CN110256574A - Fused polypeptide and the application in preparation antidepression, neurodegenerative disease drug - Google Patents

Abstract

The disclosure belongs to field of biotechnology, and in particular to 9R-eIF4A-VI fused polypeptide and its application in preparation antidepressant.Open research confirms that PDCD4 can combine to inhibit the expression of brain-derived neurotrophic factor (BDNF) with Eukaryotic initiation factor (eIF4A), injured neurons cell repair and cynapse transmission effect are hindered, depressive patient symptom is aggravated.Disclosure screening has determined structural domain of several PDCD4 in conjunction with eIF4A, pass through the segment structure domain on fusion cross-film sequence and eIF4A, obtain the fused polypeptide with interference effect, experiments verify that, the fused polypeptide can effectively interfere the combination of PDCD4 and eIF4A, and there is good cross-film efficiency and stability, it can be applied to preparation antidepressant, be of great significance.

Description

Fused polypeptide and the application in preparation antidepression, neurodegenerative disease drug

Technical field

The disclosure belongs to field of biotechnology, and in particular to a kind of polypeptide of the interference PDCD4 in conjunction with eIF4A and merges more Application of the peptide 9R-eIF4A-VI as antidepression and anti-neurodegenerative disease drug.

Background technique

The information for disclosing the background technology part is merely intended to increase the understanding to the general background of the disclosure, without certainty It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art Art.

Depression is with depressed a kind of phrenoblabia disease for cardinal symptom.Severe patient even commits suiside Behavior seriously threatens human health.A large amount of Clinical Evidences show that microglia overactivity causes synaptic plasticity to be Depression generate pathological basis, brain-derived neurotrophic factor (BDNF) can Effective Regulation synaptic plasticity, repair by Neuron is damaged, signal transmits between reinforcing cynapse, alleviates the course of disease of depression.The target gene of regulation BDNF expression is found to depression Treatment have important meaning.Apoptosis gene 4 (programmed cell death 4, PDCD4) is in recent years Newfound one kind gene relevant to Apoptosis is a kind of new tumor suppressor gene, gos deep into inventor's discovery with research PDCD4 plays a significant role in a variety of inflammatory diseases.(-/-) mouse can be with the result shows that PDCD4 for the previous experiments of inventor It is effective against the generation of depression caused by chronic stress.

Sojima et al. isolates the PDCD4 gene of people from the cDNA library of human glioma cells, research shows that There are two αhelix region-MA3, respectively 164th~275 area and 329th~440 area for the amino side of PDCD4.PDCD4 is by being somebody's turn to do Functional domain and Eukaryotic initiation factor (eukaryotic translation initiation factor 4A, Isoform 2, eIF4A) it combines, to inhibit the formation of ribose nanocrystal composition and the synthesis of protein.Experiment in vivo card In real WT chronic stress mouse model, PDCD4 is expressed in hippocampus height and BDNF expression quantity is substantially reduced.Further research table Bright, long-term chronic stress causes the phosphorylation-degradation approach of PDCD4 to be suppressed, excessive PDCD4 by conjunction with eIF4A from And the starting for inhibiting BDNF to translate inhibits its expression in turn.Therefore, the high expression of PDCD4 means the reduction of BDNF content, with this Meanwhile injured neurons are difficult to be timely repaired, cynapse transmission effect is obstructed, making patients' depression state of an illness.

Polypeptide is relatively broad as medicinal application, has many advantages, such as physiological activity is strong, immunogenicity is low, curative effect is high. It is well known that the characteristics of polypeptide drug is due to itself, there are shortcomings, as Oral availability is low, enzymatic hydrolysis property height and half Phase of declining is extremely short etc., it is made to receive many restrictions as drug development.

Summary of the invention

For the studies above situation, inventor thinks to find eIF4A and PDCD4 binding site, and endogenous PDCD4 is interfered in design Interference peptide in conjunction with eIF4A can alleviate PDCD4 to the inhibiting effect of BDNF, while promote the expression of BDNF, be that one kind is controlled Treat the new treatment thoughts of depression.Inventor has carried out correlative study under the guidance of the studies above thinking, to eIF4A with The structural domain that PDCD4 is combined is screened, can by the polypeptide that co-immunoprecipitation and immunoblotting demonstrate disclosure screening With the efficiently combination of interference PDCD4 and eIF4A in vitro, and promote the expression of IIc-BDNF, improves depressive symptom.By primary Neuronal cell and depressed cell model experiments have shown that, the polypeptide is with good stability and cross-film efficiency, can be in neuron It is intracellular to retain more than for 24 hours, there is significant advantage compared to traditional polypeptide drugs.

