CN105802945B - The purposes of ubiquitin-specific protease 49 - Google Patents
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Abstract
The present invention provides a kind of purposes of ubiquitin-specific protease 49 in the drug of preparation prevention or treatment Pancreatic neoplasms, and described goes the amino acid sequence of ubiquitin-specific protease 49 as shown in SEQ ID NO:1.The present invention provides the deubiquitinating enzymes USP49 with FKBP5 protein-interacting, specify FKBP5 protein degradation and stable mechanism, and its molecular regulation to FKBP5-AKT access, provide effect and molecular mechanism of the access during cancer of pancreas occurrence and development, new basic basis is provided for the treatment of cancer of pancreas, there is direct directive significance to the personalized medicine of cancer of pancreas.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of drug prevented or treat Pancreatic neoplasms, specifically
It is a kind of purposes of ubiquitin-specific protease 49 in the drug of preparation prevention or treatment Pancreatic neoplasms.
Background technique
Pancreatic neoplasms (PDAC) are big most fatal one of the malignant tumours in the whole world five, and the ratio between the death rate and disease incidence are
0.99:1,5 years survival rate < 5%.For Pancreas cancer patients, in addition to surgical radical treatment, it is also necessary to take comprehensive including radiotherapy, chemotherapy
Treatment is closed to improve curative effect.Up to the present, gemcitabine is still the front-line chemotherapeutic agents for treating cancer of pancreas, although its curative effect
Only 20% or so, but at present still without the drug that can surmount gemcitabine, even if drug combination also fails to significantly improve treatment
Effect.
Since the 21th century, although molecular targeted therapy has achieved significant progress, chemotherapy is still current treatment
The therapeutic scheme that tumour is widely used.However, also having very since Different Individual has different genes background to the reaction of chemotherapy
Big difference, this becomes the successful major obstacle of chemotherapy.Therefore, deeply understand cancer of pancreas mechanism of drug resistance, improve tumour cell pair
The responsive type of chemotherapy, it will help exploitation is directed to the personalized chemotherapeutic strategy of Different Individual, reduces toxicity and drug resistance, mentions
High success rate.
Serine/threonine protein kitase AKT(is also referred to as PKB), it is the central regulator factor of cell growth, in cell
It plays a significant role in the regulation of numerous cellular activities such as apoptosis, proliferation, differentiation, aging.Existing research shows that AKT can inhibit
Apoptosis promotes cell survival.Firstly, AKT phosphorylation apoptotic proteins BAD, prevents it in conjunction with target protein.Secondly,
AKT phosphorylation FOXO transcription factor, makes itself and downstream targets Protein transport go out core；Third, AKT phosphorylation MDM2, makes it thin
It is positioned in karyon, MDM2 is promoted to inhibit the activity of P53；In addition, AKT phosphorylation can inhibit the activity of GSK isomers, GSK passes through
Inhibit anti-apoptotic proteins MCL-1, plays the effect for promoting apoptosis.It can be seen that AKT regulated and controled it is a plurality of related to Apoptosis
Signal pathway, activity is abnormal can not only to lead to malignant transformation of cells, and with the migration of tumour cell, stick, tumour blood
Pipe generates and the correlations such as the degradation of extracellular matrix, its superactivation acts on the drug resistance side of tumour generation and tumour cell
Face has played important function.
Recently, it is found in the detection to pancreatic cancer cell and tumor sample, compared with normal cell and tissue, FKBP5
Expression be greatly decreased, and associated AKT-Ser473Phosphorylation level is increase accordingly.When in certain pancreatic cancer cells
When the expression quantity of FKBP5 is relatively high, AKT phosphorylation level reduce, and pancreatic cancer cell chemotherapeutics is shown it is higher
Sensibility.Conversely, AKT phosphorylation level increases, and table in the considerably less pancreatic cancer cell of the expression quantity of other FKBP5
Reveal very strong drug resistance.These results all show that the variation of FKBP5 expression may be used to determine patient to the resistance to of chemotherapy
By degree, assist a physician as the therapeutic scheme of different patient selection personalizations.Therefore, FKBP5 is as important molecular labeling
Object, future will will play a significant role in the research of drug resistance of tumor.
