CN106928370A - A kind of screening system of REG γ 20S proteasome inhibitors and its application - Google Patents
A kind of screening system of REG γ 20S proteasome inhibitors and its application Download PDFInfo
- Publication number
- CN106928370A CN106928370A CN201610850405.3A CN201610850405A CN106928370A CN 106928370 A CN106928370 A CN 106928370A CN 201610850405 A CN201610850405 A CN 201610850405A CN 106928370 A CN106928370 A CN 106928370A
- Authority
- CN
- China
- Prior art keywords
- reg
- lucn
- linker1
- fusion protein
- lucc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of screening system of REG γ 20S proteasome inhibitors, the screening system includes the lucC (linker1 394) of fusion protein lucN (416 linker1) REG γ and α 7；Based on the inherent mechanism that REG γ 20S proteasomes are combined by REG γ with α 7, using luciferase fragment complementation technology, REG γ 20S proteasome inhibitors are screened.The present invention build comprising the two kinds of screening systems of fusion protein of lucC (linker1 394) of LucN (416 linker1) REG γ and α 7, with good screening effect.The present invention is optimized by screening system, improves the validity and specificity of the screening system, can efficiently carry out the screening of REG γ 20S proteasome micromolecular compound inhibitors.
Description
Technical field
The present invention relates to biochemistry and technical field of biological materials, and in particular to REG γ are combined with 20S proteasomes
Molecular mechanism and using the mechanism carry out REG gamma inhibitors screening and REG γ relevant diseases treatment.
Background technology
Proteasome is present in eucaryote, archeobacteria and some bacteriums, most protein in regulating cell
Degraded and renewal, it is very crucial to maintaining the normal vital movement of cell to play the role of.Proteasome complex is generally by two
Part constitutes：One columnar 20S core granule and proteasome activity factor (19S regulations particle, 11S activation because
Son and Blm10/PA200).
20s proteasomes as proteasome complex core position, be according to α by two kinds of subunits of α, β7β7β7α7Mould
28 subunit complex that type is constituted.The ring being made up of 7 α subunits is located at the columnar structured two ends of 20s, 7 β subunit structures
Into ring be located at inner side.α subunits similar to " entrance guard ", only it constituted it is ring-like in open mode when target protein just can be with
Into, and the N-terminal of β subunits contains activation site, plays a part of to cut off polypeptide.Three-dimensional structural analysis show, in no protease
In the presence of body activity factor, β rings are in closure state, are blocked by the polypeptide of α subunit N-terminals；And work as proteasome activation
When the factor is with the N-terminal combination of β subunits, the polypeptide of α subunit N-terminals is removed, and substrate protein can enter 20s proteasomes.β subunits
As catalytic subunit, wherein β1、β2、β5It is avtive spot, respectively with specific caspase, trypsinlike enzyme and class
Chymotrypsin activity, for the protein substrate being cut into inside core granule.
Proteasome activity factor mainly includes two classes：19S proteasomes activity factor and 11S proteasomes activation because
Son.19S proteasomes activity factor can combine to form the 26S proteasome complex of 19S-20S-19S with proteasome, with
The mode that ubiquitin and ATP are relied on is degraded substrate protein.And 11S proteasomes activity factor then combines to form 11S- with proteasome
The proteasome complex of 20S-11S, substrate protein of being degraded in the way of non-ubiquitin and ATP are relied on.
There are three different members in 11S proteasome activity factors family：REG α, REG β and REG γ.REG α/REG β are
Immunoproteasome, its effect is the antigen presentation for participating in MHC-1 class parts.REG γ (alias PA28 γ or PSME3) are initially
It is found in a kind of serum of lupus erythematosus patients, it is a kind of highly conserved to be detected by autoantibody
The nuclear protein for arriving, therefore it is named as Ki antigens.Report REG γ only have the ability of degraded small peptide class substrate before, until
The 20S proteasomes that Li Xiao great waves in 2006 et al. disclose REG γ mediations for the first time are directly dropped by non-ubiquitin and ATP Dependents
The solution auxiliary regulatory protein 3 of steroid receptor, so that find that REG γ have the function of degraded intact proteins, further investigations have shown that
It has important effect in various diseases.In recent years, deepened continuously with to REG γ researchs, REG γ occur in cancer
The effect of development also seems more and more crucial.There are some researches show REG γ are in the carcinoma of the rectum, thyroid cancer and breast cancer and pouring
Expression high, recent studies have shown that REG γ can also promote cutaneum carcinoma and colon cancer to develop in bar cancer, and REG γ are pernicious swollen
Expression high in knurl, recurrent tumor and inflammation associated tumour discloses REG γ may during tumor development
Play particularly important effect.Above result of study implies that REG γ are probably a potential target protein in treatment of cancer how
The function of suppressing REG γ -20S proteasomes will be as an effective oncotherapy new method.
Effect of the REG γ-proteasome degradation system in tumour and various diseases is demonstrated by above-mentioned elaboration, because
This, finds out the inhibitor (micromolecular compound) based on REG γ-proteasomal system extremely important by medicament sifting motion system.
The proteasome inhibitor bortezomib (Bortezomib) of food and medicine Surveillance Authority of the U.S. (FDA) approval, Carfilzomib
Carfilzomib and Ixazomib are mainly used in treating Huppert's disease, but in the treatment of entity tumor, do not appoint also
The report of what proteasome inhibitor.Between the more than ten years in past, how researcher's research suppresses or activator protein enzyme body
Function is with therapy-related disease.But these proteasome inhibitors can treating cancer be to rely on these inhibitor and can adjust
The cancer cell of cancer cell multiplication and some types has specific dependence to the function of proteasome.However, due to protease
Body has extremely important effect in vivo, therefore these can completely inhibit egg as the proteasome inhibitor of medicine
The activity of white enzyme body, so the side effect that the use of this proteasome inhibitor also can be stronger to patient's producing ratio, for example
Patient might have the adverse reactions such as serious insomnia, headache, nausea, diarrhoea.Therefore, it is specific based on REG γ-protease
The inhibitor (micromolecular compound) of system system will likely be a kind of more preferable research direction, prevention of disease and diagnosis and treatment work
It is of great importance.
