CN106309461A - Purpose of epirubicin hydrochloride in preparation of inhibitor for blocking interaction p97 and Npl4 - Google Patents
Purpose of epirubicin hydrochloride in preparation of inhibitor for blocking interaction p97 and Npl4 Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and more specifically relates to a purpose of epirubicin hydrochloride in preparation of an inhibitor for blocking interaction p97 and Npl4. The function of epirubicin hydrochloride which can effectively block the interaction of p97 and Npl4 is firstly found, a RIG-I mediated IFNbeta signal pathway is promoted so as to increase the anti-virus infection capability, so that the treatment effect for virus infection is enhanced, and the epirubicin hydrochloride has clinical and market value.
Description
Technical field
The present invention relates to biological technical field, be specifically related to epirubicin hydrochloride and can block the phase of p97 with Npl4 in preparation
Purposes in the inhibitor of interaction.
Background technology
Virus infects the antiviral immunity reaction that body can be caused strong, and the natural immunity is that body supports the first of viral infection resisting
Road barrier, by the cause of disease identification receptor in body, including toll sample receptor (TLR), RIG-I sample receptor (RLRs) can
To identify viral nucleic acid.The receptor being activated can start downstream transcription factor NF-κ B and the letter of interferon regulatory factor IRF
Number transduction pathway, produces a series of pro-inflammatory cytokine and I type interferon (type I interferons, type I IFN).I type
The release of interferon can promote transcribing thus the further duplication of effectively suppression virus of downstream gene.Meanwhile, I type interference
The secretion of element can also activate and include dendritic cell, and NK cell, mononuclear cell and macrophage are at interior participation inherent immunity
The cell of response.Additionally, the secretion of I type interferon also is able to directly activate T cell and B cell, and then generation adaptability is exempted from
Epidemic disease responsing reaction.
Infection or body that autarcetic accuracy controlling for viral infection resisting and prevents autoimmune disease produce immunity
Inflammation is most important.Ubiquitination is the major regulatory mode of the receptor-mediated signal path of RLR, with its representative receptor RIG-I
As a example by, activate RIG-I in conjunction with viral RNA and produce conformation change, recruit ubiquitin ligase, make RIG-I occur K63 chain general
Elementization, activates transcribing of downstream NF-κ B and IRF3, promotes proinflammatory factor and the expression of I type interferon, strengthens it disease-resistant
Cytotoxic activity.On the other hand, ubiquitin ligase c-Cbl and RNF125 makes RIG-I that K48 chain ubiquitination to occur, and terminates this path
Immunoreation, suppress its antiviral activity.
P97 belongs to II type ATP enzyme family, and it participates in including the protein degradation that endoplasmic reticulum and mitochondrion participate in, autophagy, cell membrane
Restructuring, DNA repairs, a series of physiological process such as cell cycle and Sex determination.P97 and its cofactor Npl4, Ufd1,
P47, UBXD7 and FAF1 combine, and utilize its activity separating enzyme to be peeled off from its cellular environment by substrate, the most
Number enters proteasomal degradation.Existing research shows that p97 and its cofactor Npl4 composition complex participates in endoplasmic reticulum and mitochondrion
A series of physiological process such as degradation pathway, p97-Npl4 complex can promote what TNF α was induced by the degraded of mediation I κ B α
NF-κ B signal path.Recently also studies have found that p97 participates in the virus neutralization of TRIM21 mediation.
Hydrochloric acid soft ratio table star (epirubicin hydrochloride), CAS numbering 56390-09-1, molecular formula:
C27H29NO11.HCl-, belongs to anthracycline antibiotics.It is currently known hydrochloric acid soft may be inserted into than table star and DNA suppresses DNA
With the synthesis of RNA, for listing antitumor drug, it is used for treating breast carcinoma, acute leukemia, lymphoma of feeling sick, pulmonary carcinoma,
The Several Kinds of Malignancies such as ovarian cancer gastric cancer.
