Polypeptide with PINK1 kinase agonist activity, polypeptide mixture and application thereof

Abstract

The invention belongs to the field of biochemistry, and particularly relates to a polypeptide with PINK1 kinase agonistic activity and application thereof. According to the technical scheme, the polypeptide mixture is composed of a polypeptide 2 and a polypeptide 3, the amino acid sequence of the polypeptide 2 is shown as SEQ ID NO: 2, and the amino acid sequence of the polypeptide 3 is shown as SEQ ID NO: 3. Four polypeptide compounds are separated and identified from the earthworm extract, have PINK1 agonist activity, have no obvious cytotoxicity, can rescue MPP + damaged nerve cells, and have the prospect of preparing the anti-Parkinson's disease medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to a polypeptide, a polypeptide mixture having PINK1 kinase agonist activity, and their applications. Background Art

[0002] Parkinson's disease (PD) is the second most common chronic neurodegenerative disease of the central nervous system globally. The pathogenesis of PD is complex and has not been fully elucidated yet. The existing anti-PD drugs are mainly divided into two categories: drugs that affect dopaminergic neurons and anticholinergic drugs. Among them, the representative drug that affects dopaminergic neurons is levodopa, which can be catalyzed by dopa decarboxylase in the brain and converted into dopamine to supplement dopamine and produce therapeutic effects. The anticholinergic drug is represented by trihexyphenidyl, which can block cholinergic receptors in the striatum and inhibit the excitability of cholinergic nerves. In addition, it can also inhibit the reuptake of dopamine in the synaptic cleft and enhance the function of dopaminergic neurons, thereby achieving an antispasmodic effect. However, these drugs cannot fundamentally improve the degenerative process of dopaminergic neurons, and their clinical applications are limited. Therefore, searching for anti-PD compounds with novel structures, high efficiency, and low side effects has become an important research direction in the field of neurodegenerative diseases.

[0003] PINK1 is a serine / threonine protein kinase located in mitochondria. Mutations in its encoding gene lead to a decrease or loss of PINK1 kinase activity, causing early-onset PD. PINK1 plays a key role in clearing damaged mitochondria and maintaining the homeostasis of dopaminergic neurons by mediating mitochondrial quality control and mitophagy processes to decompose damaged mitochondria and remove them from the cell, and is a potential target for anti-Parkinson's drugs.

[0004] Therefore, if a compound with PINK1 agonist activity can be provided, it is expected to develop new anti-Parkinson's drugs. Summary of the Invention

[0005] The object of the present invention is to provide a polypeptide, a polypeptide mixture having PINK1 kinase agonist activity, and their applications.

[0006] To achieve the above object of the invention, the technical solution adopted by the present invention is: a polypeptide mixture, which is composed of polypeptide 2 and polypeptide 3. The amino acid sequence of polypeptide 2 is as shown in SEQ ID NO: 2, and the amino acid sequence of polypeptide 3 is as shown in SEQ ID NO: 3.

[0007] Correspondingly, a polypeptide mixture comprising polypeptide 2 and / or polypeptide 3, wherein the amino acid sequence of polypeptide 2 is as shown in SEQ ID NO: 2, and the amino acid sequence of polypeptide 3 is as shown in SEQ ID NO: 3.

[0008] Preferably, the polypeptide mixture comprises polypeptide 1 and / or polypeptide 4, wherein the amino acid sequence of polypeptide 1 is as shown in SEQ ID NO: 1, and the amino acid sequence of polypeptide 4 is as shown in SEQ ID NO: 4.

[0009] Correspondingly, the use of the polypeptide mixture in enhancing PINK1 agonist activity for non-diagnostic or non-therapeutic purposes.

[0010] Correspondingly, the use of polypeptide 2 in enhancing PINK1 agonist activity for non-diagnostic or non-therapeutic purposes, wherein the amino acid sequence of polypeptide 2 is as shown in SEQ ID NO: 2.