Protein fragment complementation assay technology can quickly and accurately be studied mutual between protein by various reporter genes
Effect, is a very strong instrument of Way for Studying Protein-Protein Interactions.It is proteasome inhibitor (micromolecular compound)
Screening provide a strong technology platform.Wherein luciferase is one of its most frequently used reporter gene.Fluorescein
Enzyme is widely used in organism optical imagery, and common luciferase has two kinds, is respectively renilla luciferase and firefly
Luciferase, the substrate of renilla luciferase is coelenteron fluorescein, typically produces blue light (λ max=475nm), firefly fluorescence
The substrate of plain enzyme is D- fluoresceins, and general to produce gold-tinted to feux rouges (λ max=575-600nm), their action principle is glimmering
Under the catalytic action of light element enzyme, substrate be oxidized it is luminous, when substrate is excessive, the light quantity subnumber of generation and the concentration of luciferase
Being proportionate property.But it is difficult to be absorbed by bio-tissue because the wavelength of firefly luciferase launching light is longer, so this
It is more extensive that kind luciferase is used in organism.Luciferase fragment complementation technology can real-time monitoring be located at cell
Interactions between protein on matter, nucleus or cell membrane, is not to be limited among the same area.Luciferase fragment complementation technology
It is also a kind of reversible and highly sensitive technology, can be used to monitor body and intracellular transient response and screening protein phase
The inhibitor of interaction.With the development of the technology in recent years, nowadays these technologies are prison in real time in cell and living animal
Survey and research small molecule induction interactions between protein provide a technology platform, while also improve the technology drug development,
The novelty in the fields such as chemical genetics, proteomics and the actual application value of feasibility.
The content of the invention
It is mutual using luciferase fragment the invention provides a kind of screening system of REG γ -20S proteasome inhibitors
Benefit technology is screened to REG γ -20S proteasome inhibitors, and screening system of the invention has efficient detectability, and
The characteristics of high sensitivity, high specific.
The invention provides a kind of screening system of REG γ -20S proteasome inhibitors, the screening system includes melting
Hop protein lucN (416-linker1)-REG γ and α 7-lucC (linker1-394)；Wherein, the fusion protein lucN
(416-linker1)-REG γ are built by 1-416 N-terminal for being connected to REG γ by connecting peptides of luciferase N-terminal fragment
Form, the fusion protein α 7-lucC (linker1-394) pass through connecting peptides for 394-550 by luciferase C-terminal fragment
It is connected to the C-terminal of α 7.
Present invention also offers a kind of screening system of REG γ -20S proteasome inhibitors, the screening system includes
Fusion protein lucN-REG γ (K85QK86Q) and α 7-lucC (linker1-394)；Wherein, the fusion protein lucN-REG
γ (K85QK86Q) is mutated by the 85th lysine of nuclear localization sequence of lucN-REG γ and is obtained, the fusion protein
LucN-REG γ (K85QK86Q) are located in cytoplasm, can dramatically increase the activity of luciferase；The fusion protein α 7-
LucC (linker1-394) is by 394-550 C-terminal that α 7 is connected to by connecting peptides of luciferase C-terminal fragment.
Present invention also offers a kind of fusion protein lucN (416-linker1)-REG γ, wherein, the fusion protein
LucN (416-linker1)-REG γ are by 1-416 N-terminal that REG γ are connected to by connecting peptides of luciferase N-terminal fragment
It is built-up.
Present invention also offers a kind of fusion protein α 7-lucC (linker1-394)；Wherein, the fusion protein α 7-
LucC (linker1-394) is by 394-550 C-terminal that α 7 is connected to by connecting peptides of luciferase C-terminal fragment.
Present invention also offers a kind of fusion protein lucN-REG γ (K85QK86Q), the fusion protein lucN-REG γ
(K85QK86Q) it is mutated by the 85th lysine of nuclear localization sequence of lucN-REG γ and is obtained, the fusion protein
LucN-REG γ (K85QK86Q) are located in cytoplasm, can dramatically increase the activity of luciferase.
Present invention also offers a kind of fusion protein lucN-REG γ 244 (K85QK86Q), the fusion protein lucN-
REG γ 244 (K85QK86Q) carry out mutation and form by the 86th lysine of nuclear localization sequence of lucN-REG γ, the fusion
Albumen is located in cytoplasm.
Present invention also offers a kind of fusion protein LucN (416-linker1)-REG γ 244, by lacking REG γ C ends
10 amino acid in end are built-up, and 10 amino acid of REG γ C-terminals are particularly important with the interaction of α 7 for it, fusion protein
LucN (416-linker1)-REG γ 244 can not interact with α 7-lucC (linker1-394), can as REG γ-
The negative control of the screening system of 20S proteasome inhibitors.
Present invention also offers a kind of REG gamma inhibitors, the REG gamma inhibitors are REG γ (NL), and it passes through REG γ
Lack that the 146th to the 153rd active ring is built-up, the REG γ (NL) can suppress REG γ；In REG γ -20S albumen
In the screening system of enzyme body inhibitor, REG γ (NL) can simulate REG gamma inhibitors, can play similar REG gamma inhibitors
Function, with best simulation effect, so as to carry out the validity of the screening system constructed by inflammation.
