CN110256574B

CN110256574B - Fusion polypeptide and application thereof in preparing medicament for resisting depression and neurodegenerative diseases

Info

Publication number: CN110256574B
Application number: CN201910421651.0A
Authority: CN
Inventors: 张利宁; 贾玉峰; 李媛; 王群; 朱法良; 郭春; 李艳
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2020-12-04
Anticipated expiration: 2039-05-21
Also published as: CN110256574A

Abstract

The disclosure belongs to the technical field of biology, and particularly relates to a 9R-eIF4A-VI fusion polypeptide and application thereof in preparation of antidepressant drugs. Published studies prove that PDCD4 can be combined with eukaryotic cell translation initiation factor (eIF4A) to inhibit the expression of brain-derived neurotrophic factor (BDNF), block the effects of damaged neuronal cell repair and synaptic transmission, and aggravate the symptoms of depressed patients. The fusion polypeptide with the interference effect is obtained by fusing a transmembrane sequence and a segment of structural domain on eIF4A, and the fusion polypeptide can effectively interfere the combination of PDCD4 and eIF4A, has good transmembrane efficiency and stability, can be applied to preparation of antidepressant drugs, and has important significance.

Description

Fusion polypeptide and application thereof in preparing medicament for resisting depression and neurodegenerative diseases

Technical Field

The disclosure belongs to the technical field of biology, and particularly relates to application of a polypeptide and a fusion polypeptide 9R-eIF4A-VI which interfere combination of PDCD4 and eIF4A as an antidepressant and neurodegenerative disease resistant drug.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Depression is a type of mental disorder with mood depression as the main symptom. Severe patients even have suicidal behavior and seriously threaten human health. A large amount of clinical research evidence indicates that synaptic plasticity caused by excessive activation of microglia is the pathological basis of depression, and the brain-derived neurotrophic factor (BDNF) can effectively regulate and control the synaptic plasticity, repair damaged neurons, strengthen signal transmission among synapses and relieve the course of depression. Finding the target gene for regulating and controlling the expression of BDNF has important significance for treating depression. Programmed cell death gene 4 (PDCD 4) is a gene related to apoptosis newly discovered in recent years, is a novel cancer suppressor gene, and is discovered by the inventor along with the research that PDCD4 plays an important role in various inflammatory diseases. Previous experimental results of the inventors indicate that PDCD4(-/-) mice are effective against the development of depression caused by chronic stress.

Sojima et al isolated human PDCD4 gene from cDNA library of human glioma cells, and their studies showed that there were two alpha helical structural regions-MA 3, 164-275 region and 329-440 region, respectively, on the amino side of PDCD 4. PDCD4 binds to eukaryotic translation initiation factor (eukaryotic translation initiation factor 4A, isoform 2, eIF4A) through the functional domain, thereby inhibiting the formation of ribosome complex and the synthesis of protein. In vivo experiments prove that in a WT chronic stress mouse model, PDCD4 is highly expressed in a hippocampus and the expression level of BDNF is obviously reduced. Further studies indicate that chronic stress causes the phosphorylation degradation pathway of PDCD4 to be inhibited, and excessive PDCD4 inhibits the initiation of BDNF translation and further inhibits the expression of the BDNF translation by combining with eIF 4A. Therefore, high expression of PDCD4 means that BDNF levels are reduced, and at the same time, damaged neurons are difficult to repair in a timely manner, synaptic transmission effects are hindered, and the patient's depressive condition is aggravated.

The polypeptide has wide application as medicine, and has the advantages of strong physiological activity, low immunogenicity, high curative effect, etc. However, it is known that polypeptide drugs have many disadvantages due to their own characteristics, such as low oral availability, high enzymatic hydrolysis, and very short half-life, so that their development as drugs is limited.

Disclosure of Invention

Aiming at the research situation, the inventor thinks that finding binding sites of eIF4A and PDCD4 and designing interference peptides interfering the combination of endogenous PDCD4 and eIF4A can relieve the inhibition effect of PDCD4 on BDNF and promote the expression of BDNF, and the method is a new treatment idea for treating depression. The inventor conducts related research under the guidance of the research thought, screens a structural domain of eIF4A combined with PDCD4, and verifies that the polypeptide screened by the method can efficiently interfere the combination of PDCD4 and eIF4A in vitro through co-immunoprecipitation and immunoblotting, promotes the expression of IIc-BDNF and improves depression symptoms. Primary neuron cell and depressive cell model tests show that the polypeptide has good stability and transmembrane efficiency, can be retained in neuron cells for more than 24 hours, and has remarkable advantages compared with the traditional polypeptide drugs.