But in prior art and have no p97 virus infection immunity in effect.Meanwhile, in the middle of prior art, the most not
Once reported that epirubicin hydrochloride (the numbered 56390-09-1 of Epirubicin Hydrochloride, CAS) was at virus infection immunity
In effect.
Summary of the invention
It is an object of the invention to the inhibitor that open epirubicin hydrochloride can block the interaction of p97 Yu Npl4 in preparation
In purposes.
The nucleotides sequence of p97 gene is listed in the sequence of Serial No. NM_007126.3 in Genebank.
The present invention is by extensively in-depth study discovery, and epirubicin hydrochloride can effectively block p97 and cofactor Npl4
Between interaction, the K48 chain ubiquitination of RIG-I of suppression RNF125 mediation, and then suppression RIG-I degraded, thus
Promote the IFN β signal path of RIG-I mediation, finally play the effect improving viral infection resisting ability.
For realizing the purpose of the present invention and other relevant purposes, the present invention by the following technical solutions:
A first aspect of the present invention, it is provided that epirubicin hydrochloride can block pressing down of the interaction of p97 with Npl4 in preparation
Purposes in preparation.
Preferably, described inhibitor can suppress the K48 chain ubiquitination of RIG-I that RNF-125 mediates.
Preferably, described inhibitor can suppress the proteasomal degradation of RIG-I.
Preferably, described inhibitor can promote the IFN β signal path that RIG-I mediates.
Preferably, described inhibitor can play and improves viral infection resisting ability and then play the effect that treatment virus infects.
Further, a second aspect of the present invention, it is provided that the drug regimen that epirubicin hydrochloride infects in preparation treatment virus
Purposes in thing.
Preferably, described virus infects for picornavirus infection.
Infect for VSV virus or the infection of SeV virus it is furthermore preferred that described virus infects.
Preferably, described epirubicin hydrochloride is one of active ingredient of pharmaceutical composition of described treatment virus infection.
It is furthermore preferred that unique active ingredient that described epirubicin hydrochloride is the pharmaceutical composition that described treatment virus infects.
Preferably, described epirubicin hydrochloride is to improve antiviral sense by promoting the IFN β signal path of RIG-I mediation
Dye ability and then play the effect that treatment virus infects.
Preferably, described epirubicin hydrochloride is the proteasomal degradation by suppression RIG-I thus plays promotion RIG-I and be situated between
The effect of the IFN β signal path led.
Preferably, described epirubicin hydrochloride is the K48 chain ubiquitination by suppression RNF-125 mediation RIG-I thus plays
The effect of the proteasomal degradation of suppression RIG-I.
Preferably, described epirubicin hydrochloride be by suppression RNF125 and RIG-I between interaction thus play and press down
The effect of the K48 chain ubiquitination of RNF-125 processed mediation RIG-I.
Preferably, described epirubicin hydrochloride is to suppress p97-Npl4 to be combined by the interaction of blocking-up p97 Yu Npl4
Interaction between the formation of thing, and then suppression RNF125 and RIG-I.
Further, the pharmaceutical composition that treatment virus infects also comprises pharmaceutically acceptable excipient.
Term " comprises " and refers to " containing " and " by constituting ", and the compositions such as " comprising " X can be made up of X completely, or
Can be containing the material outside X, such as X-Y.Term used herein " therapeutically effective amount " refer to therapeutic agent treats, alleviation or
Prevention target disease or the amount of situation, or show the amount of detectable treatment or preventive effect.This effect such as can be passed through
Chemical labeling or antigen levels detect.Therapeutic effect also includes alleviating of physical symptoms.For accurately having of a certain object
Effect dosage depends on build and the health status of this object, therapeutic agent that the character of disease or degree and selecting gives and/or
The combination of therapeutic agent.Therefore, it is useless for preassigning effective dose accurately.But, for the symptom that certain is given,
Can determine this effective dose with normal experiment, clinicist can interpolate that out.