Screening system present invention also offers the REG γ -20S proteasome inhibitors is in screening REG γ -20S eggs
Application in white enzyme body inhibitor.
Present invention also offers application of the fusion protein in REG γ -20S proteasome inhibitors are screened.
Present invention also offers applications of the REG γ (NL) in REG γ -20S proteasome inhibitors are screened.
The screening system of REG γ -20S proteasome inhibitors of the invention is combined based on REG γ -20S proteasomes
The screening system of the micromolecular compound inhibitor of mechanism, screens to the fragment selected by luciferase, constructs screening
Best fusion protein LucN (416-linker1)-the REG γ and α 7-lucC (linker1-394) of effect；Screening system is entered
Row optimization,
Luciferase fragment complementation technology has been used in screening system of the invention, can quickly study protein it
Between interaction, fluorescein plum complementary technology is a very strong instrument of Way for Studying Protein-Protein Interactions.
One aspect of the present invention illustrates the inherent mechanism of REG γ -20S proteasomes combination, i.e. REG γ -20S proteasomes
With reference to indeed through the combination between REG γ and α 7, on the other hand, the what is more important present invention utilizes luciferase piece
Section complementary technology constructs the screening system for screening REG γ -20S proteasome inhibitors.
The present invention, through being screened, finally found that luciferase N-terminal fragment 1-416 to the fragment selected by luciferase
Position is connected to the N-terminal of REG γ by connecting peptides, and luciferase C-terminal fragment 394-550 is connected to α's 7 by connecting peptides
C-terminal, i.e., constructed fusion protein lucN (416-linker1)-REG γ and α 7-lucC (linker1-394) screening effect is most
It is good.Then the present invention is optimized to the screening system again, and the present invention is by the nuclear localization sequence the 85th of lucN-REG γ and the
86 lysines are mutated, and prevent the albumen from entering in nucleus after synthesizing, the lucN-REG γ (K85QK86Q) after mutation
With lucN-REG γ 244 (K85QK86Q) be located at cytoplasm in (the REG γ after mutation can go out nucleus and enter cytoplasm, with
α 7-lucC common locations in cytoplasm), the lucN-REG γ (K85QK86Q) after simultaneous mutation can dramatically increase luciferase
Activity.In order to verify the validity of the screening system, the validity of the screening system is verified, build negative control LucN
(416-linker1)-REG γ 244, LucN (416-linker1)-REG γ 244 are missing from 10 amino acid of REG γ C-terminals
Albumen, 10 amino acid of REG γ C-terminals are particularly important for the interaction of REG γ and α 7), LucN (416-linker1)-REG γ
244 find that it can not interact with α 7-lucC (linker1-394), can be as the feminine gender of screening system of the present invention
Compare to verify the validity of screening system；Meanwhile, the REG γ fragments that the present invention constructs many different lengths go to simulate REG
Gamma inhibitors, the 146th REG γ to the 153rd active ring of experiment display missing is that there is REG γ (NL) best simulation to imitate
Really, and subsequent experiment demonstrates REG γ (NL) and can play the function of similar REG gamma inhibitors, REG γ (NL) can press down
Interaction between fusion protein LucN (416-linker1)-REG γ and α 7-lucC (linker1-394) processed, Neng Goumo
Intend the effect of REG gamma inhibitors, further illustrate the validity of constructed screening system.
The beneficial effects of the present invention are the screening system of the REG γ -20S proteasome inhibitors that the present invention is provided, bag
Include fusion protein lucN (416-linker1)-REG γ and α 7-lucC (linker1-394)；Fusion protein lucN (the 416-
Linker1)-REG γ are built-up by the N-terminal that connecting peptides are connected to REG γ by luciferase N-terminal fragment 1-416；
Screening system of the invention is screened using luciferase fragment complementation technology to REG γ -20S proteasome inhibitors, tool
There is efficient detectability, and the characteristics of high sensitivity, high specific.
Brief description of the drawings
Fig. 1 is the co-immunoprecipitation figure of the subunit of proof REG γ specific binding 20S proteasome Alphas 7 in embodiment 1.
Fig. 2 is the reverse co-immunoprecipition figure of the subunit of proof REG γ specific binding 20S proteasome Alphas 7 in embodiment 1.
Fig. 3 is the yeast two-hybrid result figure of the subunit of proof REG γ specific binding 20S proteasome Alphas 7 in embodiment 2.
Fig. 4 is the molecular imaging system schematic diagram of structure research REGr and a7 interactions in embodiment 3.
Fig. 5 has highest to verify lucN (416-lk1)-REG γ and α 7-lucC (lk1-394) combination in embodiment 3
Uciferase activity result figure.
Fig. 6 is LucN (416-lk1)-REG γ and α 7-LucC (lk1-394) immunofluorescence common location figure in embodiment 4.
Fig. 7 is mutation LucN (416-lk1)-REG γ (K85QK86Q) and LucN (416-lk1)-REG γ in embodiment 4
(244) (K85QK86Q) immunofluorescence common location figure.
Fig. 8 is mutation LucN (416-lk1)-REG γ (K85QK86Q) and α 7-lucC (lk1-394) interaction in embodiment 4
Dramatically increase the result figure of uciferase activity.
Fig. 9 is for REG γ (NL) can suppress mutation LucN-REG γ (K85QK86Q) and α 7-lucC are mutual in embodiment 5
Exercising result figure.
Figure 10 can be with LucN-REG γ (K85QK86Q) interaction result for REG γ (NL)-lucC in embodiment 5
Figure.
Figure 11 be embodiment 5 in REG γ (NL) competitive binding REG γ cause REG γ combined with α 7 reduce result figure.
