as a basic value, and each sample is compared with the basic value, and the obtained value is the change multiple of the relative expression quantity.

Example 2: increasing B2M content in brain increases beta-amyloid plaque deposition in brain

The hippocampus of the brain of a3 month old 5×fad mouse was cannulated by brain stereotactic technique, and then the mouse was injected with 1 μl (1 μg/μl) of purified B2M protein or PBS every 7 days via the cannula for 2 months. Mice were anesthetized with 5% chloral hydrate after injection, heart perfused with phosphate buffer, brain tissue was taken, fixed overnight in 4% paraformaldehyde, dehydrated with 25% and 30% sucrose solution, brain tissue embedded with OCT, frozen sections, immunofluorescent staining was performed, dye 4',6-diamidino-2-phenylindole (DAPI) labeled nuclei, antibody 6E10 (Biolegend, cat No. 803016) labeled aβ, and images were acquired by laser confocal fluorescent microscopy.

As a result, as shown in fig. 1D to 1F, injection of B2M protein significantly increased the deposition of β -amyloid plaques in the brain compared to injection of PBS.

The results indicate that the level of B2M protein is elevated in the brain of AD mice, and that increasing the B2M content in the brain can further increase the deposition of β -amyloid plaques in the brain of AD mice.

Example 3: B2M and Aβ _{1 -42} There is an interaction between

Under in vitro conditions, the purified B2M protein is reacted with purified Abeta _1-42 Proteins were co-incubated, co-immunoprecipitated with anti-Abeta (BioLegend, 6E 10) or anti-B2M (abcam, # 75853) and then analyzed by immunoblotting for the presence of direct interactions in vitro.

The experimental results are shown in fig. 2A to 2B. The results showed that Abeta was co-precipitated either with the anti-Abeta antibody (6E 10) or with the anti-B2M antibody _1-42 And B2M precipitated together.

Further, after the 5×fad mouse brain cortex tissue of 12 months old was taken and subjected to tissue lysis, co-immunoprecipitation was performed with an anti-aβ antibody (6E 10), and then the presence or absence of direct interaction between the two was analyzed by immunoblotting.

The experimental results are shown in fig. 2C. The results show that co-immunoprecipitation with anti-Abeta antibody (6E 10) can precipitate Abeta in vivo _1-42 And B2M precipitated together.

The results show that B2M and Abeta under in vivo and in vitro conditions _1-42 Interaction exists between the two.

Example 4: B2M and Aβ _{1 -4 2} Interaction between them can promote Abeta _{1 -42} A kind of electronic deviceOligomerization

Sulfur, sulfur and its preparation methodThe element-T is a fluorescent dye which can be combined with beta-sheet-rich protein, and after combination, the fluorescence intensity of the thioflavin T can be enhanced, so that the element-T is an effective index for detecting the formation of fibrinogen. Purified B2M is combined with Abeta in the presence of thioflavin-T _1-42 The two proteins were incubated at 37℃and simultaneously fluorescence values were read with a fluorescence microplate reader every 5min (excitation light λ=448 nm, absorption light λ=488 nm) and continuously detected for 12-14 hours. Drawing a graph based on the change condition of fluorescence value along with time after the experiment is finished, and using the graph in the reaction system for Abeta _1-42 Fibril formation.

The experimental results are shown in fig. 2D. The results show that the B2M protein incubates Abeta _1-42 After that, Aβ can be promoted _1-42 Fibril formation and exhibits a certain concentration dependence.

In addition, the inventors performed thioflavin-T experiments on purified B2M protein alone under the same experimental conditions, since the B2M protein itself also had a certain oligomerization ability.

The experimental results are shown in fig. 2E. The results show that under the same experimental conditions, the B2M protein alone does not oligomerize, does not generate fibrils, and does not interfere with the experimental results in fig. 2D.

The results show that B2M and Abeta _1-42 Interaction between them can promote Abeta _1-42 Is an oligomerization of (a).

_1-4 Example 5: the B2M truncated peptide can be used as a blocking peptide for blocking B2M and Abeta ₂ Binding, inhibition of B2M-induced Aβ1- 42Oligo(s)Polymerization

To clarify B2M and Abeta _1-42 The amino acid regions where interactions occur, as shown in FIG. 3A, the present inventors truncated the full-length human B2M sequence into 4 small peptides of non-overlapping sequence, designated B2M-1, B2M-2, B2M-3, B2M-4, respectively (see Table 1 for detailed sequences below). The polypeptides in Table 1 were synthesized.