In order to realize the above technical purpose, the disclosure the following technical schemes are provided:

The disclosure is in a first aspect, provide the polypeptide of the amino acid sequence as shown in SEQ ID NO:4 as PDCD4 and eIF4A The application of binding inhibitors.

The amino acid sequence as shown in SEQ ID NO:4 is that a segment length is 12 amino acid in eIF4A full length sequence Structural domain, i.e. eIF4A-VI.Show the structural domain compared to other PDCD4 in conjunction with eIF4A, above-mentioned ammonia through disclosure screening The polypeptide of base acid sequence can interfere significantly with the combination of PDCD4 and eIF4A, and promote the expression of BDNF, can be used as PDCD4 Substance is repaired with eIF4A binding inhibitors and neuronal cell to be applied.

Disclosure second aspect provides a kind of fused polypeptide, and the fused polypeptide is mainly by 9R cross-film sequence and SEQ ID Amino acid sequence shown in NO:4 is formed by connecting.

The fused polypeptide that the disclosure provides uses the 9R cross-film sequence as shown in SEQ ID NO:5.

Preferably, in above-mentioned fused polypeptide, N sections are 9R cross-film sequence, and C sections are amino acid sequence shown in SEQ ID NO:4.

Preferably, the fused polypeptide has amino acid sequence shown in SEQ ID NO:6 or the fused polypeptide is SEQ Amino acid sequence shown in ID NO:6 is substituted, lacks and/or increases one or several amino acid derived and has identical function Polypeptide.

The disclosure third aspect provides the nucleotide sequence of fused polypeptide described in coding second aspect, the nucleotides sequence Column include the nucleotide sequence for translating to obtain same amino acid sequence due to Codon degeneracy.

It is more to provide fusion described in the polypeptide of amino acid sequence shown in SEQ ID NO:4, second aspect for disclosure fourth aspect Nucleotides sequence described in peptide and/or the third aspect is listed in preparation antidepressant or treats answering in neurodegenerative disease drug With.

Preferably, the polypeptide of amino acid sequence shown in the SEQ ID NO:4 or fused polypeptide are as BDNF agonist.

It is further preferred that the polypeptide or fused polypeptide of amino acid sequence shown in the SEQ ID NO:4 are as IIc- BDNF agonist.

The 5th aspect of the disclosure, provides a kind of antidepressant, which includes amino shown in SEQ ID NO:4 Fused polypeptide described in the polypeptide or second aspect of acid sequence.

Preferably, in above-mentioned antidepressant, the concentration of the fused polypeptide is 12.5nM or more；It is further preferred that Concentration is 12.5~50nM.

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

The disclosure the utility model has the advantages that

1. the disclosure has obtained the structural domain that several eIF4A and PDCD4 combine closely, inventor's plan pair by screening EIF4A carries out interference in conjunction with PDCD4 to promote the expression of BDNF.The corresponding interference of binding site design that screening is obtained Sequence, and screened after being detected to the interference effect of the sequence and obtained the amino acid sequence as shown in SEQ ID NO:6, That is eIF4A-VI, the polypeptide of the sequence can interfere the combination of PDCD4 and eIF4A in vitro and can promote IIc-BDNF's Expression.

2. by immunoblotting analysis experimental verification, when 9R-eIF4A-VI concentration reaches 12.5nM or more, as free PDCD4 And IIc-BDNF content express increase, and present concentration dependent, it was demonstrated that the fused polypeptide can be used as drug for treat suppression Strongly fragrant symptom, and the adjustment to therapeutic effect can be realized by adjusting the dosage of fused polypeptide.

3. the disclosure also passes through the cross-film efficiency and stability that immunofluorescence method has detected 9R-eIF4A-VI, fluorescence inspection The result shows that 9R-eIF4A-VI is with PDCD4, there are apparent common location situations for survey, it was demonstrated that 9R-eIF4A-VI interferes result good. Also, the problems such as often low with utilization rate, degradable compared to traditional polypeptide drugs, 9R-eIF4A-VI has good stability, can Retain more than for 24 hours in neuronal cell, and there is good cross-film efficiency.

4. by studying 9R-eIF4A-VI interference effect mechanism, it was demonstrated that the fused polypeptide lures the degradation of PDCD4 It is main by lysosomal pathway progress to lead effect, has further clarified the mechanism that the fused polypeptide is used as drug.Also, it passes through The disclosure studies have shown that the fused polypeptide on the expression of IL-10 in primary neuronal cell and IL-6 without influence, inflammation will not be caused Disease reaction, use are safe.

