Polypeptide with function of regulating aggregation behavior of A beta fragment polypeptide and application of polypeptide

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a polypeptide with an A beta fragment polypeptide aggregation behavior regulation function and application of the polypeptide. A core sequence A beta 16-37 fragment in an A beta 1-42 polypeptide system forming a beta-cross-linked aggregate is used as a basis; the invention relates to a novel polypeptide fragment formed by respectively replacing loop region sequences in the fragment with BG modules for inducing beta-turning, the name of the polypeptide is Ab16BG, the sequence is KLVFFAEBGIIGLMVG, and the sequence is shown as SEQ ID No: 1 in a sequence table. According to the present invention, the aggregation process of Alzheimer's disease pathology-related polypeptide fragment Ab16-37 and Ab16BG sequences is tracked by using the external standard fluorescence spectrum, the microscopic morphology analysis, the vibration spectrum and the circular dichroism, such that the conclusion that the modification sequence uses the different aggregation path from the original sequence is obtained so as to obtain the Ab16-37 sequence stable oligomer; the research means provided by the invention is at the international frontier level, and the research results provide basic theoretical support and constructive guidance for the cross fields of biophysics, pharmacology and drug design related to the'folding disease '.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to polypeptide modification technology, and specifically to a polypeptide having the function of regulating the aggregation behavior of Aβ fragment polypeptides and its application. Background Art

[0002] Amyloid fiber structure is closely related to a variety of neurodegenerative diseases. Its characteristic β-fold cross-linked aggregate structure appears in the anatomical results of different diseases, such as Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, chorea, etc. The pathological proteins all fold abnormally to form amyloid aggregates with similar morphology under transmission electron microscopy observation. Therefore, these diseases are collectively referred to as "folding diseases". The thermodynamic stability of this β-fold cross-linked aggregate structure is extremely high and almost irreversible once formed. It is worth noting that the polypeptide oligomers that form the precursors or intermediates of β-fold cross-linked aggregates are more toxic than their polymers. Current theoretical studies believe that their structure is highly flexible and has a stronger ability to induce abnormal folding of normal protein molecules. They also have a small molecular weight and strong membrane penetration ability, and can remotely "infect" other tissues. Therefore, the study of this type of oligomer structure, especially the exploration of its formation mechanism, aggregation behavior regulation and characterization methods, is undoubtedly the basis and focus of the diagnosis and treatment of "folding diseases".

[0003] However, the stability of oligomer structure is poor, and it is difficult to directly analyze it using the instrumental methods for studying polymers. Modifying the monomer polypeptide sequence to stabilize the oligomer conformation has become a prerequisite for studying its structure. There are roughly three modification methods reported so far, specifically: destroying the hydrogen bond system of polypeptide polymers, reorganizing the arrangement of side chains, and adding turning points.

[0004] Members of the Eisenberg research group modified the short peptide sequence with the artificial amino acid Hao group, and took advantage of the lack of hydrogen bond donors in the group to prevent the extension of the hydrogen bond system and stabilize the misaligned oligomers, and analyzed a series of crystal structures. Nilsson et al. conducted a systematic study on the effects of side chain aromaticity, hydrophobic interaction and volume on the structure of aggregates, pointing out that side chains with strong hydrophobic interaction will stabilize the conformation of oligomers; Sawaya et al. concluded that the complementary arrangement of side chain groups is the main factor in stabilizing the β-barrel intermediate after analyzing the oligomer crystal structure. Pande et al. implanted D-proline residues that promote β-turning in the Αβ1-42 sequence through site-directed mutagenesis technology, obtained oligomers that are stable in solution, and analyzed the structure of the intermediate using two-dimensional nuclear magnetic resonance technology.

[0005] Screening stable oligomers and studying their aggregation mechanism provide a meaningful theoretical basis for the design of drugs for "folding diseases"; the design of such drug molecules often integrates the dual mechanism of having a high affinity for oligomers and inhibiting their further cross-linking aggregation. Peptide molecules have become excellent templates for anti-cross-linking and aggregation drugs due to their high biological affinity and mature design and modification methods. However, peptide drugs have large molecular weights and are prone to enzymatic hydrolysis, resulting in low bioavailability, which makes clinical applications difficult. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a polypeptide having the function of regulating the aggregation behavior of Aβ fragment polypeptides. 1-42 Core sequence Aβ in the peptide system 16-37 Based on the fragments, the loop (irregular link) region sequences in the fragments are replaced with BG modules that induce β-turns to form new polypeptide fragments, thereby obtaining Ab16-37 sequence-stabilized oligomers; the polypeptide provided by the present invention is expected to provide a basis for the research and development of anti-cross-linking aggregation drugs.

