CN109880811A - A kind of polypeptide promoting cell autophagy and/or endocytosis or the derivative of the polypeptide and application thereof - Google Patents

Abstract

The invention discloses polypeptides or the derivative of the polypeptide of a kind of promotion cell autophagy and/or endocytosis and application thereof, the polypeptide is the peptide fragment on the catalytic subunit p110 β of phosphoinositide 3-kinase, the peptide fragment is using the 596th glutamine of p110 β peptide chain and the 597th isoleucine as Key residues, the N-terminal of the glutamine is connected at least seven amino acid residue, the C-terminal of the isoleucine is connected at least five amino acid residue, and the polypeptide includes at least 14 amino acid residues；The derivative connect with cell-penetrating peptide for the polypeptide and is formed by chimeric peptide.The polypeptide and its derivative can efficiently induce or the autophagy and/or endocytosis of active cell；And the polypeptide and its derivative can be as the agonist of Small GTPases Rab5.

Description

A kind of derivative of the polypeptide for promoting cell autophagy and/or endocytosis or the polypeptide and Its purposes

Technical field

The invention belongs to protein and peptide chemical fields, and in particular to a kind of polypeptide for promoting cell autophagy and/or endocytosis Or derivative of the polypeptide and application thereof.

Background technique

Autophagy is the process that cytosolic components and organelle are swallowed and degraded by two film construction, can be considered as a kind of special Multistep film transportational process.It starts from the part of double film autophagy phagocyte matter, is followed by melting for these vesicas and lysosome Close the degradation included with autophagy.Other than its important stable state effect, this degradation pathway further relates to various human diseases, including Metabolic disease, neurodegenerative disease, cancer and infectious disease.It is under conditions of the nutrition of cell or growth factor are deprived and various Autophagy is induced under stress conditions.It has shown to play a significant role in terms of maintaining cell homeostasis and adjusting cell survival.Autophagy Inhibition it is related with the nosopathology of many types, including but not limited to cancer, Parkinson's disease, Alzheimer disease and Heng Ting Disease, pancreatitis, obesity and diabetes, infectious diseases such as bacterium infection, AIDS virus, hepatitis and native pyreticosis (referring to Rubinsztein DC et al., Nat Rev Drug Discov.11 (9), 709-30 (2012)).It has been known that there is several molecules The starting and process of autophagy are participated in, including but not limited to 1,2- palmityl phosphinositides -3- phosphoric acid (PI3P) contains dual FYVE Albumen (DFCP1), AMP dependant kinase (AMPK) and the Group III PI3 kinases of structural domain, such as Vps34-Vps15- Beclin1-Atg14L compound.In addition, the Rab GTP enzyme of some adjusting secretions and endocytosis film flow has been demonstrated in autophagy Play crucial or booster action, including but not limited to Rab1, Rab5, Rab7, Rab9, Rab24 and Rab33.

Endocytosis refers to the process of that mammalian cell absorbs extracellular substances.Endocytosis is taken in many important cell function Can, including intake extracellular nutrients, cell surface receptor expression is adjusted, cell polarity and antigen presentation are maintained.Endocytosis way Diameter is also viral, and toxin and symbiotic microorganism are utilized, to enter cell.There is the endocytic pathway of several types, i.e., it is receptor-mediated Pass through the coated cell endocytic of clathrin, it is cave-shaped virus or film bud, macrophage increase disease and phagocytosis.It has illustrated Effect of the endocytosis in physiology and pathologic process, including maintain polarization, antigen presentation, glucose transport, artery Encytosis (Mukherjee, S.et al., the Physiol Rev of atherosis, Alzheimer's disease and toxin and virus 77 (3), 759-803,1997)

In general, p110 β is the catalytic subunit of PI3K, can be with the pure and mild phosphatidylinositol diphosphate of phosphorylation phosphatidyl-4 It generates 3,4,5- triphosphoric acid phosphatidylinositols (PIP3).PIP3 contains the domain PH by raising to the film including AKT1 and PDPK1 The protein of (Pleckstrin homeodomain) plays key effect, activation participate in cell growth, survival, proliferation, movement and The signal cascade of form.In addition, p110 β is by g protein coupled receptor (GPCR) ligand such as CXCL12, sphingosine 1-phosphate and molten The activation of AKT1 is participated in after the stimulation of serium inorganic phosphorus resin acid.P110 β be also required in different signal paths stable platelet adhesion reaction and Aggregation, and by GPCRs, α IIb/ β -3 integrin (ITGA2B/ITGB3) and ITAM (based on immunity receptor tyrosine Activation motif) cause platelet activation signal in work.In addition, p110 β is adjusted necessary to cell transmitting convergent force The adhesion strength of ITGA2B/ITGB3 activated receptor.Need the platelet aggregation induced by F2 (fibrin ferment) and thromboxane A2 (TXA2) Collect and works in cell survival.Have shown that p110 β is formed in cell migration and autophagosome and acted as in encytosis With.P110 β also adjusts the Intracellular levels of phosphatidylinositols 3- phosphoric acid, and activates PIK3C3 kinase activity, and can be used as bracket, Independently of its lipid kinase activity actively to adjust autophagy.In addition, p110 β may have in insulin signal transduction as not Need the effect of the scaffolding protein of lipid kinase activity.P110 β may adjust cell Proliferation and clathrin-mediated endocytosis There is the function unrelated with kinases in effect.Medium of the p110 β as oncogenic signals in the cell line for lacking PTEN.Finally, P110 β function is necessary to ERBB2 and RAS driving tumour growth.