5.BDNF is as the multinomial life such as a kind of brain-derived neurotrophic factor and cynapse transmitting, reparation injured neurons cell Reason activity is related.Present disclose provides eIF4A-VI and 9R-eIF4A-VI to interfere PDCD4 in conjunction with eIF4A, improve BDNF table Up to the effect of aspect, which is equally expected to the treatment applied to neurodegenerative disease, including alzheimer's disease, Parkinson Disease, Huntington disease, amyotrophic lateral sclerosis, spinocerebellar ataxia etc..

Detailed description of the invention

The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.

Fig. 1 is in embodiment 1 to Domain Polypeptide the selection result figure；

Wherein, Fig. 1 a is the four kinds of fused polypeptides prepared after screening in embodiment 1；

Fig. 1 b is that four kinds of fused polypeptides and GFP are expressed as fusion protein；

Fig. 1 c is the histogram that four kinds of fused polypeptides interfere PDCD4 to be combined in vitro with eIF4A；

Fig. 1 d is the histogram that four kinds of fusion proteins induce BDNF expression.

Fig. 2 is that screening obtains fused polypeptide 9R-eIF4A-VI bioactivity testing result figure in embodiment 2；

Wherein, Fig. 2 a is that screening obtains fused polypeptide and control peptide；

Fig. 2 b is the histogram that fused polypeptide raises BDNF expression；

Fig. 2 c is the effect histogram of fused polypeptide and control peptide to BDNF；

The diminished induction in peptide sequence to BDNF is compareed, shows that the fused polypeptide has specificity；

Fig. 2 d is the inducing action histogram that various concentration fused polypeptide expresses BDNF；

Show that fused polypeptide has apparent concentration dependent to the induction of BDNF；

Cross-film efficiency and intracellular stability fluorogram of Fig. 2 e for immunofluorescence mode detection fusion polypeptide；

Show that the fused polypeptide can enter endochylema in 1h across cell membrane, and can be stabilized in the cell 24h；

Fig. 2 f is the histogram for the combination that co-immunoprecipitation experiment detection fusion polypeptide interferes PDCD4 and eIF4A；

Fig. 2 g is that double fluorescent molecule complementation tests prove that fused polypeptide interferes the combination histogram of PDCD4 and eIF4A；

Fig. 2 h is that immunofluorescence experiment proves that fused polypeptide interferes fluorogram of the PDCD4 in conjunction with eIF4A.

Fig. 3 is cross-film efficiency and study on the stability result figure of the 9R-eIF4A-VI in neuronal cell in embodiment 3；

Wherein, Fig. 3 a is fused polypeptide cross-film efficiency and stability fluorogram in the primary hippocampal neurons of mouse；

Fig. 3 b is fused polypeptide and PDCD4 fluorogram in Immunofluorescence test mouse primary neuronal cell；

Fig. 3 c is the histogram that fused polypeptide raises BDNF expression in primary neuron and microglia；

Fig. 3 d is the expression result chart that ELISA detects BDNF, IL-10, IL-6 in cell conditioned medium.

Fig. 4 is 9R-eIF4A-VI in embodiment 4 to neuronal cell depression model effect picture；

Wherein, Fig. 4 a is the expression schematic diagram that rapamycin raises PDCD4；

Fig. 4 b is that rapamycin raises the expression of PDCD4 to inhibit BDNF, building cell depression model histogram；

Fig. 4 c is the inhibiting effect histogram that fused polypeptide alleviates that rapamycin expresses BDNF.

Fig. 5 is the result of study figure of 9R-eIF4A-VI degradation mechanism in embodiment 5.

Wherein, Fig. 5 a is the band of expression figure that fused polypeptide inhibits PDCD4 in SHSY5Y cell；

Fig. 5 b is the influence result figure that fused polypeptide expresses mRNA in PDCD4；

Fig. 5 c is that fused polypeptide will not cause miRNA-21 expression up-regulation result histogram；

Fig. 5 d is that fused polypeptide promotees degradation not by proteasome pathway progress to PDCD4；

Fig. 5 e is that fused polypeptide promotees degradation mainly by lysosomal pathway progress to PDCD4；

Fig. 5 f is the histogram that fused polypeptide inhibits PDCD4 expression in mouse primary neuron.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the disclosure.Unless another It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field The identical meanings of understanding.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.

