CN113045626B - Polypeptide for inhibiting amyl-beta focusing and application thereof - Google Patents
Polypeptide for inhibiting amyl-beta focusing and application thereof Download PDFInfo
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- CN113045626B CN113045626B CN202110362946.2A CN202110362946A CN113045626B CN 113045626 B CN113045626 B CN 113045626B CN 202110362946 A CN202110362946 A CN 202110362946A CN 113045626 B CN113045626 B CN 113045626B
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Abstract
The invention relates to a polypeptide for inhibiting the focusing of an amyl-beta and application thereof, which searches a polypeptide ligand P2696 with the best binding mode and affinity with a target protein in a virtual polypeptide database by means of a molecular docking virtual screening technology on the basis of an amyl-beta crystal structure by means of computer aided design, wherein the polypeptide sequence is YEKKGM. Solid phase synthesis of YEKKGM, utilizing Abeta 1‑42 Protein standard is respectively subjected to thioflavin T ThT fluorescence, surface plasmon resonance and cytotoxicity experiments, and the results show that the polypeptide P2696 is specific to Abeta 1‑42 The protein aggregation has good binding capacity, thus proving that the polypeptide designed by the invention can be used for inhibiting Abeta 1‑42 Toxicity of aggregation.
Description
Technical Field
The invention relates to a polypeptide for inhibiting amyl-beta focusing and application thereof, belonging to the field of polypeptide design and drug screening development.
Background
With the continuous development of technology in recent years, computer application is increasingly important in the field of biology, and the molecular docking virtual screening technology based on computer simulation is a research hotspot for rational design and screening of affinity peptides in recent years. The method realizes continuous butt joint of polypeptide small molecules on target protein molecule active sites by means of rapid computer operation, the polypeptides are mostly derived from prepared virtual peptide libraries, then the small molecule polypeptides which can be combined with target proteins are found through virtual butt joint with the target proteins, the mode of combining the polypeptides with the target proteins is calculated through computer software, and scoring is carried out, ligands which are well combined with the target proteins are selected according to scoring results, and after synthesis, in vitro experiment screening and verification are carried out.
The economic burden and social problems caused by alzheimer's disease (Alzheimer disease, AD) are becoming increasingly serious and one of the great challenges facing all humans in common. The excessive aggregation of the amyoid-beta structure (β -amyloid) and the excessive phosphorylation of Tau protein are considered two major hypotheses for the occurrence of AD. Many scholars believe that senile plaques formed by the deposition of aβ formed after cleavage of APP are the leading cause of AD pathogenesis, and thus preventing aβ aggregation is a promising approach to treat AD. The prior researches prove that the toxicity of the soluble Abeta oligomer is maximum, and the soluble Abeta oligomer mainly activates the glial cells to trigger inflammatory reaction and other approaches by influencing cell membrane ion channels to generate oxidative stress. Abeta exists in various forms in human body, mainly by Abeta 1-42 And Aβ 1-40 Mainly, wherein Abeta 1-42 Because of its greater toxicity and greater susceptibility to aggregation, it is becoming an important point of research.
Disclosure of Invention
The invention searches the polypeptide ligand P2696 with the best binding mode and affinity with the target protein in the virtual polypeptide database by means of the computer aided design and based on the amyl-beta crystal structure through the molecular docking virtual screening technology, and the polypeptide sequence is YEKKGM. Solid phase synthesis of YEKKGM, utilizing Abeta 1-42 Protein standard is respectively subjected to thioflavin T (ThT) ThT fluorescence, surface plasmon resonance (Surface Plasmon Resonance, SPR) and cytotoxicity experiments, and the results show that the polypeptide P2696 is specific to Abeta 1-42 The protein aggregation has good binding capacity, thus proving that the polypeptide designed by the invention can be used for inhibiting Abeta 1-42 Toxicity of aggregation.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a polypeptide sequence P2696 for inhibiting the focusing of the amyoid-beta, wherein the polypeptide sequence P2696 is YEKKGM.
The polypeptide sequence P2696 takes the polypeptide sequence P2696 as a core, and the corresponding modification is carried out on the polypeptide sequence P2696; the modifying material comprises nano material, fluorescent material, enzymes and biotin.
The polypeptide sequence P2696 is applied to inhibiting the toxicity of the amyl-beta structure.