Figure 12 is that overexpression REG γ (NL) can suppress p21 and be degraded result figure in embodiment 5.
Specific embodiment
With reference to specific examples below and accompanying drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following examples.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and with appending claims as protection domain.Implement process of the invention,
Condition, reagent, experimental technique etc., in addition to the following special content for referring to, are the universal knowledege and common knowledge of this area,
The present invention is not particularly limited content.
The co-immunoprecipitation experiment of embodiment 1 proves the subunit of REG γ specific binding 20S proteasome Alphas 7
The α subunits of 20S proteasomes are its regulation subunit, are opened or closed by the regulation and control of activity factor, REG γ energy
Enough promoting the degraded of substrate protein to be likely to REG γ may open the gate of 20S by being combined with α subunits, and the present invention is utilized
The method of co-immunoprecipitation have detected the interaction of REG γ and 20S proteasome Alpha 1- α 7.
Specific experiment method：
1st, before transfecting by HeLa cell seedings in six orifice plates, concentration reaches 60%, cultivates 12 hours.
2nd, 300 μ L plasma-free DMEM mediums are added in 1.5mL centrifuge tubes, purpose plasmid is subsequently added and is transfected with Young
Reagent, the two ratio corresponds to 1 μ g：2 μ L, six orifice plates can turn 1 μ g plasmids per hole.
3rd, vortex viberating 10S is mixed, after being stored at room temperature 15min, with liquid-transfering gun by solution six orifice plates of addition.
4th, after cell growth 48h, start to do co-immunoprecipitation.
5th, culture medium is suctioned out from six orifice plates, with PBS one time to the cold
6th, cell is scraped into 1.5mL EP pipes from culture plate with cell spatula to 1mL PBS are added in each hole again,
5000rpm, is centrifuged 5min, abandons supernatant.
7th, to 100 μ L 1%NP40 cell pyrolysis liquids are added in every solencyte, 40min is cracked on ice, according to experiment needs,
The dephosphorylation enzyme inhibitors such as sodium vanadate, sodium fluoride are added, EP pipes are put into 4 DEG C of centrifuges, 12000rmp is centrifuged 10min.
8th, in supernatant being moved into new 1.5mL EP pipes, take the 10% i.e. cell lysate supernatant of 30 μ L and managed in new EP
It is interior, 6 μ L 5X albumen loading are added, it is well mixed, processed according to the albuminoid method of above-mentioned receipts, as Input.
9th, in remaining 250 μ L of supernatant being moved into new 1.5mL EP pipes, (beads is added to add 5 μ L flag beads
Before should be washed 3 times using 1%NP40 cell pyrolysis liquids), be incubated 4 hours in 4 DEG C of Mute mixers.
10th, EP pipes are put into 4 DEG C of centrifuges, 3500rpm is centrifuged 1min, reverses EP pipes, then is centrifuged 10 seconds, abandons
Clearly, 500 μ L cell pyrolysis liquids are resuspended, clean three times, finally rejoin 30 μ L cell pyrolysis liquids, add 6 μ L 1X albumen
Loading, is processed according to the albuminoid method of above-mentioned receipts, finally uses protein immunoblot experiment testing result.
Experimental result only has phase interaction as shown in figure 1, showing that REG γ do not have interaction with α 1-6 subunits with the subunits of α 7
Tested Fig. 2 such as and also demonstrated REG γ and α 7 with (Flag-REG γ/nip30 is used as positive control), and reverse co-immunoprecipition
Interaction.
The yeast two-hybrid assay of embodiment 2 proves the subunit of REG γ specific binding 20S proteasome Alphas 7
The subunit of REG γ specific binding 20S proteasome Alphas 7 is demonstrated again that using different experiments.
By PSMA1-PSMA7 (α in experiment1-α7) be building up on pGADT7 carriers, trial utilizes yeast two-hybrid assay
The further interaction of checking REG γ and α 7.
As shown in figure 3, only when PGAD- α 7/pGBK-REG γ corotation, yeast just can be for the result of yeast two-hybrid
Four lack growth on plate, that is to say, that REG γ only have interaction with the subunits of α 7, and this is consistent with the result of co-immunoprecipitation
(nip30 is used as positive control).
The specific experimental technique of yeast two-hybrid assay：
1st, take out yeast strain room temperature from -80 DEG C of refrigerators to melt, then oese is burnt to red heat, cooling on alcolhol burner
A small amount of bacterium solution is dipped in the 1.5mL centrifuge tubes equipped with saccharomycete with oese afterwards, then in the full culture medium of YPDA yeast solids
Upper line.
2nd, will streak in Yeast Cultivation plate 30 DEG C of constant incubators of placement of line, culture grows monoclonal in 2-3 days.
3rd, monoclonal is inoculated into the 1mL full culture mediums of YPD liquid, resuspended thalline is transferred in 30mLYPDA, 30
DEG C, 250rpm shakes 14-16h, OD600>1.5.
4th, the bacterium of 20mL incubated overnights is transferred in 200mL YPDA, OD600 is reached 0.2-0.3,30 DEG C,
230rpm, cultivates 3h, and its OD600 is reached within 0.4-0.6 (exponential phase) 3h should determine OD600, in order to avoid miss right
The number phase.
5th, saccharomycete is fitted into 50mL centrifuge tubes, 1000g, 5min centrifugation, remove supernatant, share 40mL sterilizing TE or
The resuspended saccharomycete of H2O, 1000g, 5min, room temperature centrifugation.Supernatant is abandoned, saccharomycete is resuspended in 1 × TE/1 of the fresh configurations of 1.5mL
× LiAc, competent yeast cells are prepared and completed.