It as background technique is introduced, discloses in the prior art, there are two αhelix areas by amino side by PDCD4 Domain-MA3 is in conjunction with Eukaryotic initiation factor eIF4A, to inhibit BDNF expression, aggravates Damaged nerve cell and depressive symptom.For the studies above as a result, inventor develops the knot for interfering endogenous PDCD4 and eIF4A The interference peptide of conjunction is applied to the treatment of depression.Under the guidance of above-mentioned technical thought, it is more that a kind of fusion is prepared in the disclosure 9R-eIF4A-VI, N sections of peptide are 9R cross-film sequence, and C sections are the obtained eIF4A-VI structural domain of screening.By Diagnosis of Sghistosomiasis document Confirmation is tested, which can effectively interfere the combination of PDCD4 and eIF4A, and concentration dependent is presented in interference effect, and should Polypeptide has good cross-film efficiency and stability, can retain more than for 24 hours in neuron system cell, overcome traditional peptide drug Object stablizes poor, half-life short technological deficiency.

In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool The technical solution of the disclosure is described in detail in the embodiment and comparative example of body.

Embodiment 1

In the present embodiment, by consulting literatures to screen four eIF4A and PDCD4 tight by inventor in previous experiments design The structural domain (eIF4A-Q, eIF4A-I, eIF4A-Ia, eIF4A-VI) of close combination, amino acid sequence is as shown in table 1.Pass through The method invention people of molecular cloning constructs the fusion protein (Fig. 1 a Fig. 1 b) containing target structure domain.Co-immunoprecipitation (Fig. 1 c) The result shows that eIF4A-VI group IB:HA band is substantially less than other structures domain, it is almost colourless, it was demonstrated that eIF4A-VI interference group EIF4A and PDCD4 there's almost no interaction, and eIF4A-VI has good jamming effectiveness.Meanwhile immunoblot experiment (Fig. 1 d), which demonstrates eIF4A-VI, can induce the expression of IIc-BDNF, and it is more that inducing effect is equally significantly higher than other structures domain Peptide, eIF4A-VI meets the effect of combination and the induction BDNF expression of interference eIF4A and PDCD4 simultaneously, as antidepression ingredient And other neurodegenerative disease therapeutic components have good development significance.

1 domain amino acid of table and nucleotide sequence

Embodiment 2

1 result of study in conjunction with the embodiments, selection eIF4A-VI is accordingly modified in the present embodiment, is increased at N sections 9R cross-film sequent synthesis polypeptide 9R-eIF4A-VI.As shown in Figure 2 a, N sections of the fused polypeptide are 9R cross-film sequence, and C sections are sieve Select obtained eIF4A-VI structural domain.9R cross-film sequence and 9R-eIF4A-VI sequence are as shown in table 2 below:

2 9R and 9R-eIF4A-VI amino acid sequence of table

In order to detect the effect that the fused polypeptide interferes the combination of PDCD4 and eIF4A, inventor designs 9R-mueIF4A- VI is tested as control peptide, and the 9R-mueIF4A-VI is compared to the 9R-eIF4A-VI random mutation amino in one site Acid.(Fig. 2 b/2c/2d) confirms that 9R-eIF4A-VI can promote the expression of IIc-BDNF, and the rush by immunoblot experiment Concentration dependent is presented into effect, as shown in Figure 2 d, as loading concentrations increase, the content expression of IIc-BDNF also rises therewith It is high.

It is illustrated by taking the control peptide as shown in SEQ ID NO:9 as an example in the present embodiment, the 9R- after amino acid mutation MueIF4A-VI loses facilitation (Fig. 2 c), it was demonstrated that the 9R-eIF4A-VI has good specificity, any in the sequence The replacement of amino acid is difficult to realize identical effect.Amino acid sequence used in the specific test is as shown in table 3:

Each group amino acid sequence in 3 specific test of table

As shown in Figure 2 e, the 9R-eIF4A-VI of fluorescent marker is added to HEK-293 cell and is detected, 1h or so is i.e. Polypeptide can be observed and enter cell, the polypeptide fluorescent marker until being still able to observe that cell for 24 hours.Demonstrate 9R-eIF4A- Stability of the VI with good cross-film efficiency and in the cell.(Fig. 2 f/2g/2h) proves that 9R-eIF4A-VI can be interfered The combination of PDCD4 and eIF4A.

Embodiment 3

Embodiment 2 have confirmed that the 9R-eIF4A-VI in HEK-293 cell model have good cross-film effect and Stability, the present embodiment further study the effect of neuronal cell for the polypeptide.

As shown in Figure 3a, in primary neuronal cell inventor equally demonstrate the cross-film efficiency of 9R-eIF4A-VI with And stability.Such as Fig. 3 b, in primary neuron, fluorescence observation shows 9R-eIF4A-VI, and there are apparent common location feelings with PDCD4 Condition further illustrates the polypeptide sequence and interferes the specificity of PDCD4.Fig. 3 C shows in primary neuron, 9R-eIF4A- VI can promote the expression of BDNF.(Fig. 3 d) has detected the table of BDNF in primary neuronal culture supernatant by way of ELISA It reaches, realizes combination of inventor's design by interference PDCD4 and EIF4A, improve the expression of BDNF to reinforce believing between cynapse Number transmitting, alleviate the purpose of depressive symptom.