The polypeptide sequence P2696 is applied to detection of the amyoid-beta structure.
Application of the polypeptide sequence P2696 in preparation of medicines with an amyoid-beta structure.
The invention has the beneficial effects that:
1. the invention obtains a polypeptide sequence P2696 specifically combined with beta-amyloid based on an amyloid-beta crystal structure (PDB ID: 6 SHS) through a molecular docking virtual screening technology, and the polypeptide sequence is YEKKGM. Solid phase synthesis of polypeptide and affinity identification with amyl-beta, P2696 sequence and Abeta 1-42 Equilibrium dissociation constant K for interactions between proteins D 2.693 ×10 -6 M, indicates better affinity.
2. The P2696 polypeptide sequence has no toxicity to PC12 cells to a certain extent and is also toxic to Abeta 1-42 The PC12 cell injury caused by the protein has better protection effect. The polypeptide designed and synthesized by the invention has the advantages of convenience, high efficiency and low cost.
Drawings
FIG. 1 shows the sequence P2696 and Abeta 1-42 And displaying the docking result of the protein.
FIG. 2 shows inhibition of Abeta by P2696 sequence 1-42 Graph of the effect of protein aggregation.
Wherein Abeta represents that it contains Abeta only 1-42 A sample; p812696, P2696 and Abeta 1-42 Is a sample of the mixture.
FIG. 3 shows the sequence P2696 and Abeta 1-42 LSPR affinity assay results for proteins.
Wherein the ordinate represents the signal value detected by the sensor; the abscissa represents the time of interaction of the sample in the sensor.
FIG. 4 shows the effect of the P2696 sequence itself on PC12 cell viability.
Wherein the abscissa indicates the polypeptide concentration and the ordinate indicates the cell viability.
FIG. 5 shows P2696 sequences and Abeta at different concentrations 1-42 Protein co-incubation followed by incubation with Abeta 1-42 Effects of protein toxicity.
Description of the embodiments
The following describes the embodiments of the present invention in further detail with reference to examples.
Example 1 molecular docking and screening of virtual peptide libraries
1、Aβ 1-42 Preparation of protein Crystal Structure
Ass by means of computer aided design software 1-42 The crystal structure of the protein (PDB ID: 6 SHS) was analyzed, and amino acid residues 16 to 23 were selected as a designated docking region for molecular docking.
2. Design of virtual polypeptide library
The invention adopts a mode of extending amino acid residues one by one, firstly, the amino acid libraries with the highest scores are butted with the structure of the target protein one by one, the optimal amino acid residues are selected as cores according to the butting condition, and then the corresponding amino acid numbers are sequentially increased until the optimal butting result is achieved. The peptide sequences generated by the virtual polypeptide library are preferably amino acid residues 3-12.
3. Evaluation of docking results
Respectively calculating the free energy of the combination of the polypeptide and the protein, comprehensively evaluating the mechanical parameters such as hydrogen chain, van der Waals force and the like, judging the screening result, and screening to obtain P2696, wherein the polypeptide sequence is YEKKGM, and the polypeptide sequence is connected with Abeta 1-42 The results of the interaction of the protein docking are shown in FIG. 1.
Example 2P 2696 sequence and Abeta 1-42 Identification of fluorescent intensity of protein binding
1. First is Aβ 1-42 Preparation of monomers, Aβ 1-42 1mg/mL of the solution was dissolved in Hexafluoroisopropanol (HFIP), allowed to stand for 1 hour, and then sonicated for 10 minutes after sufficient dissolution, after which the HFIP was completely volatilized in a fume hood, stored in a-80℃refrigerator, and dissolved in PBS buffer (containing 100mM PB,10mM NaCl,pH 7.4) when used.
2. Dissolving the obtained P2696 sequenceIn PBS buffer (containing 100mM PB,10mM NaCl,pH 7.4). Will A beta at the time of experiment 1-42 Mixing the monomer solution and the P2696 solution, and Aβ 1-42 The final concentration was 25. Mu.M and the final concentration of P2696 was 50. Mu.M.
3. The mixed samples were incubated in an incubator at 37℃for 24 hours, and 20. Mu.L of the samples were taken. After dilution 20-fold with the ThT dye solution (25. Mu.M of ThT,25mM PB) the intensity of the ThT fluorescence was measured at an excitation wavelength of 440nm and an emission wavelength of 480 nm. Will contain Abeta only 1-42 The fluorescence intensity of the sample was set to 100% and normalized (see FIG. 2).