6th, 1 μ g pGBK-REG γ plasmids are added, 0.5 μ g pGAD-PSMA3 and 10 μ L carrier DNAs (10mg/mL) are new one
In 1.5mL EP pipes, it is experimental group to mix pGBK-REG γ/pGAD-PSMA3 here, negative control group be pGAD (0.5 μ g)/
PGBK (1 μ g), checking self-activation control group is pGBK (1 μ g)/pGAD-PSMA3 (0.5 μ g), pGAD (0.5 μ g)/pGBK-REG
γ (1 μ g), has verified that pGAD-nip30 (0.5 μ the g)/pGBK-REG γ (1 μ g) of interaction for positive control, i.e., before
Need exist for preparing five EP pipes.
7th, often pipe adds 100 μ L competent cells, Vortex to mix, and often pipe adds the PEG/ of 0.6mL high-temperature sterilizations
LiAc, vortex, fully mix, 30 DEG C, 30min, 200rpm.8th, 70 μ L DMSO are added, gently overturning mixing (should not
vortex)。
9th, 42 DEG C of water-baths, heat shock 15min takes out and is placed in 1-2min on ice at once.
10th, 14000rpm, room temperature centrifugation 5S, abandons supernatant, with the resuspended thalline of 1 × TE of 0.5mL
11st, take 100 μ L coated plates (SD/-Leu/-Trp), 30 DEG C, be inverted culture to growing clone (about 2-4 days),
12nd, the clone of maximum is chosen, is scoring on same selective SD (two is scarce scarce with four) plate, sealed membrane is sealed up,
Put to 4 DEG C of refrigerators.According to yeast growth situation, judge pGAD-PSMA3 and pGBK-REG γ whether there is interaction.
Embodiment 3 is using luciferase fragment complementation technique construction REG γ and the screening system of the interaction inhibitors of α 7
By the Molecular imaging techniques of relatively more several conventional research protein-protein interactions, such as energy resonance turns
Shifting technology, gfp fragment complementary technology and luciferase fragment complementation technology etc..The present invention have finally chosen height
Imitate the system base of the easily inhibitor that luciferase fragment complementation technology interacts as present invention screening REG γ and α 7
Plinth.In contrived experiment, the present invention considers three problems of key：First key issue is the cutting of (1) luciferase
How site, cut two fragments that luciferase is allowed to produce and can be good at recombining, and the fluorescein for newly combining
Enzyme can regain the activity of luciferase.As shown in figure 4, the present invention have selected the most commonly used luciferase N-terminal fragment：
LucN (1-398) and LucN (1-416), and the C-terminal fragment of luciferase is than more consistent, generally LucC (394-550).In addition
One important influence factor (2) is that the connecting peptides between albumen and luciferase fragment are also crucial influence factor, this
Invention builds two kinds of connecting peptides of different length：Linker1 is that-GGGGSGGGGS-and Linker2 is-GGGGSG-
GGGSGGGGS-, to prevent because of the interaction between connecting peptides too short influence REG γ and α 7.(3) connecting peptides are connected
Two end proteins relative position problem because the difference of albumen relative position, it is more likely that cause originally two interaction
Albumen can not interact, so in the experiment designed by the present invention, the present invention has attempted REG γ and LucN and has connected
Connect, α 7 is connected all possible relative position, i.e. REG γ with LucC and is connected to the N-terminal of LucN, REG γ are connected to the C of LucN
End, the N-terminal and α 7 that α 7 is connected to LucC is connected to the C-terminal of LucC.3 points of considerations according to more than, in order that the screening system is obtained
Best interaction result, the present invention devises 18 kinds of different collocation modes altogether, and every kind of collocation has its corresponding negative control.
As shown in figure 4, with its negative control be total to the fusion protein plasmid of this 18 kinds collocation by the method for embodiment 1 by the present invention
With being transfected into HeLa cells, whether whether detection fusion albumen has interaction and produces active fluorescence after 48 hours
Plain enzyme.The present invention is detected using Promega companies luciferase cell pyrolysis liquid cell lysis with Luciferase Assay Reagent
The uciferase activity of cell pyrolysis liquid.
Result is as shown in figure 5, present invention discover that fusion protein lucN (416-linker1)-REG γ and α 7-lucC
(linker1-394) interaction significant difference compared with negative control lucN (416-linker1)-REG γ (244), and
And, that is, the collocation selected is that luciferase N-terminal 1-416 bit slices section is connected by Linker1 than other several groups of arranging effects more preferably
The C-terminal of α 7, both fusion proteins are connected to by Linker1 in N-terminal and luciferase C-terminal 394-550 the bit slices section of REG γ
The interaction of lucN-REG γ and α 7-lucC can produce a large amount of active luciferases, and such present invention can basis
The activity of luciferase judges the combined amount of REG γ and α 7, and then judges whether added compound can suppress REG
Whether the interaction of γ and α 7, the i.e. compound are likely to become the specific inhibitor for suppressing REG γ functions.
Embodiment 4 is mutated the screening effect that lucN-REG γ (K85QK86Q) improves system
REG γ are primarily targeted in nucleus as a kind of proteasome activity factor, according to former report α 7 thin
All it is distributed in karyon and cytoplasm.In order to detect whether two kinds of fusion proteins constructed by the present invention with intrinsic protein have phase
Same positioning, the present invention detects the Subcellular Localization of both fusion proteins using immunofluorescence experiment.
