CN102643350A - Polypeptide for detecting activity of ATG4B (Autophagy-related protein 4B) in living cell and application thereof - Google Patents

Abstract

The invention relates to a polypeptide for detecting the enzymatic activity of ATG4B (Autophagy-related protein 4B) in a living cell and an application thereof, in particular to the design and synthesis of a novel small peptide and the application of the small peptide in detecting the autophagy level of cells related to the activity of the ATG4B. The polypeptide disclosed by the invention is used for detecting the activity of the ATG4B in the living cell, and the detection has the advantages that the operation is simple and quick and the detection result is objective and accurate.

Description

Active polypeptide of ATG4B and application thereof in a kind of detection viable cell

Technical field

The present invention relates to a kind of can the specific detection viable cell in the preparation and the application of novel polypeptide of ATG4B enzymic activity, particularly a kind of design of novel little peptide, synthetic and this little peptide are detecting the application of the cell autophagy relevant with the ATG4B activity aspect horizontal.

Background technology

Autophagy GAP-associated protein GAP 4B (Autophagy-raleted protein 4B; ATG4B) be a kind of key enzyme in the cell autophagy process; At first equal discovery in 2003 and report by

; Taichi etc. has resolved its structure subsequently, and has illustrated its effect in the cell autophagy generating process and mechanism gradually.

1.ATG4B essentially consist and structure

The ATG4B total length is made up of 394 amino-acid residues, comprises 8 αLuo Xuanjiegou and 13 βZhe Die structures (Fig. 1) in its structure, forms flexible and 2 structural domains of proteolytic enzyme altogether.D278 wherein, H280 and C74 amino acids residue are the critical sites (Fig. 2) in its performance function.

2.ATG4B effect in the cell autophagy process and mechanism

Cell autophagy (Autophagy) is a kind of basic survival mechanisms of cell; Its major function is components such as the interior organoid that damages of timely scavenger cell, protein; The cleaning that keeps cell interior is with stable; And can will decompose the raw material of the products such as amino acid of generation as the cell growth, therefore be called as intracellular " destructor plant ".The process of cell autophagy can be divided into 3 stages: at first generate one section double-deck immobilized artificial membrane; Secondly duplicature constantly extends, and the cellular constituent that needs are degraded is wrapped to form cytolysosome; Cytolysosome and lysosome merge to form the autophagy lysosome then, and by the proteolytic enzyme in the lysosome with the degraded of the cellular constituent in the cytolysosome (Fig. 3).

Wherein, in the generative process of cytolysosome, ATG4B is playing the part of important role.As shown in Figure 4; In the cytolysosome forming process; ATG4B at first cuts one section with its substrate protein LC3; Make it become LC3-II (16Kda), connect the preceding paragraph fat chain by Atg3/Atg7 and Atg5/Atg12 at the C of LC3-II end then, and LC3-II is anchored on the film of cytolysosome by LC3-I (18Kda).The variation in this course of LC3 albumen is the significant incident of cell autophagy process, therefore is used to the height of autophagy level in the identification of cell, and promptly the high more representative autophagy of the amount of LC3-II level is high more, means that also the ATG4B activity is high more.

3.ATG4B enzymolysis to LC3

LC3 is called autophagy GAP-associated protein GAP 8 again, and (Autophagy related protein 8 ATG8), is the key protein in the cell autophagy generating process, is the specificity enzymolysis substrate of ATG4B.Research shows that the enzymolysis site of ATG4B in LC3 is positioned at its glycine residue of 116, and TFG tripeptides wherein is the basic substrate sequence (Fig. 5) of ATG4B enzymolysis identification, thus the TFG tripeptides can be separately as the substrate use of ATG4B.

4.ATG4B activity test method

Because the vital role of ATG4B in the cell autophagy generating process, usually with the activity level of ATG4B in the cell index as the cell autophagy level, detection method commonly used comprises following several kinds at present:

(1) protein immunoblotting method (Western blot)

Through isolated cell and prepare protein example, use the variation between the LC3-I and LC3-II in the antibody test cell of LC3 to judge the generation and ATG4B activity of cell autophagy after the electrophoretic separation.Be most popular method during present cell autophagy detects, advantage is directly perceived, clear, and shortcoming is can't accurate quantification, trivial operations.

(2) activity based on the TFG tripeptides detects

Because the substrate that the TFG tripeptides can be used as ATG4B directly acts on, therefore the C-terminal of TFG tripeptides can be used for detecting the ATG4B activity after with fluorescein-labelled (like AFC).But get into cell owing to the TFG tripeptides can not pass freely through cytolemma, therefore need, hatch cell lysate with detecting substrate, and then detect the variation of fluorescence associated value, thereby reflection ATG4B activity level with after the lysis.This method advantage be can the direct quantitative cell in ATG4B active, method is simple relatively; Shortcoming is that the ATG4B that can't directly measure in the viable cell is active.