In order to achieve the above technical object, the present disclosure provides the following technical solutions:

in a first aspect of the disclosure, there is provided use of a polypeptide having an amino acid sequence as set forth in SEQ ID No. 4 as an inhibitor of binding of PDCD4 to eIF 4A.

The amino acid sequence shown as SEQ ID NO. 4 is a structural domain with the length of 12 amino acids in the full-length sequence of eIF4A, namely eIF 4A-VI. The screening of the disclosure shows that compared with other structural domains of PDCD4 and eIF4A, the polypeptide of the amino acid sequence can significantly interfere the combination of PDCD4 and eIF4A, promote the expression of BDNF, and can be used as a combination inhibitor of PDCD4 and eIF4A and a neuron cell repair substance.

In a second aspect of the disclosure, a fusion polypeptide is provided, which is formed by connecting a 9R transmembrane sequence and an amino acid sequence shown in SEQ ID NO. 4.

The fusion polypeptides provided by the present disclosure employ the 9R transmembrane sequence shown in SEQ ID NO 5.

Preferably, in the fusion polypeptide, the N terminal is a 9R transmembrane sequence, and the C terminal is an amino acid sequence shown in SEQ ID NO. 4.

Preferably, the fusion polypeptide has an amino acid sequence shown in SEQ ID NO. 6, or the fusion polypeptide is derived by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 6 and has the same function.

In a third aspect of the disclosure there is provided a nucleotide sequence encoding a fusion polypeptide according to the second aspect, said nucleotide sequence comprising a nucleotide sequence which translates to the same amino acid sequence due to codon degeneracy.

In a fourth aspect of the present disclosure, there is provided a polypeptide having an amino acid sequence shown in SEQ ID NO. 4, a fusion polypeptide of the second aspect, and/or a nucleotide sequence of the third aspect for use in the preparation of an antidepressant or a medicament for treating a neurodegenerative disease.

Preferably, the polypeptide or fusion polypeptide of the amino acid sequence shown in SEQ ID NO. 4 is used as the BDNF agonist.

Further preferably, the polypeptide or fusion polypeptide of the amino acid sequence shown in SEQ ID NO. 4 is used as IIc-BDNF agonist.

In a fifth aspect of the present disclosure, there is provided an antidepressant drug comprising the polypeptide of amino acid sequence shown in SEQ ID No. 4 or the fusion polypeptide of the second aspect.

Preferably, in the antidepressant drug, the concentration of the fusion polypeptide is 12.5nM or more; further preferably, the concentration is 12.5 to 50 nM.

Preferably, the antidepressant further comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

The beneficial effect of this disclosure:

1. the present disclosure obtained several domains of eIF4A tightly bound to PDCD4 by screening, and the inventors propose to interfere with the binding of eIF4A to PDCD4 to promote the expression of BDNF. The corresponding interference sequence is designed for the screened binding site, and the amino acid sequence shown in SEQ ID NO. 6, namely eIF4A-VI is screened after the interference effect of the sequence is detected, and the polypeptide of the sequence can interfere the binding of PDCD4 and eIF4A in vitro and can promote the expression of IIc-BDNF.

2. An immunoblotting experiment proves that when the concentration of 9R-eIF4A-VI reaches more than 12.5nM, the content expression of free PDCD4 and IIc-BDNF is increased and concentration dependency is presented, which proves that the fusion polypeptide can be used as a medicament for treating depression symptoms, and the adjustment of the treatment effect can be realized by adjusting the dosage of the fusion polypeptide.

3. The transmembrane efficiency and stability of 9R-eIF4A-VI are detected by an immunofluorescence method, and fluorescence detection results show that the 9R-eIF4A-VI and PDCD4 have obvious co-localization conditions, so that the 9R-eIF4A-VI interference result is good. And compared with the problems of low utilization rate, easy degradation and the like of the traditional polypeptide medicine, the 9R-eIF4A-VI has good stability, can be reserved in the neuron cell for more than 24h, and has good transmembrane efficiency.