[0007] The present invention is achieved through the following technical solutions:

[0008] A polypeptide having the function of regulating the aggregation behavior of Aβ fragment polypeptides, named: Ab16BG, with a sequence: KLVFFAEBGIIGLMVG, as shown in SEQ ID No: 1 in the sequence table.

[0009] A further improvement of the present invention is:

[0010] An Ab16-37 sequence stable oligomer is formed by the above-mentioned polypeptides with the function of regulating the aggregation behavior of Aβ fragment polypeptides being aggregated in an antiparallel β-folding conformation.

[0011] Furthermore, a polypeptide derivative is a derivative produced by chemically modifying the above-mentioned polypeptide sequence having the function of regulating the aggregation behavior of Aβ fragment polypeptides.

[0012] A further improvement of the present invention is:

[0013] The application of the above-mentioned polypeptides, oligomers or polypeptide derivatives in the design and preparation of drugs for "folding diseases".

[0014] Furthermore, the "folding diseases" include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and chorea.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention targets the modification in the loop (irregular link) region of the non-β-ribbon structure. A BG module that induces β-turn is introduced into the loop region to form a new polypeptide fragment, and the aggregation behavior of the modified sequence is studied. It is found that the Aβ fragment polypeptide provided by the present invention can regulate the aggregation behavior and induce stable oligomers. The oligomers aggregate in an antiparallel β-folding conformation and have strong supramolecular chirality.

[0017] The present invention uses circular dichroism to measure the supramolecular chirality of polypeptide aggregates with extremely high sensitivity, and has a wide range of sample applications. It can test liquid, solid film or suspension samples, and effectively solves the sample preparation problem of poor solubility of polypeptide aggregates and changes in solubility properties during aggregation. Among them, the theoretical model of the spectrum of vibrational spectroscopy-aggregate structure has been published in the literature, which greatly simplifies the work of analyzing the aggregate structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Predict peptide conformations for PEP-FOLD;

[0019] Among them, A) Ab16-37 is a β-hairpin connected by a long loop; B) Ab16BG is a twisted β-hairpin locked by a BG turn,

[0020] like Figure 1 As shown, the PEPFOLD program predicts that the Ab16-37 monomer is a β-hairpin structure connected by long loops, while Ab16pG is a twisted β-hairpin locked by a pG turn. At the same time, the β-band of Ab16BG is longer than that of Ab16-37, suggesting that its β-hairpin conformation is more stable.

[0021] Figure 2 The aggregation kinetics profile of Ab series peptides tracked by ThT;

[0022] The initial concentration was 100 M, and the solvent was 2% thionyl chloride / pH 7 phosphate buffer (a) without additives; (b) 150 mM sodium chloride; (c) 150 mM sodium chloride + 8% dextran.

[0023] Figure 3 This is the UV CD spectrum characterization diagram of Ab series peptides;

[0024] Figure 4 TEM images of Ab series peptides at different incubation times;

[0025] Figure 5 Comparison of IR and VCD spectra of Ab1637 and Ab16BG during incubation. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with specific embodiments.

[0027] The experimental materials and reagents used in the present invention are of the highest purity that can be purchased and are used directly without treatment. The polypeptide samples were purchased from Beijing Zhongke Yaguang Biotechnology Co., Ltd., with a purity greater than 95%.

[0028] Example 1: Design of polypeptide sequence

[0029] Solid-state NMR experiments confirmed that most of the Aβ 1-42 In the peptide system, Aβ 16-37 The fragment is a core sequence, and its monomer sequence contains two β-ribbons connected by a loop region, arranged in a U shape. The monomers extend longitudinally to form fiber precursors through hydrogen bonding between β-ribbon molecules and geometrical complementary arrangement of side chains, and stack horizontally to complete the aggregation of amyloid fibers through weak forces between molecules in the loop region. In order to obtain the aggregation intermediate, this example 16-37 The loop region sequence in the fragment was replaced with the BG module that induces β-turn to obtain the polypeptide of the present invention, which was named Ab16BG.

[0030] In order to analyze the difference in aggregation behavior between the modified sequence and the original sequence, the present invention also 16-37 The loop sequence in the fragment was replaced with pG and Gp modules, involving Aβ 16-37 The fragment sequences and their modified sequences are shown in Table 1.