Described pharmaceutically acceptable excipient (or carrier) refers to the excipient for Therapeutic Administration, itself and use thereof
Amount should not induce the antibody that the individual generation accepting said composition is harmful, and does not has undue toxicity after administration.Pharmaceutically can connect
The excipient being subject to generally includes nontoxic solid, semisolid or liquid filler, diluent, lapping or any conventional class
The formulation auxiliary of type.Suitably excipient includes but are not limited to water, glucose, glycerol, saline, ethanol or a combination thereof.
Described excipient also includes other reagent such as wetting agent or emulsifying agent, pH buffer agent or strengthens the adjuvant of preparation effect.Other
Material such as antioxidant, wetting agent, viscosity stabiliser and similar reagents can be added as needed on.Liposome is also included within pharmacy
In the definition of upper acceptable excipient.
Generally, pharmaceutical composition can be made injectable agent, such as liquid solution or suspension;May also be fabricated which and fit before the injection
Conjunction is allocated in solution or suspension, the solid form of liquid-carrier.
The invention have the benefit that
Present invention firstly discovers that, epirubicin hydrochloride can effectively block the interaction of p97 Yu Npl4, promotes that RIG-I is situated between
The IFN β signal path led thus improve viral infection resisting ability and then play the effect that treatment virus infects, great clinic and city
Field is worth.
Accompanying drawing explanation
Fig. 1 .p97 is by the antiviral response of its atpase activity suppression IFN β mediation.(A) HEK293T cell transfects
After p97 siRNA or comparison RNA, SeV stimulate 12h, IFN β (left), PRDIII-I (in) and ISRE (right)
The transcriptional activity of reporter gene.(B) in the cell that p97 knocks out, after SeV stimulates, IFNB (left), RANTES (in)
And the transcriptional level of ISG56 (right).(C-D) in the cell that p97 knocks out, poly (I:C), poly (dA:dT), SeV
Or VSV-GFP process after reporter gene (C) and the result of Q-PCR (D).(E-F) p97 siRNA has been transfected
MEF cell infection VSV-GFP virus after, fluorescent phase-contrast microscopic examination (E) and cell streaming experimental result (F).
(G-H) after p97 knocks out cell infection SeV, carrying out rescue experiment with p97 or its mutant, detection IFN β is transcribed sharp
(G) and the transcriptional level (H) of IFNB alive.N.c., represent comparison siRNA;Scr, represents comparison shRNA;WT, wild type;PH,
Difference micro measurement;FL, fluorescence microscopy detection (lower same).Data analysis uses: the standard error (mark of three independent parallel experiments
Accurate poor), variance analysis and t-test.N.s., without notable change;* P < 0.05 is represented;* represents P < 0.01;* * represents P < 0.001.
Fig. 2 .Npl4 works in coordination with the antiviral response of p97 suppression IFN β mediation.(A-B) the cell sense of different siRNA has been transfected
The transcriptional level (B) of transcriptional activity (A) IFNB of IFN β promoter after dye SeV.(C-D) various dose has been transfected
After the cell infection SeV of Npl4 the reporter gene of IFN β (left) and ISRE (right) promoter activity (C) and
IFNB (left) and the mRNA transcriptional level of ISG56 (right).(E-F) in the cell that Npl4 knocks out, after SeV stimulates,
Reporter gene (E) and the detection of Q-PCR (F).(G) the MEF cell that Npl4 knocks out, after infecting VSV-GFP
Rescue experiment.(H), after knocking out the different plasmid of transfection in the cell of p97, IFN β transcriptional activity and IFNB transcribe water
Flat.