4. The research on the interference action mechanism of 9R-eIF4A-VI proves that the degradation induction effect of the fusion polypeptide on PDCD4 is mainly performed through a lysosome way, and further defines the mechanism of the use of the fusion polypeptide as a medicament. Moreover, the research of the disclosure shows that the fusion polypeptide has no influence on the expression of IL-10 and IL-6 in primary neuron cells, does not cause inflammatory reaction, and is safe to use.

BDNF is used as a brain-derived neurotrophic factor and is related to a plurality of physiological activities such as synaptic transmission, damaged neuron cell repair and the like. The disclosure provides the effects of eIF4A-VI and 9R-eIF4A-VI in interfering the combination of PDCD4 and eIF4A and improving the expression of BDNF, and the polypeptide is also expected to be applied to the treatment of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia and the like.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a graph showing the results of screening for domain polypeptides in example 1;

wherein, FIG. 1a shows four fusion polypeptides prepared after screening in example 1;

FIG. 1b shows the expression of four fusion polypeptides as fusion proteins with GFP;

FIG. 1c is a band diagram of the interference of four fusion polypeptides with PDCD4 binding to eIF4A in vitro;

FIG. 1d is a histogram of the induction of BDNF expression by four fusion proteins.

FIG. 2 is a graph showing the results of the bioactivity assays for fusion polypeptide 9R-eIF4A-VI selected in example 2;

wherein, FIG. 2a shows the fusion polypeptide and the control peptide obtained by screening;

FIG. 2b is a histogram of fusion polypeptides upregulating BDNF expression;

FIG. 2c is a histogram of BDNF effects of fusion polypeptides and control peptides;

the induction effect on BDNF in the contrast peptide sequence disappears, which shows that the fusion polypeptide has specificity;

FIG. 2d is a histogram of induction of BDNF expression by different concentrations of fusion polypeptides;

the fusion polypeptide has obvious concentration dependence on the induction of BDNF;

FIG. 2e is a fluorescence plot of transmembrane efficiency and intracellular stability of the fusion polypeptides detected by immunofluorescence;

the fusion polypeptide can cross a cell membrane to enter cytoplasm within 1h and can stably exist in the cell for 24 h;

FIG. 2f is a band diagram of co-immunoprecipitation assay detecting fusion polypeptides interfering with the binding of PDCD4 to eIF 4A;

FIG. 2g is a graph of the binding band of fusion polypeptide interference PDCD4 and eIF4A, which is shown in a double fluorescent molecule complementation experiment;

FIG. 2h is a fluorescence plot of a fusion polypeptide interfering with the binding of PDCD4 and eIF4A as demonstrated by immunofluorescence experiments.

FIG. 3 is a graph showing the results of examining the transmembrane efficiency and stability of 9R-eIF4A-VI in neuronal cells in example 3;

wherein, FIG. 3a is a fluorescence plot of transmembrane efficiency and stability of the fusion polypeptide in primary hippocampal neurons of mice;

FIG. 3b is a photograph of immunofluorescence detecting fusion polypeptides and PDCD4 in mouse primary neuronal cells;

FIG. 3c is a histogram of fusion polypeptides upregulating BDNF expression in primary neurons and microglia;

FIG. 3d is a graph showing the results of ELISA detection of BDNF, IL-10, IL-6 expression in cell supernatants.

FIG. 4 is a graph of the effect of 9R-eIF4A-VI on a model of neuronal cell depression in example 4;

wherein, FIG. 4a is a schematic diagram of the expression of rapamycin up-regulating PDCD 4;

FIG. 4b is a histogram of the up-regulation of PDCD4 expression by rapamycin, thereby inhibiting BDNF, constructing a cellular depression model;

FIG. 4c is a histogram of the inhibition of BDNF expression by rapamycin alleviated by the fusion polypeptide. FIG. 5 is a graph showing the results of the 9R-eIF4A-VI degradation mechanism in example 5.