Rab5 is the Small GTPases albumen generated from RAB5A gene expression.There are two types of forms for it: 1) GTP is combined, also referred to as Its activated state (herein for " Rab5-GTP, active Rab5 or Rab5 ") and 2) GDP are combined or its inactive form.It is logical Often known Rab GTP enzyme adjustment secretion and endocytosis film flow, and have been demonstrated to play crucial or booster action in autophagy.Thin The Small GTPases Rab5 to play a crucial role in born of the same parents' autophagocytosis is participated in certainly by the interaction with 1 complex of Vps34-Beclin Body phagosome forms (Ravikumar, B.et al., J Cell Sci 121,1649-1660,2008).The GTP of Rab5 is combined Form be adjusting film transport active form (Barbieri, MA et al., J Biol Chem 269,18720-18722, 1994；Stenmark, H.et al., EMBO J 13,1287-1296,1994).In addition, including p110 β shortage and Rab5 inactivation It gulps down and causes some similar changes in autophagy approach, show that p110 β and Rab5 may play it in identical signal path Function (Dou, Z.et al., 2010).But both sides relation is unknown.

Summary of the invention

The present invention be overcome both p110 β and Rab5 interaction relationship in the prior art unknown, and cell autophagy and/ Or the very not specific defect of the mechanism of endocytosis, provide a kind of promotion cell autophagy and/or endocytosis polypeptide or the polypeptide Derivative and application thereof.The polypeptide and its derivative all have the function of p110 β, the research of the invention finds that the polypeptide and its Derivative and p110 β are final to promote cell autophagy and/or endocytosis competitively in conjunction with Rab5, and can be used as Small GTPases The agonist of Rab5.

The present invention provides the derivative of a kind of polypeptide for promoting cell autophagy and/or endocytosis or the polypeptide, the polypeptide For the peptide fragment on the catalytic subunit p110 β of phosphoinositide 3-kinase, the peptide fragment is with the 596th paddy ammonia of p110 β peptide chain Amide and the 597th isoleucine are Key residues, and the N-terminal of the glutamine is connected at least seven amino acid residue, institute The C-terminal for stating isoleucine is connected at least five amino acid residue, and the polypeptide includes at least 14 amino acid residues；Described Derivative connect with cell-penetrating peptide for the polypeptide and is formed by chimeric peptide.

Preferably, the N-terminal of the glutamine Key residues is connected with 7~10 amino acid residues, the isoleucine C-terminal be connected with the amino acid residue of 5~8 amino acid residues and/or the polypeptide and be no more than 20；More preferably, described more The amino acid sequence of peptide has sequence shown in SEQ ID NO.1 in sequence table；Further more preferably, the amino acid of the polypeptide Sequence is as shown in SEQ ID NO.2 in sequence table.

Such as this field routine, the cell-penetrating peptide can connect the N-terminal or C section in the polypeptide, preferably, described Cell-penetrating peptide be connected to the N section of the polypeptide；More preferably, the cell-penetrating peptide is TAT structural domain, the TAT structural domain Amino acid sequence is as shown in SEQ ID NO.3；Further more preferably, in the amino acid sequence of the polypeptide derivative such as sequence table Shown in SEQ ID NO.4 or SEQ ID NO.5.

The present invention also provides the derivative of the polypeptide or the polypeptide of a kind of above-mentioned promotion cell autophagy and/or endocytosis with Purposes in the drug for the disease that the agonist or treatment Small GTPases Rab5 that prepare Small GTPases Rab5 mediate.The disease compared with It goodly include metabolic disease, neurodegenerative disease, cancer and infectious disease；The metabolic disease be preferably Pancreas Disease, obesity or Diabetes；The neurodegenerative disease is preferably Parkinson's disease, Alzheimer's disease or Huntington disease；And/or the infection Disease is preferably AIDS, hepatitis or native pyreticosis.Preferably, the polypeptide or polypeptide derivative are unique work of the drug Property ingredient.