Early-stage study as a result, it has been found that, the drug resistance of FKBP5 expression in the cell and pancreatic cancer cell is closely related.But
The expression quantity of FKBP5 in the cell be by mRNA level in-site regulation or albumen posttranslational modification regulation it is not clear.?
It is found when consulting FKBP5 microarray data in the website oncomine, the mRNA level in-site of FKBP5 is in cancer of pancreas and normal group
It is not significantly different in knitting, then the level of FKBP5 in the cell regulates and controls possibly via posttranslational modification, and by general
The degradation of fibroin enzyme body approach.Therefore, the deubiquitinating enzymes with FKBP5 protein-interacting are identified, FKBP5 protein degradation is illustrated
With stable mechanism, and go deep into dissect deubiquitinating enzymes to the molecular regulation of FKBP5-AKT access, further confirm the access
Effect and molecular mechanism during cancer of pancreas occurrence and development, it will help understand the pathogenesis of cancer of pancreas, be cancer of pancreas
Treatment new basic basis is provided, there is direct directive significance to the personalized medicine of cancer of pancreas.
Summary of the invention
For above-mentioned technical problem in the prior art, the present invention provides a kind of ubiquitin-specific proteases 49 to prepare
Prevention or treatment Pancreatic neoplasms drug in purposes, this ubiquitin-specific protease 49 preparation prevention or
The purposes that person treats in the drug of Pancreatic neoplasms will solve chemoprophylaxis in the prior art or treat Pancreatic neoplasms
Ineffective technical problem.
The present invention provides a kind of ubiquitin-specific proteases 49 in preparation prevention or the drug for the treatment of Pancreatic neoplasms
In purposes.
Further, described to go the amino acid sequence of ubiquitin-specific protease 49 as shown in SEQ ID NO:1.
Further, the base sequence of the coded ubiquitin-specific protease 49 is as shown in SEQ ID NO:2.
The present invention is compared with prior art, and technological progress is significant.USP49 of the invention can with FKBP5 interaction,
The protein level of FKBP5 can be stablized to play prevention or treat the effect of cancer of pancreas.
Detailed description of the invention
Fig. 1 shows the interaction albumen that several FKBP5 are identified using tandem affinity purification and mass spectral analysis (TAP-MS).
Fig. 2 shows that USP49 can stablize the protein level of FKBP5.
Fig. 3, which shows USP49 wild type obviously, can lower the ubiquitination level of FKBP5
Fig. 4 shows the ubiquitination level of FKBP5 after stable abatement USP49.
Fig. 5 shows that USP49 plays a role by regulating and controlling the protein level influence AKT signal path of FKBP5.
Fig. 6 shows that influence of the USP49 to Cell Proliferation of Pancreatic Cancer Cell is to rely on AKT signal path.
Fig. 7 shows that USP49 influences pancreatic cancer cell growth by direct regulation and control FKBP5.
Fig. 8 shows that USP49 can influence the formation of tumour by direct regulation and control FKBP5.
Specific embodiment
Following embodiments is all made of general experiment method below:
Versatile material and method
1. co-immunoprecipitation
1) it inhales and abandons culture medium, 1ml PBS is added and is gently scraped cell with cell scraper, is collected into the centrifuge tube of 1.5ml,
1000r/min is centrifuged 5min.；
2) it inhales and abandons PBS, add 1ml PBS and cell is resuspended, 1000r/min is centrifuged 2min；
3) it inhales and abandons PBS, every pipe is added 600ul NETN protein lysate (protease inhibitors is added) and hangs cell precipitation
Floating, 4 DEG C of shaking tables crack 20min.；
4) 4 DEG C of centrifuges, 12000r/min are centrifuged 10min.It first takes out 60ul supernatant to be put into new centrifuge tube, be added
15ul 5 × Loading, 100 DEG C boil be centrifuged after 10min it is spare；Remaining 580ul supernatant is put into another centrifuge tube,
The primary antibody of destination protein is added, is placed in 4 DEG C of shaking tables and is incubated overnight；
5) 25ul albumen agarose A/G pearl is taken out in 1.5ml centrifuge tube, and 1ml NETN protein lysate is added,
8000r/min is centrifuged 1min, inhales and abandons supernatant, and pearl is spare；
6) supernatant that antibody yesterday and target protein are incubated for is added to and is managed containing the albumen sepharose 4B EP washed
In, 4 DEG C of shaking tables are incubated for 2h again；
7) 8000r/min is centrifuged 1min, abandons supernatant, and new NETN protein lysate washing pearl is added, is repeated 3 times；
8) supernatant is abandoned, the liquid inside EP pipe is inhaled and is abandoned completely, 1 × Loading of 60ul is added, 100 DEG C are boiled, centrifugation
Western-blot is detected later.