As shown in fig. 6, there is fusion protein lucN-REG γ and endogenous REG γ identical to position, nucleus is all located at
It is interior；But the positioning of fusion protein α 7-lucC is different from endogenous α 7, fusion protein α 7-lucC are primarily targeted in cytoplasm,
It is positioned in nucleus on a small quantity.This experiment shows because lucN-REG γ are different from the Subcellular Localization of α 7-lucC, it will lead
Causing most fusion protein can not combine because of position difference, and then can influence the sensitivity and accuracy of screening system.
In order to exclude the interference of endogenous REG γ and make the positioning of two fusion proteins more consistent, the present invention will
The nuclear localization sequence of lucN-REG γ is mutated, and prevents the albumen from entering in nucleus after synthesizing.Due to REG α/REG β masters
It is positioned in cytoplasm, so amino acid sequence of the present invention than right REG γ and REG α/REG β, finds REG γ the 85th
With the 86th lysine than more conservative, then the present invention by the amino acid mutation in the two sites into REG α corresponding sites paddy ammonia
Acid amides, then constructs the lucN-REG γ (K85QK86Q) and negative control lucN-REG γ 244 (K85QK86Q) of mutation, warp
Cellular immunofluorescence is detected, as shown in fig. 7, lucN-REG γ (K85QK86Q) and lucN-REG γ (1- after mutation
244K85QK86Q) it is positioned in cytoplasm, does not enter back into nucleus, this treatment had both eliminated the shadow of endogenous REG γ
Ring, also cause that there is both fusion proteins identical to position.
In order to detect whether the fusion protein after mutation can cause uciferase activity higher, the present invention respectively will mutation
Front and rear lucN-REG γ/lucN-REG γ (K85QK86Q) and α 7-lucC are co-expressed in HeLa cells, then detect fluorescence
Plain enzymatic activity.As shown in figure 8, on the premise of protein level is consistent, compared to the fusion protein before mutation, after mutation
LucN-REG γ (K85QK86Q) can dramatically increase the activity of luciferase, and the negative control that is mutated and unmutated
LucN-REG γ (1-244) are almost consistent, always all in reduced levels.Compared to the fusion protein for interacting before, mutation
System with interaction afterwards has lower background, and more efficient detectability further enhances the sensitivity of the screening system.
Embodiment 5REG γ (NL) proves the validity of screening system
Before the compound that REG γ and α 7 interact is suppressed using the screening system, the present invention needs checking, and this is
Whether the selection result of system is effective.In order to prove this point, the REG γ fragments that the present invention constructs many different lengths go simulation
The inhibitor of REG γ, the 146th REG γ to the 153rd active ring of experimental result display missing is that REG γ (NL) have most
Good simulation effect, present invention corotation together with screening system lucN-REG γ (K85QK86Q)/α 7-lucC by REG γ (NL)
In contaminating HeLa cells, as shown in figure 9, when REG γ (NL) are co-expressed together with lucN-REG γ (K85QK86Q)/α 7-lucC
When, on the premise of protein level is consistent, REG γ (NL) can pole significantly decrease lucN-REG γ (K85QK86Q) and α 7-
The quantity of luciferase produced by lucC interactions, this explanation REG γ (NL) can play effect of similar inhibitor.
The specific mechanism of lucN-REG γ and α 7-lucC interactions can be suppressed in order to further study REG γ (NL),
The present invention again construct lucN-REG γ (NL) and REG γ (NL)-lucC respectively, by they respectively with α 7-lucC and lucN-
REG γ (K85QK86Q) cotransfection enters in HeLa cells, then judged by detecting fluorescent value REG γ (NL) whether because with
The interaction of lucN-REG γ (K85QK86Q) or α 7-lucC and then have impact on lucN-REG γ's (K85QK86Q) and α 7-lucC
With reference to.
As shown in Figure 10-A, by contrasting the fluorescent value that negative control is produced, present invention discover that lucN-REG γ (NL) and α
7-lucC is almost produced without fluorescent value, and this explanation REG γ (NL) no may interact with α 7-lucC；But REG γ
(NL)-lucC may have stronger interaction to be combined with lucN-REG γ, as shown in figure 10-b, REG γ (NL)-lucC and lucN-
After REG γ (K85QK86Q) cotransfection, very hyperfluorescence value is able to detect that, this explanation REG γ (NL)-lucC and lucN-REG γ
(K85QK86Q) it is be combined with each other in cell, and active luciferase can be produced.The two description of tests REG γ (NL)
The combination for suppressing lucN-REG γ (K85QK86Q)/α 7-lucC is probably because lucN-REG γ (K85QK86Q) can be with REG γ
(NL) combine, so that lucN-REG γ (K85QK86Q) quantity combined with α 7-lucC tails off, cause the fluorescence of last generation
Plain enzyme is reduced.These description of tests have the inhibitor energy for suppressing system lucN-REG γ (K85QK86Q)/α 7-lucC combinations
It is enough to be emerged from by final fluorescent value within the system.REG γ (NL) suppress lucN-REG γ (K85QK86Q)/α 7-
Whether the combination of lucC can illustrate that REG γ (NL) can suppress the combination of endogenous REG γ and α 7.Therefore, it is of the invention by flag-
REG γ (NL) do co-immunoprecipitation experiment with HA- α 7 and HA-REG γ corotation HeLa cells respectively, as shown in Figure 11-A,
Flag-REG γ (NL) can interact with HA-REG γ, without being combined with HA- α 7.In order to further illustrate REG γ (NL) energy
Enough suppress the combination of endogenous REG γ and α 7, the present invention individually transfects HA- α 7, and at the same time by HA- α 7 and Flag-
REG γ (NL) cotransfections to six orifice plate HeLa cells, as shown in Figure 11-B, when HA- α 7 and REG γ (NL) are co-expressed, HA- α 7
With reference to endogenous REG γ reduce, this explanation REG γ (NL) can suppress the combination of endogenous REG γ and α 7 really, this
Equally demonstrate the validity of the screening system.