Fig. 3 .p97-Npl4 composite structure and mutation analysis.(A) the domain schematic diagram of Npl4 and p97.(B) fruit bat
The structure of Ter94-Npl4 complex.Residue on interaction interface highlights with ball-bar model, represents and fruit in bracket
The aminoacid in the people source that this site of fly is corresponding.(C) critical sites that p97 (left)-Npl4 (right) interacts.(D)
P97 mutant different for Npl4 pulldown with Strep label.(E) with His label p97 pulldown not
Same Npl4 mutant.(F) Npl4 Yu p97 or the co-immunoprecipitation of p973A mutant.(G) p97 Yu Npl4 or
The co-immunoprecipitation of Npl4 4A mutant.(H-I) in the cell of the different plasmid of transfection IFN β transcriptional activity (H) and
IFNB transcriptional level (I).3A represents p97-F52A/D55A/Y110A mutant;4A represents
Npl4-I6A/R8A/R17A/R50A mutant.
Fig. 4 .p97-Npl4 complex promotes that RIG-I suppresses RIG-I signal path by proteasomal degradation.(A)poly(I:C)
Stimulate p97 to knock out different time after cell, analyze RIG-I, MAVS, IRF3 and IRF3 phosphorus by immunoblot experiment
Acidifying.(B) poly (I:C) stimulates the cell different time of process LAN Npl4, is expressed by immunoblot experiment analyzing proteins
Level.(C) poly (I:C) stimulates the cell different time that reticent Npl4 expresses, and analyzes RIG-I by immunoblot experiment,
MAVS, p-TBK1, TBK1, IKK ε, IRF3 and IRF3 phosphorylation level.(D) MG132 process p97 knocks out
Cell in after process LAN p97, the protein level of RIG-I.(E) RIG-I after process LAN Npl4 after MG132 processes cell
Protein level.(F-G) after knocking out the cell SeV stimulation that p97 (F) knocks out Npl4 (G), the ubiquitination water of RIG-I
Flat.(H) knock out after the cell of p97 transfects p97 wild type and 3A mutant, the ubiquitination level of RIG-I.(I)
Transfect in the cell of Npl4 wild type and 4A mutant, the ubiquitination level of RIG-I.
Fig. 5 .RNF125 is necessary for p97-Npl4 complex suppression RIG-I signal path.(A) HEK293T is thin
Born of the same parents transfect matched group, knocks out RNF125, knock out c-Cbl, or the Npl4 of various dose, after SeV stimulates, IFN β
The activity of luciferase reporter gene.(B) cell transfects different RIG-I CARD domains and comparison, knock out
RNF125 or after knocking out the Npl4 of c-Cbl and various dose, the activity of IFN β luciferase reporter gene.(C) knock out
In the cell of RNF125, in the case of transfecting or not transfecting Npl4, the protein level of RIG-I.(D) cotransfection Npl4 with
The cell of RNF125, after SeV stimulates, the ubiquitination level of RIG-I.(E) cell transfecting Npl4, RNF125 that SeV infects
After.(F) mass spectral analysis in RIG-I ubiquitination site after cotransfection RNF125 and HA-K48Ub.(G) cotransfection or
Transfect in Npl4, RNF125 cell respectively, after transfected wild-type or mutant RIG-I, the immunoblotting assay of RIG-I.
(H) in the cell of cotransfection Npl4 Yu RNF125, after transfected wild-type or mutant RIG-I, the ubiquitination water of RIG-I
Flat.(I) in the cell of cotransfection Npl4 Yu RNF125, after transfected wild-type or mutant RIG-I, IFN β transcriptional activity.
(J) knocking out in the cell of Npl4 and RNF125, after transfection RIG-I or its mutant, VSV-GFP virus infects thin
The fluorescence microscope result of born of the same parents.