A kind of method that cell autophagy effect or encytosis are adjusted the present invention also provides non-therapeutic use, will be above-mentioned Promote the polypeptide of cell autophagy and/or endocytosis or the derivative and mixing with cells of the polypeptide；Preferably, the polypeptide or described Final concentration of 30 μM after polypeptide derivative and mixing with cells.

The present invention also provides the agonist of Small GTPases Rab5 a kind of, the agonist include above-mentioned promotion cell autophagy and/ Or the polypeptide or the polypeptide of endocytosis.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is that:

Polypeptide and its derivative provided by the present invention can efficiently induce or the autophagy and/or endocytosis of active cell； The polypeptide and its derivative can be as the agonist of Small GTPases Rab5.

Detailed description of the invention

Fig. 1 is p110 β/Rab5- binding peptide TAT-BR1 or TAT-BR2 Induces Autophagy, and wherein Rapamycin is thunder The English name of pa mycin.

Fig. 2 is TAT-BR1 and TAT-BR2 efficient activation autophagy function.

Fig. 3 is the agonist that polypeptide BR1 is Rab5.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

The synthesis of 1 polypeptide of embodiment

Polypeptide BR1 (SEQ ID NO.1), TAT-BR1 (SEQ ID NO.4) and TAT-BR2 (SEQ ID NO.5) by The synthesis of Peptide 2.0, Inc. company, synthetic method used are solid-state phase synthesi (Solid Phase Peptide Synthesis).In general, Solid phase peptide synthesis includes consolidating for the synthetic polymer containing the reactive group for including but is not limited to-OH Body support.Reactive group is easy to react with the carboxyl of the N-a amino acid protected, thus with polymer covalent bond.Then It can remove amino protecting group, and can be by the amino acid of second N-a protection and the amino acid couplings being connect.Repeat this A little steps, the sequence needed for obtaining.At the end of synthesis, C- end amino acid and polymerization are cut using different reagents Key between object carrier；Peptide is subsequently into solution and can obtain from solution.TAT structural domain is added so that peptide is trained in cell It supports and is easier to penetrate into cell (Wagstaff, K.M., Jans, D.A.Current Medicinal Chemistry in vivo 13,1371-1387,2006)

Embodiment 2 polypeptide derivative TAT-BR1 and TAT-BR2 induce Hela cell autophagy

Hela cell (Human cervical cancer cell lines are purchased from ATCC) stablizes expression GFP-LC3, takes 4 parts of isometric cell (cells Concentration is 1 × 10⁶A/ml) be added be separately added into dimethyl sulfoxide (DMSO), TAT (30 μM), rapamycin (1 μM), TAT- BR1 (30 μM) and TAT-BR2 (30 μM).After 24 hours, take pictures under the microscope in fluorescence microscopy.Inducing autophagy is by dotted (Figure 1A) that the formation of GFP-LC3 indicates.

Show that the cell of dotted GFP-LC3 is calculated and be quantified as the percentage of total cell, the results showed that TAT-BR1 and TAT-BR2 peptide can induce autophagy GFP-LC3 (Figure 1B).Wherein rapamycin (Rapamycin) is used as inducing the sun of autophagy Property control.

3 polypeptide derivative TAT-BR1 and TAT-BR2 efficient activation cell autophagy of embodiment

HeLa human cervical carcinoma cell (by mycoplasma contamination during laboratory cultures) is tied with dimethyl sulfoxide, TAT respectively Structure domain (30 μM), TAT-BR1 peptide (30 μM), (30 μM) of TAT-BR2 peptide carry out 24 hours culture (used medium be containing 10% fetal calf serum, 2mM L-Glutamine, 100 units/mL penicillin (penicillin), 100 μ g/mL streptomysins DMEM, temperature are 37 DEG C) after, with DNA dyestuff 4', 6- diamidino -2-phenylindone (DAPI) of 1 mg/ml to cell into Row dyeing.The result shows that dimethyl sulfoxide and TAT structural domain treatment cell show the cytoplasm dyeing of DNA, show mycoplasma Pollution.TAT-BR1 and TAT-BR2 cell mycoplasma is significantly removed.

4 polypeptide BR1 of embodiment is the agonist of Rab5

The non-specific peptide fragment (mispairing peptide, SEQ ID NO.6) of BR1 and random alignment with specificity are respectively added to P110 β+/+or -/- MEFs cell pyrolysis liquid in.GST-R5BD precipitating display (Fig. 3), polypeptide BR1 can restore p110 β and lack The activity (Rab5-GTP) for falling into Rab5 in cell pyrolysis liquid illustrates that polypeptide BR1 is the agonist of Rab5.