[0031] Table 1 Based on Aβ 16-37 Name and sequence of modified peptide

[0032]

[0033] Example 2: Characterization of Ab16BG aggregation kinetics by ThT fluorescence enhancement and comparison of sequences

[0034] The aggregation process of Ab16BG and the comparison sequence in different media was tracked with ThT as an external standard. The instrument used for fluorescence spectrum measurement was Horiba Fluoromax3, and the measurement temperature was controlled by the PELTIER component. The sample concentration was 100 μM, the incident light path of the quartz sample cell was 1 mm, and the structure was 10×1 mm with four sides transparent (Starna, Inc.). The width of the entrance and exit slits was 1 / 1 nm. The tryptophan excitation wavelength was 280, and the FRET efficiency (E) was calculated by formula (3).

[0035] E=1-I DA / I D (3)

[0036] Among them I DA is the integrated value of tryptophan fluorescence intensity in BPD from 300 to 450 nm, and I Dis the integral value of the corresponding peak of BP peptide. The FRET distance (R) is calculated by formula (4), where

[0037]

[0038] For the ThT fluorescence enhancement experiment, the sample was added to the ThT stock solution for determination, and the final concentration was 300 μM ThT and 100 μM peptide sample. The excitation wavelength was 450 nm, and the entrance and exit slit width was 1 / 1 nm.

[0039] Test results see Figure 2 .

[0040] like Figure 2 As shown in Figure 1, within the 12-hour kinetic experiment, only the Ab16-37 sequence can significantly enhance the fluorescence of ThT. The peptide monomer sequence dissolved in pure DMSO was diluted into 50 times the volume of neutral sodium chloride-free phosphate buffer and the timing was started ( Figure 2 a) Ab16-37 enhances ThT fluorescence more than other modified sequences at the beginning of incubation. This may be due to hydrophobic collapse and aggregation of polypeptide monomers when entering the aqueous phase from the organic phase. ThT binds to the aggregates and increases the fluorescence quantum yield. Within 2 hours, its fluorescence gradually decreases. Possible explanations are that the aggregates are partially disaggregated, the conformation is adjusted, or the aggregation reaction products do not bind to ThT. After 7 hours of incubation, when Ab16-37 is added to the system, ThT fluorescence increases sharply until it reaches equilibrium around 10 hours. Overall, this is consistent with Aβ 1-42 The nucleation and aggregation mechanism is consistent with that of the oligomers formed by monomeric polypeptides and then into fiber precursor seeds, which is the rate-controlling step and presents an S-curve characteristic with the subsequent growth and maturation stages.

[0041] Ab16BG also showed an S-curve characteristic in the fluorescence enhancement of ThT, reaching a peak at about 3h and then gradually weakening. Its binding degree to ThT was one order of magnitude smaller than that of Ab16-37. Ab16Gp and Ab16pG enhanced the fluorescence of ThT during the incubation time, but did not change with time. Among them, Ab16pG was the sequence with the smallest fluorescence enhancement of ThT among all sequences. This phenomenon shows that Ab16pG may only stay in the β-oligomer stage, or its aggregate structure is special and cannot continue to bind to ThT. When physiological concentrations of NaCl are added to the system, or the macromolecular crowding agent dextran is further added, Ab16-37 can still enhance the fluorescence of ThT to a degree equivalent to that without external additives, but the nucleation and growth rates are different. Nucleation is completed in about 2h, and maturity is achieved in 10h without crowding agents, while maturity can be achieved in 6h under the condition of 8% dextran. The fluorescence enhancement of ThT by other modified sequences in the presence of additives hardly changes over time, and the enhancement degree is only about 1% of that of the mature fibers of Ab16-37.

[0042] Example 3: UV CD Characterization of Ab16BG and Comparative Sequences

[0043] After the ThT tracking Ab series peptide aggregation experiment, the UV CD spectra of each system were measured. The UV CD spectra were measured on Jasco J-810, and the sample solution concentration was about 100μM. The quartz cuvette had an optical path of 1mm and was purchased from Starna, Inc. The measurement range was from 185 to 250nm, the scanning speed was 50nm / min, the bandwidth was 1nm, and the reaction time was 2s. Temperature control was completed by the built-in sensor of the PELTIER component. The final spectrum is the average of 8 scans after baseline calibration. See the spectrum Figure 3 .