TABLE 1

With B2M truncated peptides and Abeta _1-42 Pre-incubation, small peptides that bind efficiently can block full-length B2M protein from Aβ _1-42 Thereby inhibiting B2M-induced Abeta _1-42 Oligomerization. The inventors separately prepared 10. Mu.M of four truncated peptides and nonsensical control peptide with 10. Mu.M of Abeta _1-42 Preincubation was performed at 37℃for 3 hours, after which 1. Mu.M full-length B2M protein was added for thioflavin-T detection.

The experimental results are shown in FIG. 3B, which shows that only the third B2M-3 truncated peptide can effectively block B2M and Abeta _1-42 Binding, inhibition of B2M-induced Abeta _1-42 Oligomerization.

Further, the present inventors used B2M-3 truncated peptides at different concentrations (5. Mu.M, 10. Mu.M, 20. Mu.M) with 10. Mu.M of Abeta _1-42 Pre-incubation was performed at 37℃for 3 hours, after which 1. Mu.M full length B2M was added and thioflavin-T assay was performed again.

The experimental results are shown in fig. 3C. The results show that the B2M-3 peptide can effectively block B2M induced Abeta _1-42 Oligomerizes and exhibits a certain concentration dependence.

Aβ _1-42 Fibrous oligomers are formed by oligomerization, followed by deposition to form amyloid plaques. Will 10 mu M A beta _1-42 The polypeptide was pre-incubated with 10. Mu. M B2M-3 small peptide at 37℃for 3 hours, then co-incubated with purified 1. Mu.M B2M protein at 37℃for 72 hours, then the samples were spotted on a carbon-coated grid, stained with 1% uranyl acetate, and images were acquired by Hitachi HT-7800 transmission electron microscopy (Hitachi New technology, japan). The experimental results are shown in fig. 3D. The results show that, compared with the control group, the B2M-3 small peptide can effectively inhibit the B2M full-length protein from inducing Abeta after pre-incubation _1-42 Forming fibrous oligomers.

As shown in FIG. 3E, the present inventors further shortened the amino acid sequence to narrow the effective binding range based on B2M-3. The truncated details of B2M-3 are shown in Table 2 below.

TABLE 2

SEQ ID NO:	Naming the name	Sequence(s)
			6	B2M-3-1	HSDLSFSKDW
7	B2M-3-2	SFYLLYYTEFTPTEK
			8	B2M-3-3	SFYLLYYTE

After the synthesis of small peptides, the inventors of the present invention truncated three small peptide sequences (B2M-3-1, B2M-3-2 and B2M-3-3) with different concentrations (1. Mu.M, 5. Mu.M, 10. Mu.M) of B2M-3 with 10. Mu.M of Abeta, respectively, using the same experimental method as described above _1-42 Preincubation followed by addition of 1. Mu.M of B2M full-length protein for thioflavin-T detection. The experimental results are shown in fig. 3F to 3H. The results show that both the B2M-3-2 and B2M-3-3 small peptide sequences can effectively inhibit the B2M induced Abeta _1-42 Oligomerizes and has a certain concentration dependence.

The results show that B2M and Abeta _1-42 The interactive amino acid region is mainly located in the third truncated range, and B2M-3 peptide, B2M-3-2 or B2M-3-3 can be used as blocking peptide to effectively inhibit B2M induced Abeta _1-42 Oligomerization, and effective inhibition of B2M-induced β -amyloid plaque formation.

_1-42 Example 6: B2M-3 blocking peptides block B2M-induced AβOligo(s) _1-42 Aggregation, thereby inhibiting the neurotoxicity of Abeta

Oligomerized aβ _1-42 Has strong neurotoxicity, and B2M promotes Abeta _1-42 Thereby further enhancing its neurotoxicity. b2M-3 blocking peptide-based inhibition of B2M-induced Abeta _1-42 Oligomerization, it is therefore necessary to investigate whether the B2M-3 blocking peptide is capable of inhibiting B2M-induced oligomerized Abeta _1-42 Is a neurotoxicity of (a).

With 10. Mu.M of B2M-3 peptide or nonsensical control peptide and 10. Mu.M of Abeta, respectively _1-42 Preincubation for 3 hours at 37℃followed by addition of 1. Mu.M full-length B2M protein for a further incubation for 12 hours, yielding two Abeta respectively _1-42 Incubation products with different degrees of oligomerization.

