Polypeptide with PINK1 kinase agonist activity 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 or the polypeptide mixture comprises polypeptide 1, and the amino acid sequence of the polypeptide 1 is shown as SEQ ID NO: 1. The novel polypeptide compound is separated and identified from the earthworm extract, has PINK1 agonist activity, has no obvious cytotoxicity, can save MPP + damaged nerve cells, and has 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 having PINK1 kinase agonist activity and its application. 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. Currently, 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 replenish 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, thus 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 purpose of the present invention is to provide a polypeptide having PINK1 kinase agonist activity and its application.

[0006] To achieve the above object of the invention, the technical solution adopted by the present invention is: a polypeptide, the polypeptide is polypeptide 1, and the amino acid sequence of the polypeptide 1 is as shown in SEQ ID NO: 1.

[0007] Correspondingly, a polypeptide mixture, the polypeptide mixture includes the polypeptide 1 described in claim 1.

[0008] Preferably, the polypeptide mixture further comprises any one or more of polypeptides 2, 3, and 4. 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. The amino acid sequence of polypeptide 4 is as shown in SEQ ID NO: 4.

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

[0010] Correspondingly, the use of the polypeptide mixture in enhancing PINK1 agonist activity in non-diagnostic or non-therapeutic applications.

[0011] Correspondingly, an anti-Parkinson's disease drug prepared using the polypeptide.

[0012] Correspondingly, an anti-Parkinson's disease drug prepared using the polypeptide mixture.

[0013] Correspondingly, an anti-Parkinson's disease drug prepared using polypeptide 2 or polypeptide 3 or polypeptide 4. 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. The amino acid sequence of polypeptide 4 is as shown in SEQ ID NO: 4.

[0014] Correspondingly, the use of polypeptide 2 or polypeptide 3 or polypeptide 4 in enhancing PINK1 agonist activity in non-diagnostic or non-therapeutic applications. 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. The amino acid sequence of polypeptide 4 is as shown in SEQ ID NO: 4.

[0015] The present invention has the following beneficial effects: The present invention isolates and identifies a new polypeptide compound from earthworm extracts, which has PINK1 agonist activity, no obvious cytotoxicity, and can rescue nerve cells damaged by MPP + and has the prospect of preparing 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 is the LC-MS / MS spectrum of polypeptide 4;

[0020] Figure 5 Schematic diagram of the effect of polypeptide 1-4 on the survival rate of SH-SY5Y cells;

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

[0022] Figure 7 For polypeptide 1-4 on MPP + Schematic diagram of the effect on the survival rate of MPP-damaged SH-SY5Y cells. Specific implementation mode

[0023] The present invention provides a new polypeptide: polypeptide 1, whose amino acid sequence is shown in SEQ ID NO.1. Polypeptide 1 has certain PINK1 agonist activity, has a neuroprotective effect, can relieve apoptosis caused by mitochondrial damage, and is expected to be used in the preparation and development of anti-Parkinson drugs.

[0024] 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 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.

[0025] Example 1: Isolation and purification of polypeptide components

[0026] 1. Isolation and purification

[0027] Guided by the PINK1 enzyme activity assay, 4 polypeptides with potential anti-Parkinson activity were isolated and purified from the earthworm extract. The specific isolation method is as follows: The dried earthworms are crushed and refluxed with 0.9% sodium chloride solution (1×10L) at 100°C for 1h. After filtration, 0.9% sodium chloride solution (1×8L) is added again, and secondary extraction and reflux are carried out at 100°C for 30min. The filtrates are concentrated together to obtain a crude extract.

[0028] The crude extract is dissolved in 50% methanol, and the supernatant is obtained by centrifugation. The supernatant is loaded onto a Sephadex LH-20 gel filtration column (200cm×2.5cm), eluted with 50% methanol solution, and monitored by thin layer chromatography (TLC). The components containing the same ingredients are combined, and their cell activities are evaluated using the CCK-8 assay method. Then, elution is carried out with ultrapure water (A) and methanol (B) as the mobile phase, and the part with higher neuroprotective activity is further purified using an RP-C18 column. The eluate is collected.

[0029] 2. Structure identification

[0030] Using Q Exactive TM Hybrid quadrupole - orbitrap TM A liquid chromatography - tandem mass spectrometry (LC - MS / MS) analysis of the eluate components was performed using a mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). After reducing 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 before LC - MS / MS analysis. The mobile phase consisted of 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.

[0031] Table 1 Comparison table of sequencing results of each polypeptide

[0032] 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

[0033] 3. Test for PINK1 agonist activity

[0034] Ubiquitin is an effective substrate of PINK1. Under the catalysis of PINK1, ubiquitin is phosphorylated, and ADP is generated simultaneously; 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, thereby calculating the PINK1 agonist activity of the polypeptide sample.

[0035] The specific operation method is as follows: The enzymatic reaction was carried out on a white 96 - well plate, and the buffer solution was set as: 50 mM Tris - HCl (pH = 7.5), 0.1 mM EDTA (metal chelator), 10 mM MgCl2 (catalyst), and 4% 2 - hydroxy - 1 - ethanethiol (to prevent oxidation of disulfide bonds and protect proteins from being damaged). 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) were added to the buffer solution and incubated at 25 °C for 10 min.

[0036] A drug control group was set: the rest was the same, and 10 μL of resveratrol was used to replace the polypeptide sample.

[0037] After the reaction, the ATP consumption was detected using the Kinase - lumi TM Luminescent kinase detection kit, and the PINK1 agonist rate was calculated using the following formula.