Internal deubiquitination experiment
1) cell is inoculated into culture dish by certain density, second day density of cell is made to reach 70%-80%.By purpose
Plasmid-transfected cells；
2) after transfecting 48h, 50uM MG132 is added and handles cell 4h；
3) cell is collected to wash one time into the centrifuge tube of 1.5ml, and with PBS；
4) inhale and abandon PBS, every pipe be added 1ml NETN protein lysate (containing cocktail protease inhibitors, NEM and
IOD deubiquitination protease inhibitors) cell precipitation is suspended, 4 DEG C of shaking tables crack 20min.
5) 25ul albumen agarose A/G pearl is taken out in 1.5ml centrifuge tube, and 1ml NETN protein lysate is added,
8000r/min is centrifuged 1min, inhales and abandons supernatant, and pearl is spare.
6) 4 DEG C of centrifuges, 12000r/min are centrifuged 10min.It first takes out 80ul supernatant to be put into new centrifuge tube, be added
20ul 5 × Loading, 100 DEG C boil be centrifuged after 10min it is spare；Remaining 900ul supernatant is put into containing having washed
In the EP pipe of sepharose 4B, 4 DEG C of shaking tables are incubated for 3h.
7) 8000r/min is centrifuged 1min, discards supernatant, and new NETN protein lysate washing pearl is added.It is repeated 5 times.
8) supernatant is abandoned, the liquid inside EP pipe is inhaled and is abandoned completely, 1 × Loading of 60ul is added, 100 DEG C are boiled, centrifugation
Western-blot is detected later.
Experiment
1) SDS- polyacrylamide gel electrophoresis
A) separation gel is prepared, and separation sol solution is gently injected in layer glass plate after mixing, on liquid level
It is carefully added into dehydrated alcohol covering gel, is stored at room temperature, is completely polymerized into line to glue.
B) concentration glue is prepared in proportion.The dehydrated alcohol above separation gel is sucked, concentration sol solution is gently injected,
Careful insertion comb avoids being stored at room temperature and polymerizeing completely to glue there are bubble in tooth tip.
C) gel having polymerize is placed in electrophoresis tank, carefully extracts comb, electrophoretic buffer is added.In order successively
Molecular weight Marker and protein sample is added.It is filled with 1*SDS buffer in no sample hole.
D) sample-adding finishes, and electrophoretic apparatus power supply is connected, and 80 V of constant pressure is after bromophenol blue removes concentration glue, constant pressure 150
V electrophoresis is until gel end, stopping electric current being adjourned in bromic acid indigo plant forward position.
2) transferring film
A) after electrophoresis, stripping device carefully opens offset plate, and gel is put in the transferring film being pre-chilled containing 4 °C and is buffered
It is impregnated in the container of liquid about 10 minutes.
B) every piece of glue prepares an appropriately sized nitrocellulose filter, and two appropriately sized thick filter paper are also placed in and turn
It is impregnated in film buffer.
C) transferring film article is put well layer by layer in the following order: filter paper, gel, nitrocellulose filter, filter paper.
D) determine between each layer there is no bubble.
E) under room temperature, the transferring film time is set according to albumen size.
3) film to take a turn for the better is placed in 5% milk TBST solution at room temperature, is put on shaking table and shakes closing 1h.
4) a corresponding antiantibody is diluted by appropriate thinner ratio with 5% milk TBST, addition is put in the box of film.
5) it is put under the conditions of 4 DEG C to shake on shaking table and be incubated overnight.
6) TBST is cleaned at least 3 times, at least 5min every time.
7) it is incubated in container with 5% milk TBST by appropriate thinner ratio dilution 3-5mL or so corresponding two antiantibodys, film is put
Enter, it is ensured that antibody diluent covers film, shakes on room temperature shaker and is incubated for 1 h.
8) TBST cleaning at least 3 times is used, every time at least 5min.