Under normal circumstances, the combination of endogenous REG γ and α 7 shows as the combination of endogenous REG γ and 20S proteasomes,
And REG γ -20S proteasomes have the ability of degraded p21, if REG γ (NL) can suppress the knot of endogenous REG γ and α 7
Conjunction would potentially result in increasing for p21 contents.The present invention transfects into HeLa cells unloaded and different amounts of REG γ (NL) respectively
In, the content of p21 is detected after 48 hours.As shown in figure 12, as overexpression REG γ (NL), the content of intracellular p21 substantially increases
Plus.The inhibitor out that this explanation lucN-EG γ (K85QK86Q)/α 7-lucC system is screened can suppress endogenous REG
The combination of γ and α 7, and also with the ability of suppression REG γ -20S proteasome functions, it was demonstrated that lucN-REG γ
(K85QK86Q) the selection result of/α 7-lucC screening systems is reliable and effective.
Claims (11)
1. a kind of screening system of REG γ -20S proteasome inhibitors, it is characterised in that the screening system includes fusion egg
White lucN (416-linker1)-REG γ and α 7-lucC (linker1-394)；Wherein, the fusion protein lucN (416-
Linker1)-REG γ are built-up by the N-terminal that connecting peptides are connected to REG γ by luciferase N-terminal fragment 1-416,
The fusion protein α 7-lucC (linker1-394) are connected for 394-550 by luciferase C-terminal fragment by connecting peptides
In the C-terminal of α 7.
2. a kind of screening system of REG γ -20S proteasome inhibitors, it is characterised in that the screening system includes fusion egg
White lucN-REG γ (K85QK86Q) and α 7-lucC (linker1-394)；Wherein, the fusion protein lucN-REG γ
(K85QK86Q) it is mutated by the 85th lysine of nuclear localization sequence of lucN-REG γ and is obtained；The fusion protein α 7-
LucC (linker1-394) is by 394-550 C-terminal that α 7 is connected to by connecting peptides of luciferase C-terminal fragment.
3. a kind of fusion protein lucN (416-linker1)-REG γ, it is characterised in that the fusion protein lucN (416-
Linker1)-REG γ are built-up by the N-terminal that connecting peptides are connected to REG γ by luciferase N-terminal fragment 1-416.
4. a kind of fusion protein α 7-lucC (linker1-394), it is characterised in that the fusion protein α 7-lucC
(linker1-394) by 394-550 C-terminal that α 7 is connected to by connecting peptides of luciferase C-terminal fragment.
5. a kind of fusion protein lucN-REG γ (K85QK86Q), it is characterised in that the fusion protein lucN-REG γ
(K85QK86Q) it is mutated by the 85th lysine of nuclear localization sequence of lucN-REG γ and is obtained.
6. a kind of fusion protein lucN-REG γ 244 (K85QK86Q), it is characterised in that the fusion protein lucN-REG γ
244 (K85QK86Q) are mutated and are obtained by the 86th lysine of nuclear localization sequence of lucN-REG γ.
7. a kind of fusion protein LucN (416-linker1)-REG γ 244, it is characterised in that by lacking REG γ C-terminals 10
Individual amino acid is built-up.
8. a kind of REG gamma inhibitors, it is characterised in that the REG gamma inhibitors are REG γ (NL), it passes through REG γ missings the
146 to the 153rd active rings build and obtain.
9. the screening system of REG γ -20S proteasome inhibitors as claimed in claim 1 or 2 is in screening REG γ -20S eggs
Application in white enzyme body inhibitor.
10. the fusion protein as described in any one of claim 4~8 is in REG γ -20S proteasome inhibitors are screened
Using.
Application of the 11. REG gamma inhibitors as claimed in claim 9 in REG γ -20S proteasome inhibitors are screened.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610850405.3A CN106928370B (en) | 2016-09-26 | 2016-09-26 | REG gamma-20S proteasome inhibitor screening system and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610850405.3A CN106928370B (en) | 2016-09-26 | 2016-09-26 | REG gamma-20S proteasome inhibitor screening system and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106928370A true CN106928370A (en) | 2017-07-07 |
| CN106928370B CN106928370B (en) | 2021-02-02 |
Family
ID=59443933
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610850405.3A Active CN106928370B (en) | 2016-09-26 | 2016-09-26 | REG gamma-20S proteasome inhibitor screening system and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106928370B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111208301A (en) * | 2018-11-22 | 2020-05-29 | 华东师范大学 | Monoclonal antibody for resisting REG gamma C terminal region, preparation and application thereof |
| CN112194729A (en) * | 2020-09-29 | 2021-01-08 | 浙江大学 | Biological probe for detecting degradation activity of ubiquitin-proteasome system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040156854A1 (en) * | 2002-12-06 | 2004-08-12 | Millennium Pharmaceuticals, Inc. | Methods for the identification, assessment, and treatment of patients with proteasome inhibition therapy |
| CN101333555A (en) * | 2007-12-25 | 2008-12-31 | 浙江大学 | Novel horizontal screening system construct by recipient cell G protein coupling and applications |
| CN101522914A (en) * | 2006-09-28 | 2009-09-02 | 国立大学法人大阪大学 | Screening method for prophylactic and/or therapeutic agent for disease accompanied by hepatitis c |
| CN102449147A (en) * | 2009-05-29 | 2012-05-09 | 国立大学法人东京大学 | Method for highly sensitive detection of protein-protein interaction |