(3) detect based on the proteic FRET of LC3

Reports such as Min, with the proteic N end of LC3 and C end difference coupling green fluorescent protein and yellow fluorescence protein, the recombinant protein of expression can be used as the detection substrate and measures the ATG4B activity.Owing to detecting the substrate marked two kinds of GFPs are arranged, energy can take place and shift (FRET) in its emission wavelength, and behind the ATG4B excision substrate C is held release and causes that change in fluorescence can be used as the detection index.This method advantage is but that ATG4B is active in the direct quantitative cell, but and high throughput testing; Shortcoming is the clone that needs preparation stably express GFP-LC3-YFP recombinant protein, and quality is uncontrollable, trivial operations.

Summary of the invention

First purpose of the present invention is to provide the active polypeptide of ATG4B in a kind of detection viable cell, and it is active that said polypeptide is used to detect in the viable cell ATG4B, has simple to operately, and fast, detected result is objective, advantage accurately.

Polypeptide according to the invention is a fusogenic peptide, and its aminoacid sequence is shown in SEQ ID NO:1.

The present invention is the basis with the substrate sequence TFG of ATG4B effect, wears film peptide RKKRRQRRR and amino-acid residue G in the coupling of the N of the substrate sequence TFG of ATG4B effect end, obtains above-mentioned fusogenic peptide.

Second purpose of the present invention provides a kind of test kit that comprises the aforementioned polypeptides that has affinity tag, and said test kit is used to detect the activity of ATG4B in the viable cell.

Said affinity tag is used micromolecular compound for detecting.

Said affinity tag is used micromolecular compound for detecting.

Said detection uses micromolecular compound to be resorcinolphthalein or oil of mirbane amine salt.

The essentially consist of test kit comprises: the polypeptide of tape label thing: use sterilized water to be made into final concentration and be 1mM; PH value is 7.5 reaction buffers (100mM Hepes, 20% glycerine, 0.5mM EDTA, 5mM DTT).

When polypeptide according to the invention is used for test kit, at the N of polypeptide end and C end additional marking thing, be used for mark background contrast signal and detection signal, affinity tag of the present invention can be to detect to use micromolecular compound, like resorcinolphthalein or pNA (p-nitrophenyl amine salt) etc.Can be different with affinity tag labeling polypeptide N end and C end color, its N end fluorescence contrast signal as a setting uses, and C end fluorescence uses as detection signal.

Also can connect the affinity tag that is used for mark detecting signal separately, use cell quantity or total protein concentration contrast signal as a setting at the peptide C end.

Polypeptide provided by the invention is used to detect ATG4B activity in the viable cell, and detection method is applicable to cell flow cytometer showed, spectrophotofluorometer check and analysis etc.

Advantage of the present invention is: 1. need not lysing cell, can directly detect ATG4B activity in the viable cell; 2. objectivity is strong, all detects and uses instrument to accomplish, and the result is a numerical value, has at utmost avoided artificial subjective factor; 3. operation is simple relatively, quick, can in 2 hours, accomplish mensuration.

The technique effect that the present invention reached: can directly detect ATG4B activity in the viable cell, and can reflect simply, fast, objectively that ATG4B is active in the viable cell.

Description of drawings

Fig. 1 is that secondary structure is formed synoptic diagram in the ATG4B albumen;

Fig. 2 is the proteic space structure synoptic diagram of ATG4B;

The cell autophagy process synoptic diagram that Fig. 3 participates in for ATG4B albumen;

Fig. 4 is the effect of ATG4B in the cytolysosome forming process;

Fig. 5 is the enzymolysis synoptic diagram of ATG4B to substrate LC3;

Fig. 6 is that Western blot analysis of cells autophagy level changes;

Fig. 7 is ATG4B determination of activity in the cell lysate;

Fig. 8 wears the film function for polypeptide according to the invention;

Fig. 9 is that fluorescence spectrophotometry ATG4B is active;

Figure 10 measures the ATG4B activity for the fluidic cell method.

Embodiment

Associated materials, reagent, instrument explanation

Cell: the human cervical carcinoma Hela cell, available from Shanghai Inst. of Life Science, CAS cell resource center.