[0011] Correspondingly, the use of polypeptide 3 in enhancing PINK1 agonist activity for non-diagnostic or non-therapeutic purposes, wherein the amino acid sequence of polypeptide 3 is as shown in SEQ ID NO: 3.

[0012] Correspondingly, the use of polypeptide 4 in enhancing PINK1 agonist activity for non-diagnostic or non-therapeutic purposes, wherein the amino acid sequence of polypeptide 4 is as shown in SEQ ID NO: 4.

[0013] Correspondingly, an anti-Parkinson's disease drug prepared using the polypeptide mixture.

[0014] Correspondingly, an anti-Parkinson's disease drug prepared using polypeptide 2 or polypeptide 3 or polypeptide 4, wherein the amino acid sequence of polypeptide 2 is as shown in SEQ ID NO: 2, the amino acid sequence of polypeptide 3 is as shown in SEQ ID NO: 3, and the amino acid sequence of polypeptide 4 is as shown in SEQ ID NO: 4.

[0015] The present invention has the following beneficial effects: Four polypeptide compounds were isolated and identified from earthworm extracts in the present invention, all of which have PINK1 agonist activity, no obvious cytotoxicity, and can rescue MPP + -damaged nerve cells, and can be used alone or in combination to prepare anti-Parkinson's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the LC-MS / MS spectrum of polypeptide 1;

[0017] Figure 2 is the LC-MS / MS spectrum of polypeptide 2;

[0018] Figure 3 is the LC-MS / MS spectrum of polypeptide 3;

[0019] Figure 4 LC-MS / MS spectrum of polypeptide 4;

[0020] Figure 5 Schematic diagram of the effects of polypeptides 1-4 on the viability of SH-SY5Y cells;

[0021] Figure 6 For MPP at different concentrations + Schematic diagram of the effect on the viability of SH-SY5Y cells;

[0022] Figure 7 For polypeptides 1-4 on MPP + Schematic diagram of the effect on the viability of MPP-damaged SH-SY5Y cells;

[0023] Figure 8 For polypeptides 2 and 3 on MPP + Schematic diagram of the effect on the morphology of MPP-damaged SH-SY5Y cells;

[0024] Figure 9 For polypeptides 2 and 3 on PINK1 B9 Schematic diagram of the effect on the lifespan of Drosophila;

[0025] Figure 10 For polypeptides 2 and 3 on PINK1 B9 Schematic diagram of the effect on the locomotor ability of Drosophila;

[0026] Figure 11 For polypeptides 2 and 3 on PINK1 B9 Schematic diagram of the effect on the ATP content in Drosophila;

[0027] Figure 12 For polypeptides 2 and 3 on PINK1 B9 Schematic diagram of the effect on the dopamine content in Drosophila. Detailed implementation manners

[0028] Four polypeptides (polypeptides 1-4) were screened from earthworms in the present invention, and it was first discovered that these four polypeptides have certain PINK1 agonist activities, have neuroprotective effects, can alleviate apoptosis caused by mitochondrial damage, and are expected to be used in the preparation and development of anti-Parkinson drugs.

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all averages obtained after at least 3 repetitions, and all repetitions obtained are valid data.

[0030] Example 1: Isolation and Purification of Polypeptide Components

[0031] 1. Isolation and Purification

[0032] Guided by the PINK1 enzyme activity assay, four polypeptides with potential anti-Parkinson's activity were isolated and purified from earthworm extracts. The specific isolation method is as follows: The dried earthworms were crushed and refluxed with 0.9% sodium chloride solution (1×10 L) at 100 °C for 1 h. After filtration, 0.9% sodium chloride solution (1×8 L) was added and the second extraction was carried out at 100 °C for 30 min under reflux. The filtrates were concentrated together to obtain the crude extract.