The results indicate that the P2696 sequence is specific to the synthesized Abeta 1-42 The in vitro aggregation of the protein has good inhibition effect.
Example 3P 2696 sequence and Abeta 1-42 Affinity identification of proteins
1. The appropriate pH was determined as the coupling condition. At the step of binding Abeta 1-42 Before the protein is fixed on the CM5 chip, proper pH of buffer solution is needed to be screened, so that the ligand is enriched near the surface of the CM5 chip through electrostatic adsorption, and a better coupling effect is achieved. Dilute Abeta with sodium acetate solutions of pH 5.5, 5.0, 4.5, 4.0, respectively 1-42 The sample was 50. Mu.g/mL. Loading time 180s,50mM NaOH was used as the wash solution and pH4.5 was used as the coupling condition as determined from the results.
2. Ligand coupling. Immobilization of Abeta by amino direct coupling 1-42 Proteins to CM5 chip surface. Flow Cell 1 was selected as the reference channel and Flow Cell 2 was selected as the sample channel. The coupling mode was selected as Specify contact time. Then according to the sample position diagram, HBS-EP buffer solution, EDC/NHS solution and Abeta dissolved in sodium acetate with pH of 4.5 are correspondingly put in 1-42 The solution and the ethanolamine solution are added into a sample tray, the buffer solution is checked, and the method and the result file are stored; clicking Run to start formal coupling.
3. Measurement of affinity. Selecting Run kinetic/Affinity Assay click kinetic/Affinity to set relevant experimental parameters, selecting Flow Cell as 1,2 and chip type as CM5. Startup's solution is HBS-EP buffer, binding time 120s, dissociation time 120s, regeneration solution is 0.25% Sodium Dodecyl Sulfate (SDS), stabilization time 30s. Filling in a sample named P2696, dissolving the sample in HBS-EP buffer, diluting to obtain polypeptide solutions with the concentration of 25 mu M, 12.5 mu M, 6.25 mu M, 3.125 mu M, 1.56 mu M and 0.78 mu M, setting a sample with zero concentration and a minimum concentration, placing the sample according to the requirement, checking the buffer, storing the file, and clicking Run to start the experiment. Ligand coupling and affinity assays were all performed using Biacore X100 Control Software.
4. And (5) data processing. After the experiment is finished, the results are analyzed by using Evaluation software, and the background signal of the Flow Cell 1 is subtracted by using the Flow Cell 2 to obtain experimental results. Fitting was performed using a 1:1 binding fit (see FIG. 3). The concentration of the solution corresponding to each curve in the figure gradually decreases from top to bottom.
The results show that as the concentration of P2696 increases, the binding capacity is greater, the response time is fast, and the P2696 sequence is specific to the synthesized Abeta 1-42 The protein has better affinity binding, and the P2696 sequence is used for synthesizing Abeta 1-42 The protein has better affinity binding, and the equilibrium dissociation constant K of the interaction between the protein and the protein D 2.693 ×10 -6 M。
EXAMPLE 4 toxicity identification of P2696 polypeptide
1. Polypeptide sample preparation. The P2696 polypeptide was dissolved in DMEM complete medium (DMEM high-sugar medium containing 10% fetal calf serum, 4.5g/L glucose in high-sugar medium, L-glutamine without sodium pyruvate) to prepare polypeptide solutions of different concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200. Mu.M) for the next experiment.
2. Cell plating. Selecting PC12 cells in growth log phase, washing twice with PBS, adding 1-2mL trypsin, digesting at 37deg.C for 1min, tapping the side of culture flask, observing cell state under inverted microscope, adding DMEM complete culture medium to stop digestion when cell gap becomes large and round, transferring the gently tapped cells into centrifuge tube, centrifuging at 1000r/min for 5 min, discarding supernatant, adding new DMEM complete culture medium, counting with cell counting plate, diluting to density of 5×10 4 personal/mLIs used at 100. Mu.L/well (5X 10) 3 Individual/well) of the plate.