Cell DMEM substratum: available from U.S. Hyclone company, article No.: SH30022.01B

The hungry substratum HBSS of cell: available from U.S. Hyclone company, article No.: SH30030.02B

Pancreatin: available from U.S. Hyclone company, article No.: SH30042.01

PBS damping fluid: available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, article No.: ZLI-9061

Cell pyrolysis liquid RIPA: available from the green skies, Beijing bio tech ltd, article No.: P0013B

LC3 antibody: available from U.S. Cell signaling technology company, article No.: 2775S, working concentration 1: 1000

β-actin antibody: available from U.S. Thermo Scientific company, working concentration 1: 3000

The protein content determination test kit: BCA determination of protein concentration test kit (enhancement type), available from the green skies, Beijing bio tech ltd, article No.: P0010

Fluorescence microplate reader: Spectra Max Gemini XPS plate reader, the U.S.

Inverted fluorescence microscope: OLYMPUS IX-81, Japan

Stream type cell analyzer: Beckman XL, the U.S.

Embodiment 1

One, the design of polypeptide

Substrate sequence TFG with the ATG4B effect is the basis, borrows the direct coupling of peptide bond to wear film peptide RKKRRQRRR and amino-acid residue G for one section at its N end, forms one section novel polypeptide (fusogenic peptide) (biochemical ltd is synthetic by the Shanghai gill).Said polypeptid acid sequence is RKKRRQRRRGTFG, and molecular weight is 1701.02.

Embodiment 2

Two, the design of peptide substrate is with synthetic

N end and C end affinity tag mark with polypeptide obtain peptide substrate.Said affinity tag is used micromolecular compound for detecting, and like resorcinolphthalein or oil of mirbane amine salt (pNA) etc., the affinity tag of present embodiment adopts resorcinolphthalein.Labeling polypeptide N end is different with the resorcinolphthalein color of C end, and N end fluorescence contrast signal as a setting uses, and C end fluorescence uses as detection signal.Can also be at fusogenic peptide C end separately with fluorescein-labelled as detection signal, use cell quantity or total protein concentration be contrast signal as a setting.The N end of present embodiment polypeptide is with FITC green fluorescence mark, and C holds with AMC blue-fluorescence mark.Certainly, the N of polypeptide end and C end can also be used other marking method, and with behind the FITC mark, the C end can be used red fluorescence rhodamine mark like the N end; The N end can be used FITC or pNA or AFC mark etc. with C end behind the red fluorescence rhodamine mark; Also can use rhodamine or marks such as FITC or pNA or AFC at the C end separately.

The peptide substrate structure of present embodiment design is following:

FITC-RKKRRQRRRGTFG-AMC (substrate 1)

The molecular weight of peptide substrate is 2361.77, and biochemical ltd is synthetic by the Shanghai gill.Substrate 1 is the solid yellow powder, and detecting purity through HPLC is 97.30%.Resultant quantity 10.8mg is dissolved in the 4.573ml sterilized water, and final concentration is 1mM, deposits in-20 ℃ of refrigerators.

Be the function and the advantage of checking substrate 1, also entrust gill biochemical ltd in Shanghai to synthesize substrate 2 as experiment contrast simultaneously, the structure of substrate 2 is following:

FITC-GTFG-AMC (substrate 2)

Substrate 2 molecular weight are 1040.15, are the yellow solid powder, purity 95.67%, and resultant quantity 10.0mg is dissolved in the 9.6ml sterilized water, and final concentration is 1mM, deposits in-20 ℃ of refrigerators.

Polypeptid acid sequence in the substrate 2 is shown in SEQ ID NO:2.

Embodiment 3

Three, polypeptide can detect ATG4B activity in the cell lysate

1. cell autophagy guidance model

Whether have the active ability of the ATG4B of detection in order to detect polypeptide according to the invention, at first made up the cell autophagy guidance model.With human cervical carcinoma cell Hela with 1 * 10 ⁵The ratio of/ml is seeded in the 6 porocyte culture plates, every hole 2ml.After the incubated overnight substratum is removed, control group is still cultivated with the normal cultured base, and the experimental group cell is used HBSS (the Hepas damping fluid of calcic, mg ion and 10mM) substratum instead and cultivated respectively 2,3,4 hours after the PBS washing, as the hungry model of cell.After inducing end, collect and respectively organize cell, measure the LC3 changing conditions with protein immunoblotting method after the cracking.The result is as shown in Figure 6, increases with induction time, and the proteic expression amount of LC3-II raises, and shows the ATG4B increased activity, and the cell autophagy level increases, and is best inductive condition with hungry 3 hours.