After 2 months old Wild Type (WT) mice were anesthetized, brain tissue was rapidly removed and placed in ice-cold and oxygenated artificial cerebrospinal fluid (ACSF) for cooling, followed by transfer to an oscillating microtome for coronal sectioning, with brain slice thickness of 400 μm. The brain pieces were incubated in 32 ℃ oxygen saturated ACSF for 1 hour, after which they were transferred to room temperature for 1 hour. Oligomerizing Aβ _1-42 The brain slices were incubated for 1.5 hours at room temperature with ACSF diluted to 200 nM. The brain slice was then transferred to the recording tank, the recording electrode was placed in the CA1 region of the Schaffer collateral-commercausal pathway and the stimulation electrode was placed in the CA3 region. Stimulation intensity was 30% of the maximum value of excitatory postsynaptic field potential (field excitatory postsynaptic potential, fEPSP), after 20 minutes of fEPSP baseline stable recording, high Frequency Stimulation (HFS) induced long-term enhancement, LTP (2 bursts of stimulation, each burst containing 100 stimulation pulses, each burst of stimulation spaced 30 seconds apart), for 60 minutes of recording.

The experimental results are shown in fig. 4A to 4B. The results show that pre-incubation with nonsensical peptide resulted in oligomerized aβ compared to brain slices incubated with small peptide alone _1-42 LTP, which significantly impairs the pathway from the CA3 region of the hippocampus to CA1 region Schaffer collateral-comosural of WT mice, was preincubated with B2M-3 blocking peptide to produce Aβ _1-42 Incubation products with different degrees of oligomerization were not clear of LTP from WT miceObvious injury effect.

In summary, A.beta.is pre-incubated with B2M-3 blocking peptide _1-42 Can inhibit B2M from promoting Abeta _1-42 Neurotoxicity resulting from oligomerization.

Example 7: B2M-3 blocking peptides can hinder B2M-induced β -amyloid plaque formation in the mouse brain

Experiments of examples 5-6 demonstrate that B2M-3 blocking peptides can inhibit B2M-induced Abeta under in vitro conditions _1-42 Oligomerization, thereby attenuating the oligomeric Abeta _1-42 Is a neurotoxicity of (a). To further verify the inhibitory effect of B2M-3 blocking peptide on B2M-induced β -amyloid plaque formation in vivo, the present inventors embedded a cannula into the hippocampal region of the brain of B2M humanized 5×fad mice by brain stereotactic technique, followed by weekly injections of 1 μl (5 μg/μl) of nonsensical peptide or B2M-3 blocking peptide, respectively, into the left and right hippocampus of the mice via the cannula for 2 months. After injection, mice were anesthetized with 5% chloral hydrate, perfused with phosphate buffer, brain tissue was taken, fixed overnight in 4% paraformaldehyde, dehydrated with 25% and 30% sucrose solution, brain tissue was embedded with OCT, frozen sections, immunofluorescent staining was performed, dye 4',6-diamidino-2-phenylindole (DAPI) -labeled nuclei, antibody 6E10 (Biolegend, cat 803016) -labeled aβ _1-42 The image was acquired by confocal laser fluorescence microscopy.

The experimental results are shown in fig. 4C to 4E. The results show that injection of B2M-3 blocking peptide significantly reduced the deposition of β -amyloid plaques in the brain compared to injection of nonsensical peptide.

In summary, the B2M-3 blocking peptide was able to effectively inhibit B2M-induced deposition of β -amyloid plaques in the brain of 5×fad mice.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Xiamen university

<120> beta 2-microglobulin blocking peptides, pharmaceutical compositions and uses thereof

<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 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 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 (10)

1. An isolated polypeptide which is a polypeptide as set forth in SEQ ID NO. 3 or a truncated fragment of a polypeptide as set forth in SEQ ID NO. 3, wherein the truncated fragment comprises a polypeptide as set forth in SEQ ID NO. 8.

2. The isolated polypeptide of claim 1, wherein the truncated fragment does not comprise the 6 histidines at the N-terminus of the polypeptide shown in SEQ ID No. 3.

3. The isolated polypeptide of claim 1 or 2, which is a polypeptide represented by any one of SEQ ID NOs 7-8 or 11-15.

4. An isolated polynucleotide encoding the isolated polypeptide of any one of claims 1 to 3.

5. A recombinant expression vector comprising the isolated polynucleotide of claim 4.

6. A transformed cell comprising the recombinant expression vector of claim 5.

7. A pharmaceutical composition comprising the isolated polypeptide of any one of claims 1 to 3.

8. The pharmaceutical composition of claim 7, further comprising one or more pharmaceutically acceptable excipients.

9. Use of an isolated polypeptide according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment or prophylaxis of alzheimer's disease.

10. Use of an isolated polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for:

inhibition of B2M-induced Abeta _1-42 Oligomerizing agents, agents inhibiting B2M-induced formation of beta-amyloid plaques or inhibiting aβ _1-42 Is a neurotoxic drug.