Wherein, FIG. 5a is a graph of the expression banding of fusion polypeptide for inhibiting PDCD4 in SHSY5Y cells;

FIG. 5b is a graph showing the effect of the fusion polypeptide on the expression of mRNA in PDCD 4;

FIG. 5c is a histogram of the results that the fusion polypeptide did not cause upregulation of miRNA-21 expression;

FIG. 5d shows that fusion polypeptides do not act on PDCD 4-facilitated degradation via the proteasome pathway;

FIG. 5e shows that the fusion polypeptide mainly acts on PDCD4 degradation promotion through a lysosome pathway;

FIG. 5f is a band diagram of the inhibition of PDCD4 expression by fusion polypeptides in mouse primary neurons.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, the prior art discloses that PDCD4 inhibits the expression of brain-derived neurotrophic factor and aggravates the symptoms of nerve cell damage and depression by binding two alpha helical structural regions-MA 3 on the amino side with eukaryotic translation initiation factor eIF 4A. In response to the above results, the inventors developed an interference peptide that interferes with the binding of endogenous PDCD4 to eIF4A for use in the treatment of depression. Under the guidance of the technical idea, the fusion polypeptide 9R-eIF4A-VI is prepared and obtained in the method, the N end is a 9R transmembrane sequence, and the C end is an eIF4A-VI structural domain obtained by screening. The immunoblotting experiment confirms that the fusion polypeptide can effectively interfere the combination of PDCD4 and eIF4A, the interference effect is concentration-dependent, the polypeptide has good transmembrane efficiency and stability, can be kept in neuron cells for more than 24 hours, and the technical defects of poor stability and short half-life of the traditional polypeptide medicament are overcome.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

Example 1

In this example, the inventors screened four domains (eIF4A-Q, eIF4A-I, eIF4A-Ia and eIF4A-VI) of eIF4A closely binding to PDCD4 by consulting the literature in the previous experimental design, and the amino acid sequences thereof are shown in Table 1. The inventors constructed a fusion protein containing the domain of interest by molecular cloning (FIG. 1a FIG. 1 b). Co-immunoprecipitation (figure 1c) results showed that the eIF4A-VI group IB: HA band was significantly lower than the other domains and was almost colorless, demonstrating that eIF4A of the eIF4A-VI interference group had little interaction with PDCD4 and that eIF4A-VI had good interference efficiency. Meanwhile, an immunoblotting experiment (figure 1d) verifies that the eIF4A-VI can induce the expression of IIc-BDNF, the inducing effect is also obviously higher than that of other structural domain polypeptides, the eIF4A-VI simultaneously meets the effects of interfering the combination of eIF4A and PDCD4 and inducing the expression of BDNF, and the application of the eIF4A-VI in the preparation of the medicament has good development significance as an antidepressant component and other neurodegenerative disease treatment components.

TABLE 1 Domain amino acid and nucleotide sequences

Example 2

Combining the results of the study in example 1, eIF4A-VI was selected for corresponding modification in this example, and 9R transmembrane sequence was added at the N-terminus to synthesize polypeptide 9R-eIF 4A-VI. As shown in FIG. 2a, the N-terminal of the fusion polypeptide is a 9R transmembrane sequence, and the C-terminal is the eIF4A-VI domain obtained by screening. The 9R transmembrane sequence and the 9R-eIF4A-VI sequence are shown in Table 2 below:

TABLE 29R and 9R-eIF4A-VI amino acid sequences

To test the effect of the fusion polypeptide on interfering the binding of PDCD4 to eIF4A, the inventors designed 9R-mueIF4A-VI as a control peptide to be tested, which 9R-mueIF4A-VI randomly mutated a site of amino acids compared to 9R-eIF 4A-VI. (FIG. 2b/2c/2d) it was confirmed by immunoblotting that 9R-eIF4A-VI promotes the expression of IIc-BDNF, and that the promotion is concentration-dependent, as shown in FIG. 2d, the expression of IIc-BDNF content increases with the increase of the loading concentration.

In this example, the control peptide shown in SEQ ID NO. 9 is used as an example to illustrate that 9R-mueIF4A-VI after amino acid mutation loses the promoting effect (FIG. 2c), which proves that the 9R-eIF4A-VI has good specificity, and the same effect is difficult to achieve by replacing any amino acid in the sequence. The amino acid sequences used in this specificity assay are shown in table 3:

TABLE 3 sets of amino acid sequences in the specificity test

As shown in FIG. 2e, the HEK-293 cell was added with the fluorescently labeled 9R-eIF4A-VI and detected, and the polypeptide entering the cell was observed for about 1h, until 24h, the polypeptide fluorescence labeling of the cell was still observed. The 9R-eIF4A-VI has good transmembrane efficiency and stability in cells. (FIG. 2f/2g/2h) demonstrates that 9R-eIF4A-VI can interfere with the binding of PDCD4 to eIF 4A.