[0033] The crude extract was dissolved in 50% methanol and centrifuged to obtain the supernatant. The supernatant was loaded onto a Sephadex LH-20 gel filtration column (200 cm × 2.5 cm) and eluted with 50% methanol solution, monitored by thin layer chromatography (TLC). The fractions containing the same components were combined and their cell activities were evaluated using the CCK-8 assay. Then, elution was carried out with ultrapure water (A) and methanol (B) as the mobile phase, and the fraction with higher neuroprotective activity was further purified using an RP-C18 column. The eluate was collected.

[0034] 2. Structure Identification

[0035] Use a Q Exactive TM hybrid quadrupole-orbitrap TM mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) to perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis on the eluate components. After reduction with 10 mmol / L dithiothreitol in a 55 °C water bath for 30 min, 15 mmol / L iodoacetamide was added and the reaction was carried out in the dark at room temperature for 0.5 h, followed by LC-MS / MS analysis. The mobile phase was solvent A (0.1% (v / v) formic acid (FA) in ultrapure water) and solvent B (0.1% (v / v) FA in acetonitrile). A total of 4 components (polypeptide 1, polypeptide 2, polypeptide 3, and polypeptide 4) were separated, and their LC-MS / MS spectra are shown as Figures 1-4 shown. The amino acid sequences of the 4 polypeptides were identified as shown in Table 1.

[0036] Table 1 Comparison Table of Sequencing Results of Each Polypeptide

[0037] Polypeptide Amino acid sequence (single-letter abbreviation) Sequence number Polypeptide 1 ILLIILI SEQ ID NO:1 Polypeptide 2 GYSFTTTAER SEQ ID NO:2 Polypeptide 3 AVFPSIVGR SEQ ID NO:3 Polypeptide 4 AGFAGDDAPR SEQ ID NO:4

[0038] 3. Test for PINK1 Agonist Activity

[0039] Ubiquitin is an effective substrate of PINK1. Under the catalysis of PINK1, ubiquitin is phosphorylated while generating ADP. ADP can react with the luminol-modified phosphate group to form luminol-modified ATP, and then the residual amount of ATP in the reaction solution is measured by chemiluminescence, so as to calculate the PINK1 agonist activity of the polypeptide sample.

[0040] The specific operation method is as follows: The enzymatic reaction is carried out on a white 96-well plate, and the buffer solution is set as: 50 mM Tris-HCl (pH = 7.5), 0.1 mM EDTA (metal chelator), 10 mM MgCl 2 (catalyst) and 4% 2-hydroxy-1-ethanethiol (to prevent the oxidation of disulfide bonds and protect the protein from being damaged). Add 10 μL of polypeptide sample, 10 μL of PINK1 (6.8 μM), 10 μL of ubiquitin (0.002 pg / ml), and 20 μL of ATP (10 μM) to the buffer solution, and incubate at 25 °C for 10 min.

[0041] Set up a drug control group: The rest is the same, and 10 μL of resveratrol is used to replace the polypeptide sample.

[0042] After the reaction is completed, use Kinase-lumi TM Luminescent kinase detection kit to detect the ATP consumption, and calculate the PINK1 agonist rate through the following formula.

[0043] PINK1 agonist rate (%) = [1 - (A - B) / (C - D)] × 100%. Where, A is the absorbance of the sample group composed of the sample (polypeptide or resveratrol), PINK1, ubiquitin and ATP; B is the absorbance of the sample control group composed of the sample, PINK1 and ubiquitin (lacking ATP); C is the absorbance of the control group composed of PINK1, ubiquitin and ATP (lacking the sample); D is the absorbance of the blank control group composed of PINK1 and ubiquitin. Use GraphPad Prism software to calculate the EC 50 of each polypeptide. The results are shown in Table 2, and ND indicates not detected.