2. ATG4B determination of activity in the cell lysate

Control group and experimental group cell are removed substratum, wash 2 times, add cell pyrolysis liquid in cracking 10min on ice with PBS.With the cell pyrolysis liquid sucking-off, (2 μ l 1mM) act on 40min behind the mixing in 37 ℃ of incubators, on spectrophotofluorometer, carry out fluorometric assay with 320nm excitation wavelength, 400nm emission wavelength then respectively to get 50 μ l and substrate 1 or 2.Use the Xylene Brilliant Cyanine G method to measure protein contnt in the cell pyrolysis liquid simultaneously, the fluorescent value of measuring is carried out stdn.

Mensuration result is as shown in Figure 7; Substrate 1 and 2 all can well be measured ATG4B activity in the cell lysate; Do not have significant difference between the two, and trend and the protein immunoblotting analytical results measured match, explain that the substrate that substrate 1 can be used as ATG4B carries out determination of activity.

Embodiment 4

Four, polypeptide has the film of wearing function

Whether can pass cytolemma and get in the cell for measuring polypeptide according to the invention, with human cervical carcinoma cell Hela with 1 * 10 ⁵The ratio of/ml is seeded in the 6 porocyte culture plates, every hole 2ml.After the incubated overnight substrate 1 and substrate 2 added in the substratum respectively by the concentration of 1 μ M and cultivate; Respectively at 20min; 40min, behind the 1h substratum removed and with after the PBS washing 3 times under fluorescent microscope green fluorescence situation in the observation of cell, the result is as shown in Figure 8.

Can find out that from Fig. 8 control group substrate 2 does not have the film of wearing function fully, and the substrate 1 of experimental group has and significantly wears the film function, and increase it with incubation time and wear the film effect and strengthen.

Embodiment 5

Five, polypeptide can detect ATG4B activity in the viable cell

1. ATG4B is active in the fluorescence spectrophotometry viable cell

According to the cell autophagy guidance model, control group is the normal cultured cell, and experimental group is hungry 3 hours of Hela cell.In the cell culture medium of contrast and experimental group, add substrate 1 subsequently; Concentration is 1 μ μ M, and the continuation effect is 1 hour in 37 ℃ of incubators, removes substratum; With pancreatin cell dissociation is got off after washing 3 times with PBS; Directly on the fluorescence spectrophotofluorometer, measuring the blue-fluorescence value with 320nm excitation wavelength, 400nm emission wavelength with getting 100 μ μ l behind the PBS re-suspended cell, is excitation wavelength with 488nm simultaneously, and 525nm is that emission wavelength is measured the green fluorescence value.The mensuration result of blue-fluorescence value removes the back with blank value and carries out stdn with the green fluorescence value, compares analysis with control group then, and the result sees Fig. 9.

Can find out that from Fig. 9 after hunger was induced, ATG4B is active in the viable cell obviously raise, consistent with the result in the instance 2, explains that novel polypeptide can be used for the active detection of ATG4B in the viable cell fully.

2. ATG4B is active in the cells were tested by flow cytometry viable cell

Cell is handled with 1.Directly be used for flow cytometry after being resuspended in cell among the PBS, the excitation wavelength of blue-fluorescence is 320nm, and emission wavelength is 400nm, counts the blue-fluorescence positive cell number in experimental group and the cellular control unit respectively, and the result sees Figure 10.

Can find out that from Figure 10 hunger induces back blue-fluorescence positive cell group obviously to move to right, ratio significantly raises, and shows the active significantly rising of ATG4B in the cell, conforms to the result of fluorescent spectrophotometer assay.

Conclusion:

Compare with normal ATG4B substrate sequence (substrate 2); ATG4B was active during fusogenic peptide substrate of the present invention (substrate 1) not only can detect in the cell pyrolysis liquid; But also can get into the inner ATG4B activity of directly measuring of viable cell, detection method is applicable to spectrophotofluorometer method and flow cytometer method.

Claims (6)

1. a peptide species, it is characterized in that: this polypeptide is a fusogenic peptide, its aminoacid sequence is shown in SEQ ID NO:1.

2. a test kit is characterized in that: comprise the described polypeptide of the claim 1 that has affinity tag.

3. test kit according to claim 2 is characterized in that: said affinity tag is used micromolecular compound for detecting.

4. test kit according to claim 2 is characterized in that: said detection uses micromolecular compound to be resorcinolphthalein or oil of mirbane amine salt.

5. test kit according to claim 2; It is characterized in that, this test kit basic composition is: use sterilized water to be made into the polypeptide of final concentration as the tape label thing of 1mM: pH value is 7.5 reaction buffer: 100mM Hepes, 20% glycerine, 0.5mM EDTA, 5mM DTT.

6. test kit according to claim 2 is characterized in that: said test kit is used to detect the activity of ATG4B in the viable cell.

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