9) it is incubated in box and draws A liquid and each 1mL mixing of B liquid in ECL kit, film is entered, impregnates 10 about 1 repeatedly
min。
10) film is put into exposure magazine bottom surface, is exposed in darkroom, is placed in developing machine and develops.
11) after developing, the position of the good each band of protein standard sample of film mark carries out image scanning.
Experiment
1) inoculating cell: being in the cell of logarithmic phase growth with trypsin digestion, is made into complete medium training single
Cell suspension, 1000, every hole cell inoculation is in 96 well culture plates, every pore volume 100ul, spreads 6 plates altogether；
2) it cultivates cell: culture plate is moved into CO2In incubator, in 37 DEG C, 5% CO2And it is cultivated under the conditions of saturated humidity；
3) colour generation: MTT solution (5mg/ml) 10ul is added in every hole, continues to be incubated for 4h in 37 DEG C of incubators, terminates culture, carefully
It inhales and abandons culture supernatant in hole.Then 80ul DMSO is added in every hole, vibrates 10min, dissolves crystal sufficiently；
4) colorimetric: selection 590nm wavelength measures each hole absorbance value on enzyme-linked immunosorbent assay instrument, records result.With when
Between be horizontal axis, absorbance value is whole axis, draws cell growth curve.
Soft-agar cloning forms experiment
1) logarithmic growth phase cell is blown and beaten with trypsin digestion and gently and is allowed into individual cells, counted, adjusted
Whole cell density；
2) agarose solution of 1.2% and 0.7% two concentration is prepared respectively, maintains 37 DEG C after high pressure sterilization；
3) 1.2% agarose and 2 × DMEM culture medium are mixed in 1:1 ratio, 1.5ml mixed liquor is taken to be put into 6 orifice plates
In, room temperature cooled and solidified；
4) 0.7% agarose and 2 × DMEM culture medium containing cell suspension are mixed in 1:1 ratio, mixes well note
Enter to be covered in 1.2% agarose bottom plate, with the shape at double resin layer.After the solidification of top-layer agar sugar, it is placed in 37 DEG C of 5% CO2It incubates
It is cultivated 3-4 weeks in case；
5) number of cell clones is observed, counting is taken pictures.
Tumor formation in nude mice
1) experimental animal: 4-6 week old BALB/c male nude mouse, weight be 20 ± 2 g, 24；
2) hypodermic mode is used, every mouse each side injects a needle, and every needle injects 200ul mixing with cells
Liquid (includes 2 × 106Cell)；
3) it is inoculated with latter week, observes the growing state of tumour, is started when touching raised position with hand has hard object sense
The size of periodic measurement tumour.Measurement is primary every three days, test constantly 20 days, is calculated according to 0.5 × L × H × W；
4) nude mice is put to death using dislocation of cervical vertebra method, wins mouse oxter tumor tissues, weighing is taken pictures.
The interaction albumen of 1 tandem affinity purification mass spectral analysis of embodiment identification FKBP5
The albumen composition in conjunction with FKBP5 is separated and identified using tandem affinity purification combination mass spectral analysis (TAP-MS)
Component.FKBP5 is building up in the retrovirus vector pMSCVpuro with Flag label first, by digestion identification and
The correctness of the new support of sequence verification building.Strain is stablized using the method building FKBP5 of virus infection, is screened by puro,
The expression of western testing goal albumen FKBP5.The cell strain massive amplification of expression FKBP5, tandem affinity purification knot will be stablized
Mass spectral analysis is closed, identifies the interaction albumen in conjunction with FKBP5.
As a result as shown in Figure 1A, tandem affinity purification combination mass spectral analysis successful identification is multiple may be in conjunction with FKBP5
Protein.It sorts according to FKBP5 affinity, the interaction albumen for coming front three is respectively HSP90, AKT1 and USP49.Its
In, HSP90 and AKT1 albumen can be in conjunction with FKBP5 it has been reported that mistake, mass spectral analysis new discovery deubiquitinating enzymes USP49 energy
Enough and FKBP5 interaction.
In order to verify mass spectrometry results, detected using co-immunoprecipitation (CoIP) experiment.Interior source IP the results show that
FKBP5 can interact with USP49, and vice versa (Figure 1B-C).In addition, the FKBP5 albumen pronucleus table that GST label will be had
It reaches, and is incubated for jointly with USP49 lysate, immunoprecipitation complex is finally subjected to immunoblotting assay, extracellular Pulldown
Experimental identification result also further confirms that both FKBP5 and USP49 can direct interactions (Fig. 1 D).