[0044] Table 2 PINK1 agonist activities of each component

[0045] Sample Stimulation rate (%) <![CDATA[EC 50 (μM)]]> Polypeptide 1 17.16±0.55 ND Polypeptide 2 52.46±0.96 76.77±1.3 Polypeptide 3 50.35±0.72 88.50±0.97 Polypeptide 4 33.45±0.45 ND Resveratrol 91.45±0.34 13.75±0.58

[0046] The results show that the 4 screened polypeptides all have certain PINK1 agonist activities, and polypeptides 2 and 3 have good PINK1 agonist activities.

[0047] Example 2: Toxicity test of polypeptides on nerve cells

[0048] The SH-SY5Y cell suspension is prepared according to 1×10 4Inoculate at a density of cells / well into a 96-well plate, and add 100 μL of complete medium (DMEM / F12 medium containing 1% streptomycin-penicillin double antibody and 10% fetal bovine serum FBS) to each well. Incubate in a cell culture incubator (37 °C, 5% CO 2 ) for 24 h. Subsequently, inoculate SH-SHY5Y cells into a 96-well plate at a density of 1×10 4 cells / well, and continue to incubate for 24 h under the same medium and culture conditions. Subsequently, aspirate the initial culture, and place samples of the 4 polypeptides obtained in Example 1 at different concentrations (10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 400 μM) (dissolve the polypeptide pure product in DMSO to prepare a stock solution, pipette different volumes of the stock solution and dissolve it in DMEM / F12 medium to prepare polypeptide samples at different concentrations, with the DMSO dosage < 10%) into the wells, and incubate at 37 °C for 24 h. Finally, add 10 μL of CCK-8 solution to each well, and after incubating at 37 °C for 1 h, read the absorbance of each well. The results are as Figure 5 shown. The results show that at a concentration of 100 μM, the 4 polypeptides have no obvious toxicity to nerve cells and have the potential to be developed into drugs.

[0049] The results are as Figure 5 shown. The results show that at a concentration of 100 μM, the 4 polypeptides have no obvious toxicity to nerve cells and have the potential to be developed into drugs.

[0050] Example 3: Effect of polypeptides on MPP + -induced SH-SY5Y cell injury

[0051] 1. Inoculate the SH-SY5Y cell suspension into a 96-well plate at a density of 1×10 4 cells / well, and add 100 μL of complete medium (DMEM / F12 medium containing 1% streptomycin-penicillin double antibody and 10% fetal bovine serum FBS) to each well. Incubate in a cell culture incubator (37 °C, 5% CO 2 ) for 24 h. Subsequently, inoculate SH-SHY5Y cells into a 96-well plate at a density of 1×10 4 cells / well, and continue to incubate for 24 h under the same medium and culture conditions. Subsequently, aspirate the initial culture, and place samples of the 4 polypeptides obtained in Example 1 at different concentrations (10 μM, 25 μM, 50 μM, 100 μM, and 150 μM) (prepared in the same way as in Example 2) into the wells, and incubate at 37 °C for 2 h. Then add MPP +Cells were treated with (1-methyl-4-phenylpyridine, 2 mM) for 24 h. Finally, 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 1 h. Rasagiline in equal amount was used to replace the polypeptide as the positive control group. After the experiment, the 96-well plate was removed, and the absorbance at 450 nm was measured with a microplate reader. A positive control group was set up, that is, only rasagiline was added, and cells were treated with different concentrations of MPP + for 24 h; a blank control group was set up, without adding polypeptide or rasagiline, and without using MPP + treatment, and the other conditions were the same. The results are as Figure 6 , 7 and Table 3 show

[0052] Table 3 Protective effects of each component on nerve cells

[0053]

[0054]

[0055] Figure 6 for the effect of MPP + on the survival rate of SH-SY5Y cells. The results showed that: at a concentration of 2 mM and above of MPP + , the cell survival rate decreased significantly Figure 7 for the protective effects of the polypeptide and rasagiline on cells affected by 2 mM of MPP + . Among them, Figure 7 the first item "--" on the abscissa is the blank control group, indicating that neither MPP + nor polypeptide or other drugs were used; the second item "-+" indicates the addition of 2 mM of MPP + , without using polypeptide or other drugs. The results showed that: after treatment with 4 polypeptides, the cell survival rate could be increased to a certain extent, delaying the neurotoxic effect of MPP + , having a neuroprotective effect, being able to relieve apoptosis caused by mitochondrial damage, and having the potential to be used in the preparation and development of anti-PD drugs