| CN103849645A (en) * | 2013-02-05 | 2014-06-11 | 中国农业科学院植物保护研究所 | Luciferase complementary image detection method and carrier kit for detecting protein interaction |
-
2016
- 2016-09-26 CN CN201610850405.3A patent/CN106928370B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040156854A1 (en) * | 2002-12-06 | 2004-08-12 | Millennium Pharmaceuticals, Inc. | Methods for the identification, assessment, and treatment of patients with proteasome inhibition therapy |
| CN101522914A (en) * | 2006-09-28 | 2009-09-02 | 国立大学法人大阪大学 | Screening method for prophylactic and/or therapeutic agent for disease accompanied by hepatitis c |
| CN101333555A (en) * | 2007-12-25 | 2008-12-31 | 浙江大学 | Novel horizontal screening system construct by recipient cell G protein coupling and applications |
| CN102449147A (en) * | 2009-05-29 | 2012-05-09 | 国立大学法人东京大学 | Method for highly sensitive detection of protein-protein interaction |
| CN103849645A (en) * | 2013-02-05 | 2014-06-11 | 中国农业科学院植物保护研究所 | Luciferase complementary image detection method and carrier kit for detecting protein interaction |
Non-Patent Citations (9)
| Title |
|---|
| HUAMIN CHEN ET AL.: "Firefly Luciferase Complementation Imaging Assay", 《PLANT PHYSIOLOGY》 * |
| KATHRYN E. LUKER ET AL.: "Kinetics of regulated protein–protein interactions revealed with firefly luciferase complementation imaging in cells and living animals", 《PNAS》 * |
| R. PAULMURUGAN ET AL.: "Monitoring Protein–Protein Interactions Using Split Synthetic Renilla Luciferase Protein-Fragment-Assisted Complementation", 《ANALYTICAL CHEMISTRY》 * |
| 冯美卿: "《生物技术制药》", 31 January 2016, 中国医药科技出版社 * |
| 孙鹏等: "蛋白酶体结构和活性调节机制的研究进展", 《生物化学与生物物理进展》 * |
| 张坤: "利用荧光素酶片段互补技术构建REGγ与α7相互作用抑制剂筛选系统", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 李平: "人源20S蛋白酶体激活因子REGγ结构与功能研究", 《中国学位论文全文数据库》 * |
| 田甜等: "蛋白酶体激活因子REGγ对细胞周期及凋亡的作用", 《医学分子生物学杂志》 * |
| 翟万里: "REGγ-20S蛋白酶体组装机制探究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111208301A (en) * | 2018-11-22 | 2020-05-29 | 华东师范大学 | Monoclonal antibody for resisting REG gamma C terminal region, preparation and application thereof |
| CN112194729A (en) * | 2020-09-29 | 2021-01-08 | 浙江大学 | Biological probe for detecting degradation activity of ubiquitin-proteasome system |
| CN112194729B (en) * | 2020-09-29 | 2021-12-21 | 浙江大学 | Biological probe for detecting degradation activity of ubiquitin-proteasome system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106928370B (en) | 2021-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Nair et al. | SNARE proteins are required for macroautophagy | |
| Mehus et al. | BioID identification of lamin-associated proteins | |
| Thakurta et al. | Homolog of BRCA2‐interacting Dss1p and Uap56p link Mlo3p and Rae1p for mRNA export in fission yeast | |
| Cheng et al. | In vitro model systems to investigate bile salt export pump (BSEP) activity and drug interactions: a review | |
| CN105622757A (en) | Antibodies that bind to lysyl oxidase-like 2(LOXL2) and methods of use therefor | |
| Niu et al. | Molecular imaging with activatable reporter systems | |
| JP6189329B2 (en) | Cell high-throughput encapsulation for screening or selection | |
| Srisa‐Art et al. | Analysis of protein–protein interactions by using droplet‐based microfluidics | |
| Stokes et al. | Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication | |
| CN108752425A (en) | The method for building cell-penetrating peptide expression library using display technique of bacteriophage | |
| CN105039410B (en) | A kind of method for building up of the pancreas carcinoma animal model with inflammatory basis | |
| Uetrecht et al. | Subunit exchange rates in Hepatitis B virus capsids are geometry-and temperature-dependent | |
| Saiz-Baggetto et al. | Chimeric proteins tagged with specific 3xHA cassettes may present instability and functional problems | |
| Maradeo et al. | The Elg1-RFC clamp-loading complex performs a role in sister chromatid cohesion | |
| CN106928370A (en) | A kind of screening system of REG γ 20S proteasome inhibitors and its application | |
| US20240076315A1 (en) | Khl polypeptide, and use thereof in preparation of tabp-eic cell | |
| CN103881979B (en) | Anti-hepatitis C virus medicament high flux screening model and application thereof | |
| Kim et al. | A new generation of serotype chimeric infectivity-enhanced conditionally replicative adenovirals: The safety profile of Ad5/3-Δ24 in advance of a phase I clinical trial in ovarian cancer patients | |
| Ravalin et al. | A single-component luminescent biosensor for the SARS-CoV-2 spike protein | |
| CN109576227B (en) | Luciferase reporter virus-based autophagy cell line construction method | |
| ES2715557T3 (en) | Non-radioactive procedure for determining the cytolytic action of an agent against target cells, their use and their associated kit | |
| Nishida et al. | The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation | |
| Qiang et al. | Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago | |
| WO2007027919A2 (en) | Complementation assays utilizing complexes of heteroproteins | |
| Kong et al. | Production of encapsulated creatinase using yeast spores |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 200241 No. 500, Dongchuan Road, Shanghai, Minhang District Patentee after: EAST CHINA NORMAL University Address before: 200062 No. 3663, Putuo District, Shanghai, Zhongshan North Road Patentee before: EAST CHINA NORMAL University |