[0044] like Figure 3 As shown, the spectral features are consistent with the existence of a secondary conformation of the polypeptide dominated by β-folding, among which Ab1637 gives the strongest circular dichroism signal, which is consistent with the kinetic experimental conclusion that the system has the highest aggregation degree.

[0045] Example 4: Microscopic morphology characterization of Ab16BG and comparative sequences

[0046] Figure 4The TEM images of Ab16BG and the comparison sequence are shown at 0, 2hr and 24hr incubation time. TEM images were measured on a JEM 1220 (JEOL Co. Japan) electron microscope, operating voltage 80kV, equipped with a Gatan Es1000W 11MPCCD camera. 15μl of sample solution was dripped onto the bright side of a copper mesh (400mesh, carbonized surface, Electron Microscopy Science), left to stand for 1min, blotted with the side of filter paper, then stained with 2wt% uranyl acetate solution, and dried at room temperature for testing.

[0047] like Figure 4 As shown in (a) to (c), Ab16-37 develops from amorphous aggregates to fiber precursors and finally matures into bundle-like ribbon aggregates. In (d) to (f), Ab16pG gradually develops from larger amorphous aggregates to thick ribbon aggregates with a longitudinal length of about 300nm. Ab16BG always remains in an amorphous oligomeric state during the incubation time. As for Ab16Gp, as the incubation time increases, curved fiber precursors grow from amorphous aggregates as the core. Compared with the aggregation behavior data tracked by ThT, amorphous oligomers can bind to ThT and enhance its fluorescence, but the degree is less than that of mature bundle-like aggregates generated by Ab16-37. At the same time, the aggregation of Ab16pG and Ab16Gp into curved fibers cannot significantly enhance the fluorescence of ThT.

[0048] Example 5: Infrared and vibrational circular dichroism characterization of Ab16BG and comparative sequence Ab16-37

[0049] Figure 5 The infrared and vibrational circular dichroism spectra of Ab16-37 and Ab16BG before and after incubation are compared in Figure 2. The mid-infrared absorption spectra were measured on a Bruker Vertex 80 FTIR spectrometer equipped with a DTGS detector. The resolution was 4 cm -1 , scanned 512 times. Vibrational circular dichroism spectra were measured on a Biotools ChiralIR2X instrument equipped with dual PEM and MCT detectors with a resolution of 8 cm -1 , scan 12 sections, 6000 times each section. After the peptide sample was replaced by deuterated hydrochloric acid (0.01M), 0.5 mg was dissolved in 50 μl of heavy water and packaged in a CaF2 salt tablet for determination. The optical path was 50 μm. Pure heavy water was used to replace the sample solution for baseline determination.

[0050] like Figure 5As shown, the infrared spectra of the two before and after incubation are almost unchanged, and the infrared of the amide I region has typical antiparallel β-folded conformational aggregate spectral characteristics; the spectral shape of the vibrational circular dichroism spectra of the two has not changed during the entire experiment, and both are derivative peaks from low wavenumber to high wavenumber first positive and then negative, which is consistent with the theoretical speculation of the distorted antiparallel β-folded conformational aggregation state. The difference is that the peak intensity of the vibrational circular dichroism spectrum changes greatly before and after incubation, Ab16-37 decreases from 3E-4 at the beginning of incubation to 3E-5 at 12 hours, while Ab16BG increases from 1E-4 at the beginning of incubation to 3E-4 at 12 hours. The supramolecular chirality of the vibrational circular dichroism reaction system, combined with the TEM morphology results and the ThT fluorescence enhancement dynamics data, can be inferred that the amorphous oligomers aggregate in an antiparallel β-folded conformation with strong supramolecular chirality, while the chirality of mature fibers is weak.

[0051] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide having the function of regulating the aggregation behavior of Aβ fragment polypeptides, characterized in that: The name is: Ab16BG, and the sequence is: KLVFFAEBGIIGLMVG, as shown in the sequence listing SEQ ID No:

1.

2. An Ab16-37 sequence stable oligomer, characterized in that: The polypeptide according to claim 1 is aggregated in an antiparallel b-fold conformation.

3. A polypeptide derivative, characterized in that: A derivative produced by chemically modifying the polypeptide sequence of claim 1.

4. Use of the polypeptide, oligomer, polypeptide derivative or nanogel as described in any one of claims 1 to 3 in the design and preparation of drugs for "folding diseases".

5. The use according to claim 4, characterized in that: The "folding diseases" include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, and chorea.

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