[0038] PINK1 activation rate (%) = [1-(AB) / (CD)] × 100%. Wherein, A is the absorbance of the sample group consisting of sample (peptide or resveratrol), PINK1, ubiquitin and ATP; B is the absorbance of the sample control group consisting of sample, PINK1 and ubiquitin (lacking ATP); C is the absorbance of the control group consisting of PINK1, ubiquitin and ATP (lacking sample); D is the absorbance of the blank control group consisting of PINK1 and ubiquitin. The results are shown in Table 2.

[0039] Table 2 PINK1 agonistic activity of each component

[0040] Sample Stimulation rate (%) Polypeptide 1 17.16±0.55 Polypeptide 2 52.46±0.96 Polypeptide 3 50.35±0.72 Polypeptide 4 33.45±0.45 Resveratrol 91.45±0.34

[0041] The results showed that the four peptides screened out all had certain PINK1 agonist activity.

[0042] Example 2: Toxicity test of peptides on neural cells

[0043] SH-SY5Y cell suspension was prepared at 1×10 4 SH-SHY5Y cells were seeded at a density of 1×10 cells / well in a 96-well plate, and 100 μL of complete culture medium (DMEM / F12 culture medium containing 1% streptomycin-penicillin dual antibody and 10% fetal bovine serum FBS) was added to each well, and cultured in a cell culture incubator (37°C, 5% CO2) for 24 h. 4 The cells were seeded at a density of in a 96-well plate and incubated for another 24 hours under the same culture medium and culture conditions. The initial culture was then aspirated, and samples of the four polypeptides obtained in Example 1 with different concentrations (10 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM and 400 μM) (the pure polypeptide was dissolved in DMSO to prepare a stock solution, and different volumes of the stock solution were aspirated and dissolved in DMEM / F12 culture medium to prepare polypeptide samples of different concentrations, with a DMSO dosage of <10%) were placed in the wells and incubated at 37°C for 24 hours. Finally, 10 μL of CCK-8 solution was added to each well, and after incubation at 37°C for 1 hour, the absorbance of each well was read. The results are as follows. Figure 5 The results showed that at a concentration of 100 μM, the four peptides had no obvious toxicity to nerve cells and had the potential to be used as drugs.

[0044] Example 3: Peptide to MPP + Effects of induced SH-SY5Y cell damage

[0045] SH-SY5Y cell suspension was prepared at 1×10 4Cells were inoculated into a 96-well plate at a density of 4 cells per well, and 100 μL of complete medium (DMEM / F12 medium containing 1% streptomycin-penicillin double antibody and 10% fetal bovine serum FBS) was added to each well. The cells were cultured in a cell incubator (37 °C, 5% CO2) for 24 h. Subsequently, SH-SHY5Y cells were inoculated into a 96-well plate at a density of 1 × 10 + cells per well and incubated for another 24 h under the same medium and culture conditions. Subsequently, the initial culture was aspirated, and samples of the 4 polypeptides obtained in Example 1 at different concentrations (10 μM, 25 μM, 50 μM, 100 μM, and 150 μM) (obtained in the same way as in Example 2) were placed into the wells and incubated at 37 °C for 2 h. Then, each well was treated with MPP + (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. Finally, after adding 10 μL of CCK-8 solution to each well and incubating at 37 °C for 1 h, the absorbance of each well was read. 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. And 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 polypeptides or rasagiline, and not using MPP Figure 6 、 7 for treatment, and the other conditions were the same. The results are shown in

[0046] Table 3 Protective effects of each component on nerve cells

[0047] Sample (100 μM) Cell viability Polypeptide 1 63.08％ Polypeptide 2 70.08％ Polypeptide 3 68.77％ Polypeptide 4 65.06％ Positive control (rasagiline) 72.12％ Blank control group 100.00％ <![CDATA[MPP + Group without medication after treatment]]> 55.41％

[0048] Figure 6 is the effect of MPP + on the viability of SH-SY5Y cells. The results showed that: under the action of MPP + at a concentration of 2 mM and above, the cell survival rate decreased significantly. Figure 7 and Table 3 show the protective effects of the polypeptide and rasagiline on cells affected by 2 mM MPP + . Among them, Figure 7 the first item "--" on the abscissa is the blank control group, indicating that neither MPP + nor polypeptides or other drugs were added; the second item "-+" indicates that 2 mM MPP + was added, and no polypeptides or other drugs were used. The results showed that: after treatment with the 4 polypeptides, the cell survival rate could be increased to a certain extent, the neurotoxic effect of MPP + was delayed, and they had a neuroprotective effect, could relieve cell apoptosis caused by mitochondrial damage, and were expected to be used in the preparation and development of anti-PD drugs.

[0049] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

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

1.

2. A polypeptide mixture, characterized in that: The polypeptide mixture includes the polypeptide 1 described in claim 1.

3. The polypeptide mixture according to claim 2, characterized in that: The polypeptide mixture also includes any one or more of polypeptide 2, polypeptide 3, and polypeptide 4, 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.

4. Non-diagnostic or therapeutic use of the polypeptide according to claim 1 in enhancing PINK1 agonist activity.

5. Use of the polypeptide mixture according to claim 2 or 3 in non-diagnostic or therapeutic applications for enhancing PINK1 agonistic activity.

6. An anti-Parkinson's disease drug prepared using the polypeptide according to claim 1.

7. An anti-Parkinson's disease drug prepared using the polypeptide mixture according to claim 2 or 3.

8. An anti-Parkinson's disease drug prepared using polypeptide 2, polypeptide 3 or polypeptide 4, characterized in that: 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.

9. The use of polypeptide 2, polypeptide 3 or polypeptide 4 in non-diagnostic or therapeutic applications for increasing PINK1 agonist activity, characterized in that: 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.

Images