Fig. 6 .p97-Npl4 complex interacts with RIG-I and RNF125.(A) SeV stimulates and non-SeV stimulates feelings
Under condition, intracellular Npl4 Yu RIG-I co-immunoprecipitation.(B) SeV stimulates and in the case of non-SeV stimulation, Npl4 and RIG-I
Common location.(C) interaction of external pulldown experimental verification Npl4 Yu RIG-I.(D-E) co-immunoprecipitation is real
Test the concrete structure territory that detection Npl4 Yu RIG-I interacts.(F) external pulldown experiment detection Npl4 and RIG-I
The interaction of CARD domain.(G) SeV stimulates and in the case of non-SeV stimulation, intracellular p97 and RNF-125
Co-immunoprecipitation experiment.(H) interaction of external pulldown experiment detection p97 Yu RNF125.(I) external pulldown
The impact that RIG-I Yu RNF125 is interacted by experiment detection p97-Npl4 complex.
Fig. 7 .pulldown experimental verification micromolecular compound destroys p97-Npl4 and interacts.
Detailed description of the invention
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following spy
Fixed specific embodiments;It is also understood that the term used in the embodiment of the present invention is specifically to be embodied as to describe
Scheme rather than in order to limit the scope of the invention.
Nucleotide sequence (NM_007126.3) and the aminoacid sequence thereof of people's p97 gene can be obtained from Genebank
(NP_009057.1).Homologous genes refers to ortholog herein, originates from common ancestors' base in different plant species
Some homologous geness of cause, these genes generally remain identical or similar function.According to the result of NCBI website BLAST,
The homologous genes of people p97 includes mice VCP gene (NM_009503.4) and fruit bat TEAR94 base (NM_001103780.2).
Or, " homology " homologous genes of the application can be defined." homology " refers to two polynucleotide or two polypeptide portions
/ percentage similarity.Article two, DNA or two peptide sequences are in the molecular length determined, when sequence show to
Few about 50%, preferably at least about 75%, more preferably at least about 80-85%, the best be at least about 90%, optimal the most at least about
During for 95-98% sequence similarity, each other " substantially homology ".As described herein, basic homology also refer to specific DNA or
The identical sequence of peptide sequence.
Basic experiment method:
1. the clone of genes of interest: for required genes of interest design primer, by the method for PCR by this genes of interest from comprising
Cloning out in the plasmid of this genes of interest or the cDNA of Hela cell, genes of interest fragment both sides are with two different enzyme action
Site.The size detecting PCR primer by the method for agarose gel electrophoresis is the most in the same size with required genes of interest, and profit
Reclaim test kit with agarose gel DNA this fragment is reclaimed.Add and PCR primer both sides enzyme in glue reclaims product
Cut the corresponding enzyme in site and suitably buffer carries out double digestion, 37 DEG C of overnight incubation.Meanwhile, with identical enzyme double digestion
The carrier that this fragment is to be connected.Utilize common DNA product purification kit that the fragment after enzyme action is reclaimed, utilize fine jade
Sepharose DNA reclaims test kit and reclaims the carrier after enzyme action, then fragment has been connected with carrier with T4 ligase
Come, and this connection product is converted DH5 α.Utilize resistance screening, verify and the clone of the acquisition genes of interest that checks order.
2. the rite-directed mutagenesis of plasmid: design pair of primers near mutational site, 5 ' ends of these two primers have that 15-20bp's is anti-
To complementary region, mutational site should be positioned at the centre in this reverse complemental region.The size in the incomplementarity region that 3 ' hold is at least
10bp.Utilize this pair of primers, by the method for PCR, plasmid is carried out Inverse PCR amplification.Utilize common DNA product
The product of PCR is reclaimed by purification kit, adds DpnI, hatch 2h for 37 DEG C, by methylated in reclaiming product
Plasmid template is removed.Then, the sample after being processed by DpnI directly converts DH5 α.Resistance screening and order-checking is utilized to obtain
The clone of genes of interest.