[0056] 2. Select the groups corresponding to polypeptides 2 and 3 with the most obvious cell protection effect, observe the morphological changes of the cells in each group obtained after the treatment in step 1 with a microscope, and perform FDa and PI staining on the cells in each group, and detect the morphological changes of the cells with a fluorescence microscope. The results are as Figure 8 shown

[0057] The results showed that: as Figure 8 shown in A, for SH-SY5Y cells treated with polypeptide 2 and polypeptide 3 respectively, the phenomenon of cell shrinkage and rounding was alleviated. As Figure 8As shown in Figures B and 8C, after treatment with polypeptides 2 and 3 respectively, the number of living cells increased and the number of apoptotic cells decreased. This result further demonstrated that polypeptides 2 and 3 had a protective effect on MPP + -induced SH-SY5Y cells.

[0058] Example 4: Neuroprotective effects of polypeptides 2 and 3 on PINK1 B9 in Drosophila

[0059] Polypeptides 2 and 3 were selected for this example.

[0060] 1. Effects of polypeptides 2 and 3 on the lifespan of PINK1 B9 Drosophila. The test method was as follows: Newly hatched WT male flies and PINK1 B9 male flies were collected simultaneously and divided into 4 groups, with 4 bottles in each group and 40 flies in each bottle. The first group was WT male flies, fed with 4-24 formula instant Drosophila medium. The second group was PINK1 B9 male flies, also fed with 4-24 instant Drosophila medium. The third group was PINK1 B9 male flies, with 0.1 mM polypeptide added to the 4-24 instant Drosophila medium. The last group was PINK1 B9 male flies, with 4-24 instant Drosophila medium and 1 mM resveratrol added. Fresh medium was replaced every 2 - 3 days, and the number of flies was recorded daily. The results were as Figure 9 shown. The results showed that: Compared with WT, the lifespan of PINK1 B9 Drosophila was significantly shortened. However, polypeptide 2 (0.1 mM), polypeptide 3 (0.1 mM), and resveratrol (1 mM) could significantly extend the lifespan of PINK1 B9 Drosophila, by 13.1%, 6.6%, and 23.5% respectively.

[0061] 2. Effects of polypeptides 2 and 3 on the motor ability of PINK1 B9 Drosophila. The test method was as follows: Drosophila were anesthetized with carbon dioxide (each experimental group consisted of 30 flies). Subsequently, the flies were transferred to a vertical transparent glass tube (15 × 1.5 cm) and incubated in an incubator at 25 °C for 30 minutes. The glass was gently tapped to ensure that the flies landed on the bottom of the glass bottle, and the movement of the flies was recorded every 5 minutes using an infrared behavior recorder for at least 1 hour.

[0062] The results were as Figure 10 shown. The results showed that: Compared with the wild type (WT), the climbing ability of PINK1 B9 Drosophila was significantly reduced, being 42.13% of WT at 6 days old, 36.77% of WT at 12 days old, and 32.70% of WT at 18 days old. Adding 0.1 mM polypeptide 2 significantly improved the climbing ability of PINK1 B9The motor abilities of Drosophila melanogaster, and the motor abilities at three age stages were increased to 68.52%, 60.13%, and 54.79% of that of the WT, respectively. Similarly, the treatment with 0.1 mM of polypeptide 3 could also significantly improve PINK1 B9 The motor abilities of Drosophila melanogaster, and the percentages at three age stages reached 59.42%, 54.81%, and 50.7%, respectively.