2 USP49 of embodiment can stablize the protein level of FKBP5
Ubiquitin-Proteasome Pathway is intracellular important protein degradation systems, ubiquitin small molecule can by with target egg
White combination mediates its degradation, and removing ubiquitin enzyme (DUBs) is the protease for capableing of this converse process, can be marked by deubiquitination
The target protein of note stablizes intracellular protein level.Fig. 1 has confirmed, USP49 can with FKBP5 interaction, in the present embodiment
Further whether detection USP49 can stablize the protein level of FKBP5.
The abatement stable cell line of USP49 is established first with the shRNA of USP49, Western the result shows that, and compare
It compares, after stable abatement USP49 albumen knocks out, the protein level of FKBP5 also lowers (Fig. 2A) therewith.MG132 is a kind of
Proteasome inhibitor can prevent target protein from degrading by Ubiquitin-Proteasome Pathway, when in the stable subtractive cell line strain of USP49
After middle addition MG132 processing, FKBP5 albumen is restored to normal level again, this shows that MG132 can reverse USP49 to FKBP5 egg
The regulation (Fig. 2A) of white level.
In addition, in the cell strain for stablizing abatement USP49 after express exogenous USP49 wild type (USP49 WT) or
USP49 catalysis region saltant type (USP49CA), as shown in Figure 2 B, when external source is overexpressed USP49 WT, the albumen water of FKBP5
It is flat to restore, but when external source overexpression USP49 CA, the protein level of FKBP5 can not then restore.
It Cycloheximide(CHX) is a kind of protein synthesis inhibitor.Will stablize abatement USP49 SU86 cell strain and
Control cell strain is handled with CHX, and the processing time is respectively 0h, 1h, 4h and 8h.As a result as shown in Figure 2 C, compared with the control group, exist
In the cell strain for stablizing abatement USP49, FKBP5 protein stability is substantially reduced.
In conclusion USP49 regulates and controls and stablizes the protein level of FKBP5 in such a way that deubiquitination relies on.
The ubiquitination level of 3 USP49 of embodiment regulation FKBP5 albumen
USP49 is overexpressed plasmid or USP49 catalyst deactivation mutant plasmid and ub plasmid co-transfection 293T cell, 48 is small
When after be added MG132 handle 4h, collect cell carry out ubiquitination experiment, as a result as shown in figure 3, USP49 wild type obviously can
The ubiquitination level of FKBP5 is lowered, but USP49 catalyst deactivation saltant type loses this function.In addition, stablize abatement USP49,
The ubiquitination level of FKBP5 obviously increases (Fig. 4) for control group.
4 USP49 of embodiment influences AKT signal pathway by the protein level of regulation FKBP5
The abatement stable cell line of USP49 is established first with the shRNA of USP49, detects the variation of AKT signal pathway.
Western the result shows that, compared with the control, when stablize cut down USP49 after, the protein level of FKBP5 is lowered therewith.AKT-Ser
The up-regulation of 473 phosphorylation levels, 308 phosphorylation level of AKT-Thr is unaffected, and AKT and downstream albumen p-GSK3B and p-
The phosphorylation level of FOXO1 also obviously raises, as shown in Figure 5.
In the cell strain for stablizing abatement USP49, continue to cut down FKBP5, the results show that AKT-Ser 473, AKT-
The phosphorylation level of Thr308, p-GSK3B and p-FOXO1 are compared with the control all without variation (Fig. 5 A).If cut down stablizing
In the cell strain of USP49, it is overexpressed FKBP5, also there is no variation (figures compared with the control for 473 phosphorylation level of AKT-Ser
5B).These results explanation, USP49 are located at the upstream FKBP5, and the protein level by regulating and controlling FKBP5 influences AKT signal path hair
The effect of waving.