3. The CCK-8 method detects the cell viability. After the cells were attached, polypeptide solutions of different concentrations were added, 5 duplicate wells per concentration, as dosing group, as control group with medium solution alone, and wells with 100 μl of medium and 10 μl of CCK-8 solution but without cells added were used as blank group. After incubation at 37℃for 24h, 10. Mu.L of CCK-8 solution was added to each well, and the incubation was continued in a cell incubator for 2 hours, and absorbance at 450nm was measured. Cell viability= (dosing group-blank)/(control group-blank) ×100% (see fig. 4).
The result shows that with the increase of the concentration of the polypeptide P2696, the P2696 has certain toxicity to PC12 cells at higher concentration, the effect is better at low concentration, and the P2696 sequence has no toxicity to PC12 cells to a certain extent.
EXAMPLE 5 inhibition of Abeta by P2696 polypeptide 1-42 Identification of toxicity
1. Sample preparation. By preparing Abeta from the precursor 1-42 The monomer and the P2696 polypeptide are respectively dissolved in a DMEM complete culture medium, and the final concentration is Abeta 1-42 A25. Mu.M, P2696 polypeptide sample was incubated at 37℃for 72h.
2. Cell plating. Selecting PC12 cells in growth log phase, washing twice with PBS, adding 1-2mL trypsin, digesting at 37deg.C for 1min, tapping the side of culture flask, observing cell state under inverted microscope, adding DMEM complete culture medium to stop digestion when cell gap becomes large and rounding, transferring the cells to centrifuge tube, centrifuging at 1000r/min for 5 min, discarding supernatant, adding new DMEM complete culture medium, counting with cell counting plate, and diluting to density of 5×10 4 Each/mL of the cell suspension was used at 100. Mu.L/well (5X 10 3 Individual/well) of the plate.
3. The CCK-8 method detects the cell viability. After the cells adhere to the wall, add the solution containing only Abeta 1-42 Monomeric sample, and Abeta in the presence of P2696 polypeptide inhibitor 1-42 Monomer sample (Aβ) 1-42 The combined volumes of monomer and P2696 polypeptide such as fig. 5), 5 wells per concentration, are the dosing group; only adding culture mediumAs a control, 100. Mu.L of medium and 10. Mu.L of CCK-8 solution were added, and wells without cells were used as a blank, and after incubation at 37℃for 24 hours, 10. Mu.L of CCK-8 solution was added to each well, and incubation was continued in a cell incubator for 2 hours, and absorbance at 450nm was measured. Cell viability= (dosing group-blank)/(control group-blank) ×100% (see fig. 5, a25 indicates aβ concentration of 25 μm).
The results show that Abeta 1-42 has a larger effect on the activity of PC12 cells, and after P2696 and Abeta 1-42 are incubated together, the activity of the cells is increased, and the cell is increased in Abeta: the highest peak is reached when the polypeptide is 1:4, and the P2696 sequence pair Abeta 1-42 The PC12 cell injury has better protection effect.
Sequence listing
<110> animal immunology important laboratory of agricultural academy of sciences in Henan province
<120> polypeptide for inhibiting amyl-beta focusing and application thereof
<130> amyloid-beta
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial sequence ()
<400> 1
Tyr Glu Lys Lys Gly Met
1 5
Claims (2)
1. A polypeptide for inhibiting the focusing of amyloid-beta, which is characterized in that the amino acid sequence of the polypeptide is YEKKGM.
2. Use of the polypeptide of claim 1 for the preparation of a medicament for inhibiting toxicity of an amyl-beta structure.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104277105A (en) * | 2013-07-12 | 2015-01-14 | 国家纳米科学中心 | Polypeptide inhibitor for inhibiting aggregation and toxicity of beta amyloid protein and application of polypeptide inhibitor |
| WO2017088711A1 (en) * | 2015-11-25 | 2017-06-01 | 中国科学院过程工程研究所 | Polypeptide binding to a plurality of amyloid monomers and aggregates, and use thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104277105A (en) * | 2013-07-12 | 2015-01-14 | 国家纳米科学中心 | Polypeptide inhibitor for inhibiting aggregation and toxicity of beta amyloid protein and application of polypeptide inhibitor |
| WO2017088711A1 (en) * | 2015-11-25 | 2017-06-01 | 中国科学院过程工程研究所 | Polypeptide binding to a plurality of amyloid monomers and aggregates, and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| β-淀粉样蛋白清除障碍在阿尔兹海默症发病中的作用;陈紫晗等;《生理科学进展》;第50卷(第2期);149-152 * |
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