3. the expression and purification of albumen: take prokaryotic expression plasmid and convert BL21, cultivate 12h for 37 DEG C.First inoculate, i.e. choose gram
In the grand LB culture medium containing antibiotic to 7.5ml, in 37 DEG C, 10-12h is cultivated in 220rpm concussion.Expand training the most again
Supporting, will above-mentioned 7.5ml bacterium solution all add in the 750ml TB culture medium containing antibiotic, 37 DEG C, 220rpm shakes training
Support to OD600 be 0.6-0.8 (about 3-4h).Now, shaking table temperature is regulated to 16 DEG C, treats that bacterium solution is cooled to 16 DEG C completely
After, add IPTG (final concentration of 0.4mM) according to the ratio of 1:2000, continue concussion and cultivate 16-18h.Finally use Beckman
High speed refrigerated centrifuge collect thalline, i.e. 4 DEG C, 6000rpm is centrifuged 10min, abandons most supernatant, carries out after being weighed by thalline
Protein purification or be stored in-80 DEG C.
(1) Ni post affinity chromatograph:
Lysis buffer:20mM HEPES pH 7.0,500mM NaCl,20mM imidazole,1mM DTT;
Wash buffer:20mM HEPES pH 7.0,500mM NaCl,40mM imidazole,1mM DTT;
Elution buffer:20mM HEPES pH 7.0,500mM NaCl,500mM imidazole,1mM DTT;
Taking appropriate beads and pour in chromatographic column, (CV represents to rinse beads, generally 10CV × 5 with substantial amounts of ddH2O
Column volume, lower same).It is balanced with Lysis buffer: 10CV × 5.Obtain after the beads balanced is moved into bacterial cell disruption
Supernatant in, at Cool Room 4 DEG C Mix 1-2h.500g, 4 DEG C of centrifugal 2min, cleer and peaceful in collection flow through liquid.Add 50ml Wash
Buffer, 500g, 4 DEG C of centrifugal 2min, cleer and peaceful wash 1 in collection.Being repeated twice, the supernatant of collection is respectively wash 2 and wash
3.Being poured into by beads in chromatographic column, continuation Wash buffer washes once.With Elution buffer eluting: 0.5CV × 10,
Eluent is collected respectively with EP pipe.SDS-PAGE electroresis appraisal.
(2) MBP post affinity chromatograph:
Lysis buffer:20mM HEPES pH 7.0,500mM NaCl,1mM DTT;
Wash buffer:20mM HEPES pH 7.0,500mM NaCl,1mM DTT;
Elution buffer:20mM HEPES pH 7.0,500mM NaCl,10mM amylose,1mM DTT;
Taking appropriate beads and pour in chromatographic column, (CV represents to rinse beads, generally 10CV × 5 with substantial amounts of ddH2O
Column volume, lower same).It is balanced with Lysis buffer: 10CV × 5.Obtain after the beads balanced is moved into bacterial cell disruption
Supernatant in, at Cool Room 4 DEG C Mix 1-2h.500g, 4 DEG C of centrifugal 2min, cleer and peaceful in collection flow through liquid.Add 50ml Wash
Buffer, 500g, 4 DEG C of centrifugal 2min, is repeated twice,.Being poured into by beads in chromatographic column, continuation Wash buffer washes once.
With Elution buffer eluting: 0.5CV × 10, collect eluent respectively with EP pipe.SDS-PAGE electroresis appraisal.
(3) Strep Tactin post affinity chromatograph:
Lysis buffer/Wash buffer:100mM Tris pH 8.0,150mM NaCl,10mMβ-ME;
Elution buffer:100mM Tris pH 8.0,150mM NaCl,2.5mM desthiobiotin,10mMβ-ME;
Take appropriate beads to pour in chromatography.It is balanced with Lysis buffer: 10CV × 5.Supernatant after broken is poured into
Equipped with in the chromatographic column of beads, coutroi velocity is 1/2s, collects and flows through liquid, repeats loading once.Use Wash buffer
Rinse: 10CV × 5, collect washes.With Elution buffer eluting: 0.5CV × 6, collect eluent respectively with EP pipe.