Example 3

Example 2 has confirmed that the 9R-eIF4A-VI has good transmembrane effect and stability in HEK-293 cell model, and this example is further studied for the effect of the polypeptide on neuronal cells.

As shown in fig. 3a, the inventors also verified the transmembrane efficiency and stability of 9R-eIF4A-VI in primary neuronal cells. As shown in FIG. 3b, fluorescence observation of primary neurons revealed that 9R-eIF4A-VI was clearly co-localized with PDCD4, further indicating the specific interference of the polypeptide sequence on PDCD 4. FIG. 3C shows that 9R-eIF4A-VI can promote the expression of BDNF in primary neurons. (FIG. 3d) the expression of BDNF in the culture supernatant of primary neurons is detected by ELISA, and the aim of enhancing the expression of BDNF by interfering the combination of PDCD4 and EIF4A and improving the expression of BDNF so as to strengthen the signal transmission between synapses and relieve depression symptoms is fulfilled.

At the same time, the inventors also examined the expression of inflammatory factors in neuronal cells following interference of the fusion polypeptide, as shown in FIG. 3d, 9R-eIF4A-VI did not promote increased expression of IL-10 and IL-6. The polypeptide is proved to not cause the inflammatory reaction of neuron cells and to be safer to use.

Example 4

To further confirm the therapeutic effect of the fusion polypeptide on depression, a depression cell model was made and the effect of 9R-eIF4A-VI on the depression model was investigated.

(FIG. 4a/4b) it is known in the art that CRS can inhibit the activity of mTOR, so that the degradation of PDCD4 is inhibited, excessive PDCD4 can inhibit the expression of BDNF, and (FIG. 4b) the inventor constructs a primary neuron cell depression model by inhibiting the activity of mTOR through Rapamycin with primary neuron cells.

(FIG. 4c) the addition of 9R-eIF4A-VI and 9R-eIF4A-VI to the successfully constructed cell model can significantly promote the expression of BDNF, and the polypeptide can realize the repair of neuron cells and correct depression symptoms by inducing the expression of BDNF in depressed primary neuron cells under the depression state.

Example 5

In order to provide a mechanism research as a support for the application of 9R-eIF4A-VI as an antidepressant, a preliminary study was conducted on the interference mechanism of 9R-eIF4A-VI in this example.

As shown in FIG. 5a, when the inventors stimulated the SHSY5Y cell line with 9R-eIF4A-VI, the band result showed that there was no significant decrease in the expression of PDCD4 at low concentration, but the expression of PDCD4 was significantly decreased at high concentration (200nM), which was statistically significant.

As shown in FIG. 5b, by means of RT-PCR to detect the genome expression of related targets, the inventors found that 9R-eIF4A-VI did not cause the change of PDCD4 mRNA level. (FIG. 5d/5c) the inventors found that 9R-eIF4A-VI promoted degradation of PDCD4 primarily through the lysosomal pathway by blocking the proteasome and lysosomal pathways, respectively.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

SEQUENCE LISTING

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Claims

1. The polypeptide of the amino acid sequence shown as SEQ ID NO. 4 is applied to the diagnosis and treatment of non-diseases as a PDCD4 and eIF4A combined inhibitor.

2. The fusion polypeptide is characterized by being formed by connecting a 9R transmembrane sequence and an amino acid sequence shown in SEQ ID NO. 4; the sequence of the fusion polypeptide is Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Tyr-IIe-His-Arg-IIe-Gly-Arg-Gly-Gly-Arg-Phe-Gly.

3. A nucleotide sequence encoding the fusion polypeptide of claim 2, comprising a nucleotide sequence that translates to the same amino acid sequence due to codon degeneracy.

4, the polypeptide of the amino acid sequence shown in SEQ ID NO. 4 and the fusion polypeptide of claim 2 are applied to the preparation of antidepressant drugs; the polypeptide of the amino acid sequence shown in SEQ ID NO. 4 or the fusion polypeptide is used as IIc-BDNF agonist.

5. An antidepressant drug comprising a polypeptide having an amino acid sequence as set forth in SEQ ID No. 4 or a fusion polypeptide as set forth in claim 2.

6. The antidepressant drug of claim 5, wherein the concentration of said fusion polypeptide in said drug is greater than 12.5 nM.

7. The antidepressant drug of claim 5, characterized in that it comprises, in addition to the fusion polypeptide, a pharmaceutically acceptable carrier, excipient or adjuvant.