SEQUENCE LISTING

<110>Wuhan three is at biological medicine Co., Ltd

<120>derivative and purposes of a kind of polypeptide for promoting cell autophagy and/or endocytosis or the polypeptide

<130> P1711100C

<160> 6

<170> PatentIn version 3.5

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<213> Artificial Sequence

<220>

<223> BR1

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Ala Gln Leu Gln Ala Leu Leu Gln Ile Trp Pro Lys Leu Pro

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<213> Artificial Sequence

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Glu Asp Val Ala Gln Leu Gln Ala Leu Leu Gln Ile Trp Pro Lys Leu

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Pro Pro Arg Glu

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<223>TAT structural domain

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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Gln Leu Gln Ala Leu

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Leu Gln Ile Trp Pro Lys Leu Pro

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<223> TAT-BR2

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Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Asp Val Ala Gln Leu

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Gln Ala Leu Leu Gln Ile Trp Pro Lys Leu Pro Pro Arg Glu

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<223>mispairing peptide

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Leu Gln Leu Val Ala Asp Ala Gln Leu Ile Gln Leu Glu

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Claims (10)

1. the derivative of a kind of polypeptide for promoting cell autophagy and/or endocytosis or the polypeptide, which is characterized in that the polypeptide is Peptide fragment on the catalytic subunit p110 β of phosphoinositide 3-kinase, the peptide fragment is with the 596th glutamy of p110 β peptide chain Amine and the 597th isoleucine are Key residues, and the N-terminal of the glutamine is connected at least seven amino acid residue, described The C-terminal of isoleucine is connected at least five amino acid residue, and the polypeptide includes at least 14 amino acid residues；Described spreads out Biology connect with cell-penetrating peptide for the polypeptide and is formed by chimeric peptide.

2. the polypeptide of cell autophagy and/or endocytosis or the derivative of the polypeptide, feature is promoted to exist as described in claim 1 In the N-terminal of the glutamine Key residues is connected with 7~10 amino acid residues, and the C-terminal of the isoleucine is connected with 5 The amino acid residue of~8 amino acid residues and/or the polypeptide is no more than 20；Preferably, the amino acid sequence of the polypeptide Column have sequence shown in SEQ ID NO.1 in sequence table.

3. the polypeptide of cell autophagy and/or endocytosis or the derivative of the polypeptide, feature is promoted to exist as claimed in claim 2 In the amino acid sequence of the polypeptide is as shown in SEQ ID NO.2 in sequence table.

4. the derivative of the polypeptide as claimed in any one of claims 1 to 3 for promoting cell autophagy and/or endocytosis or the polypeptide Object, which is characterized in that the cell-penetrating peptide is connected to the N section of the polypeptide；Preferably, the cell-penetrating peptide is TAT structure Domain, the amino acid sequence of the TAT structural domain is as shown in SEQ ID NO.3；More preferably, the amino acid sequence of the polypeptide derivative Column are as shown in SEQ ID NO.4 in sequence table or SEQ ID NO.5.

5. a kind of such as the described in any item promotion cell autophagies of Claims 1 to 4 and/or the polypeptide of endocytosis or spreading out for the polypeptide Purposes of the biology in the drug for the disease that the agonist or treatment Small GTPases Rab5 for being used to prepare Small GTPases Rab5 mediate.

6. purposes as claimed in claim 5, which is characterized in that the disease includes metabolic disease, neurodegenerative disease, cancer And infectious disease.

7. purposes as claimed in claim 6, which is characterized in that the metabolic disease is Pancreas Disease, obesity or diabetes；

The neurodegenerative disease is Parkinson's disease, Alzheimer's disease or Huntington disease；

And/or the infectious disease is AIDS, hepatitis or native pyreticosis.

8. the purposes as described in claim 5~7, which is characterized in that the derivative of the polypeptide or polypeptide is sole active Ingredient.

9. adjusting to a kind of non-therapeutic use the method for cell autophagy effect or encytosis, which is characterized in that will be such as right It is required that the derivative and mixing with cells of 1~4 described in any item polypeptides for promoting cell autophagy and/or endocytosis or the polypeptide； Preferably, the derivative of the polypeptide or the polypeptide and final concentration of 30 μM after mixing with cells.

10. a kind of agonist of Small GTPases Rab5, which is characterized in that the agonist includes such as any one of Claims 1 to 4 The derivative of the polypeptide or the polypeptide for promoting cell autophagy and/or endocytosis.