Meanwhile inventor also has detected the expression of inflammatory factor in neuronal cell after fused polypeptide is interfered, such as Fig. 3 d institute Show, 9R-eIF4A-VI will not promote the expression of IL-10 and IL-6 to increase.Prove that the polypeptide will not cause neuronal cell inflammation Reaction, use are safer.

Embodiment 4

In order to further confirm therapeutic effect of the fused polypeptide to depression, the present embodiment makes depressed cell model, and Study effect of the 9R-eIF4A-VI to depression model.

(Fig. 4 a/4b) it is known in the art that CRS can inhibit the activity of mTOR cause PDCD4 degradation be suppressed, mistake More PDCD4 can inhibit the expression of BDNF, and (Fig. 4 b) inventor inhibits mTOR by Rapamycin with primary neuronal cell Activity, construct primary neuronal cell depression model.

9R-eIF4A-VI is added into the successful cell model of building in (Fig. 4 c), and 9R-eIF4A-VI can be obviously promoted The expression of BDNF, it was demonstrated that under depressive state, which can pass through the expression of BDNF in the depressed primary neuronal cell of induction It realizes the reparation to neuronal cell, corrects depressive symptom.

Embodiment 5

It is supported to be used as to 9R-eIF4A-VI as the application offer Mechanism Study of antidepressant, in the present embodiment It is conducted a preliminary study for the interference mechanism of 9R-eIF4A-VI.

As shown in Figure 5 a, inventor's addition 9R-eIF4A-VI is stimulated in SHSY5Y cell line, and slice result is shown The expression of PDCD4 is there is no being substantially reduced under low concentration, but the expression of PDCD4 is obviously to drop at high concentration (200nM) Low, there is statistical significance.

As shown in Figure 5 b, the genomic expression of related target is detected by way of RT-PCR, inventor has found 9R- EIF4A-VI will not cause the variation of PDCD4mRNA level.(Fig. 5 d/5c) inventor passes through blocks protein enzyme body and lyase respectively Body approach finds that 9R-eIF4A-VI promotes the degradation of PDCD4 mainly to carry out by lysosomal pathway.

The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.

SEQUENCE LISTING

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Claims (10)

1. application of the polypeptide of the amino acid sequence as shown in SEQ ID NO:4 as PDCD4 and eIF4A binding inhibitors.

2. a kind of fused polypeptide, which is characterized in that the main ammonia as shown in 9R cross-film sequence and SEQ ID NO:4 of the fused polypeptide Base acid sequence is formed by connecting.

3. fused polypeptide as claimed in claim 2, which is characterized in that N sections are 9R cross-film sequence, and C sections are SEQ ID NO:4 institute Show amino acid sequence.

4. fused polypeptide as claimed in claim 2, which is characterized in that the fused polypeptide has ammonia shown in SEQ ID NO:6 Base acid sequence.

5. fused polypeptide as claimed in claim 2, which is characterized in that the fused polypeptide is amino shown in SEQ ID NO:6 Acid sequence is substituted, lacks and/or increases polypeptide one or several amino acid derived and with the same function.

6. encoding the nucleotide sequence of any one of the claim 2-5 fused polypeptide, the nucleotide sequence includes due to close Numeral degeneracy is translated to obtain the nucleotide sequence of same amino acid sequence.

Polypeptide, the described in any item fused polypeptides of claim 2-5 and/or the power of amino acid sequence shown in 7.SEQ ID NO:4 Benefit require 6 described in nucleotides sequence be listed in preparation antidepressant in application.

8. the use as claimed in claim 7, which is characterized in that the polypeptide of amino acid sequence shown in the SEQ ID NO:4 or Fused polypeptide is as BDNF agonist；Preferably, the polypeptide of amino acid sequence shown in SEQ ID NO:4 or the fused polypeptide are made For IIc-BDNF agonist.

9. a kind of antidepressant, which is characterized in that the antidepressant includes amino acid sequence shown in SEQ ID NO:4 Any one of polypeptide or claim the 2-5 fused polypeptide.

10. antidepressant as claimed in claim 9, which is characterized in that the concentration of fused polypeptide described in the drug is 12.5nM or more；Preferably, the antidepressant further includes pharmaceutically acceptable carrier, excipient in addition to fused polypeptide Or adjuvant.