5 USP49 of embodiment inhibits Cell Proliferation of Pancreatic Cancer Cell to depend on AKT
MTT experiment detection cell proliferative conditions are carried out using abatement USP49 pancreas cancer cell strain is stablized, as shown in Figure 6A,
After stablizing abatement USP49, compared with the control, the proliferative capacity of pancreatic cancer cell is obviously increased.But use AKT protein inhibitor
After abatement USP49 cell is stablized in processing, cell proliferation level is not significantly different (Fig. 6 B) compared with the control.This shows USP49
Influence to Cell Proliferation of Pancreatic Cancer Cell is to rely on AKT signal path.
6 USP49 of embodiment influences pancreatic cancer cell growth by direct regulation and control FKBP5
Cut down USP49 pancreas cancer cell strain, individually stable abatement FKBP5 pancreas cancer cell strain using independent stablize, and
The cell strain that the two stablizes abatement simultaneously carries out MTT experiment, detects cell proliferative conditions.Find out from Fig. 7 A, disappears when stable respectively
When subtracting USP49 FKBP5, compared with the control, the proliferative capacity of pancreatic cancer cell is remarkably reinforced, but cuts down USP49 stablizing
On the basis of cut down FKBP5 again, the proliferative capacity of cell does not further enhance.
In addition, after external source is overexpressed FKBP5 in the pancreas cancer cell strain for stablizing abatement USP49, then MTT experiment is carried out,
The result shows that the proliferative capacity for stablizing the pancreatic cancer cell of abatement USP49 is restored to normal level after heterogenous expression FKBP5 again
(Fig. 7 B).
Finally, using individually stablizing abatement USP49 pancreas cancer cell strain, individually stablizing abatement FKBP5 pancreas cancer cell strain,
And the two stablizes the cell strain progress soft-agar cloning formation experiment of abatement simultaneously, it is as a result the same with MTT experiment result, individually
Stablize abatement USP49 or FKBP5, pancreatic cancer cell clonality is remarkably reinforced, and two albumen are cut down simultaneously, cell clone shape
Do not further enhance (Fig. 7 C, D) at ability.These results indicate that pancreatic cancer cell can be promoted by stablizing abatement USP49
Proliferation, and it is to rely on FKBP5's.
7 USP49-FKBP5 access of embodiment and the sexual intercourse of cancer of pancreas drug resistance
Tumor formation in nude mice is carried out, influence of the USP49 and FKBP5 to pancreatic tumour development process is studied.
Experiment is divided into following three groups:
First group is cancer of pancreas control cell；
Second group is cut down USP49 pancreas cancer cell strain to stablize.
Third group is that external source is overexpressed the pancreas cancer cell strain of FKBP5 again in stablizing abatement USP49 cell strain.
It is inoculated into nude mice oxter by injected s.c., after inoculation 7 days, is started every the tumour of measurement in three days
Volume after about 25 days, puts to death nude mice, carries out weighing of taking pictures to tumour.Such as Fig. 8 A, shown in B, compared with the control, second group steady
Surely cut down the pancreas cancer cell strain of USP49, the growth ability of tumour is remarkably reinforced, and the volume and weight of tumour all significantly increases,
When by 25 days, the weight of second group of tumour reaches 5 times of first group or more；However, after external source is overexpressed FKBP5, third group
The volume and weight of the tumour of mouse is not significantly different compared with the control group.The result shows that USP49 can pass through direct regulation and control
The formation of FKBP5 influence pancreatic tumour.
Claims (2)
1. a kind of purposes of ubiquitin-specific protease 49 in the drug of preparation prevention or treatment Pancreatic neoplasms, described
The amino acid sequence of ubiquitin-specific protease 49 is as shown in SEQ ID NO:1.
2. purposes according to claim 1, it is characterised in that: encode the base sequence of the ubiquitin-specific protease 49
Column are as shown in SEQ ID NO:2.
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| CN105327350A (en) * | 2014-07-23 | 2016-02-17 | 中国科学院上海巴斯德研究所 | Application of ubiquitin pathway related factor in regulating function of helper T cells |
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Non-Patent Citations (2)
| Title |
|---|
| Homo sapiens ubiquitin specific peptidase 49,mRNA(cDNA clone MGC:20741 image4579283)，complete cds;Strausberg，R.L.等;《Genbank Database》;20060811;Accession No. BC014176.2 |
| Ubiquitin carboxyl-terminal hydrolase 49 isoform b(Homo sapiens）;Taipale M等;《Genbank Database》;20151225;Accession No. NP061031.2 |
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