[0063] Example 5: Effects of polypeptides 2 and 3 on ATP and dopamine contents

[0064] Polypeptides 2 and 3 were selected for this example.

[0065] 1. Effects on ATP content. The test method was as follows: The Drosophila melanogaster of different groups in Example 4 were transferred into EP tubes and placed in liquid nitrogen for freezing, and the thoraces and heads of the Drosophila melanogaster were separated. The thoraces of 5 Drosophila melanogaster were placed in 50 μL of lysis buffer and homogenized on ice, and then centrifuged at 4 °C and 12,000 g for 15 minutes. The ATP level in the thoracic tissues of Drosophila melanogaster was quantitatively determined by using the luciferin-luciferase system and an ATP Assay Kit.

[0066] The results are as Figure 11 shown. The results showed that: The ATP content of PINK1 B9 Drosophila melanogaster decreased significantly with age. After treatment with 0.1 mM of polypeptide 2 or 3, the ATP level increased significantly at 6, 12, and 18 days.

[0067] 2. Effects on dopamine content. The test method was as follows: The heads of 30 Drosophila melanogaster obtained in the step of measuring the ATP content were collected and placed in a 1.5 mL EP tube, and 50 μL of citric acid acetate buffer (50 mM) was added. Homogenize on ice, centrifuge at 4 °C and 12,000 g for 15 minutes. Subsequently, the dopamine content was measured by using an enzyme-linked immunosorbent assay (ELISA) kit.

[0068] The results are as Figure 12 shown. The results showed that: Compared with WT Drosophila melanogaster, the dopamine content of PINK1 B9 Drosophila melanogaster decreased significantly and decreased gradually with age. After treatment with 0.1 mM of polypeptide 2 or 3, the dopamine level increased significantly on the 6th, 12th, and 18th days, respectively.

[0069] The above-described embodiments are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide mixture, characterized in that: The polypeptide mixture consists of polypeptide 2 and polypeptide 3. The amino acid sequence of polypeptide 2 is shown in SEQ ID NO: 2, and the amino acid sequence of polypeptide 3 is shown in SEQ ID NO:

3.

2. A polypeptide mixture, characterized in that: The polypeptide mixture includes polypeptide 2 and / or polypeptide 3, the amino acid sequence of polypeptide 2 is shown as SEQ ID NO: 2, and the amino acid sequence of polypeptide 3 is shown as SEQ ID NO:

3.

3. The polypeptide mixture according to claim 1, characterized in that: The polypeptide mixture includes polypeptide 1 and / or polypeptide 4. The amino acid sequence of polypeptide 1 is shown in SEQ ID NO: 1, and the amino acid sequence of polypeptide 4 is shown in SEQ ID NO:

4.

4. Use of the polypeptide mixture according to any one of claims 1 to 3 in non-diagnostic or therapeutic applications for enhancing PINK1 agonist activity.

5. A non-diagnostic or therapeutic use of polypeptide 2 for enhancing PINK1 agonist activity, wherein the amino acid sequence of polypeptide 2 is shown in SEQ ID NO:

2.

6. A non-diagnostic or therapeutic use of polypeptide 3 for enhancing PINK1 agonist activity, wherein the amino acid sequence of polypeptide 3 is shown in SEQ ID NO:

3.

7. A non-diagnostic or therapeutic use of polypeptide 4 for increasing PINK1 agonist activity, wherein the amino acid sequence of polypeptide 4 is shown in SEQ ID NO:

4.

8. An anti-Parkinson's disease drug prepared using the polypeptide mixture according to any one of claims 1 to 3.

9. An anti-Parkinson's disease drug prepared using polypeptide 2, polypeptide 3 or polypeptide 4, wherein the amino acid sequence of polypeptide 2 is shown in SEQ ID NO: 2, the amino acid sequence of polypeptide 3 is shown in SEQ ID NO: 3, and the amino acid sequence of polypeptide 4 is shown in SEQ ID NO: 4.

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