SDS-PAGE electroresis appraisal.
(4) gel permeation chromatography (Superdex 75/200):
Buffer:20mM HEPES pH 7.0,100mM NaCl,1mM DTT;
DdH2O rinses 2CV: flow velocity: 1ml/min, upper pressure limit: 0.3MPa (lower same).Buffer balances 2CV.Sample from
The heart: 12000rpm, 4 DEG C of centrifugal 40min.Rinsing loading ring: ddH2O and wash 5 volumes, Buffer washes 5 volumes.Loading:
Use syringe loading, the bubble in syringe must be drained.After Buffer eluting about 40min, start to notice that observing ultraviolet inhales
Receive the appearance at peak, once go out peak and immediately begin to collect.After sample collection, continue to rinse until ultraviolet with Binding buffer
Absorb baseline values.DdH2O rinses 2CV.Suspend after 20% alcohol flushing 2CV, close instrument.
(5) removal (TEV enzyme action) of affinity tag:
By in the sample desalination after Ni post affinity chromatograph to Lysis buffer.Measure the concentration of albumen in eluent, and according to body
The long-pending total amount estimating destination protein.Ratio with the mass ratio of TEV and destination protein as 1:15 adds enough in eluates
TEV, room temperature enzyme action 2 hours.Sample after enzyme action hangs back Ni post, and that collects outflow flows through liquid, removes cut His
The TEV enzyme of label and addition.
4. the parsing of crystal structure: employing Single wavelength anomalous scattering method (Single wavelength anomalous dispersion,
SAD) structure of Ter94-N/Npl4-UBD complex is resolved.Utilize the anomalous scattering wavelength at selenium
The diffraction data that lower collection obtains, obtains diffraction phase by the parsing of AutoSol program in Phenix software kit.Then,
Use COOT software to carry out the foundation of model, utilize the Refmac5 in Refinement and CCP4 in Phenix to carry out
The correction of structure.
5.Pull-down tests: takes the desired amount of affinity chromatograph beads, washes 3 times with appropriate pull-down buffer.According to rubbing
You mix two albumen for pull-down than the ratio for 1:1, and with pull-down buffer by molten for the albumen mixed
The final volume constant volume of liquid is to 500 μ l.20 affinity chromatograph beads pretreated for μ l are added, at 4 DEG C in this mixed liquid of protein
Slowly rotate on the rotary mixer of freezer and hatch 2h.At 4 DEG C with the centrifugation 2min of 500g, by affinity chromatograph beads
It is centrifuged at the bottom of EP pipe, abandons most supernatant.Add 1ml pull-down buffer and clean beads, 4 DEG C with the speed of 500g from
Heart 2min, abandons most supernatant.Repeated washing twice.Add 40 μ l elution buffer and carry out eluting, be incorporated on beads
Albumen elute.5 × SDS sample-loading buffer of 10 μ l, 100 DEG C of degeneration 10min are added in eluates.Utilize
SDS-PAGE analyzes the combination situation of two albumen.
Pull-down buffer:
His pull-down buffer:100mM Tris pH 8.0,150mM NaCl, 40mM imidazole, 10mM β-ME;
His elution buffer:100mM Tris pH 8.0,150mM NaCl, 500mM imidazole, 10mM β-ME;
Strep pull-down buffer:100mM Tris pH 8.0,150mM NaCl, 0.1%Trition X-100;
Strep elution buffer:100mM Tris pH 8.0,150mM NaCl, 2.5mM desthiobiotin, 0.1%Trition
X-100。
6. luciferase reporter gene experiment: luciferase reporter gene experiment is first with fluorescein for substrate to detect firefly
The activity of fireworm luciferase, after detect the activity of renilla luciferase with coelenteron fluorescein for substrate, and add follow-up
The substrate entering renilla luciferase has been simultaneously introduced the material of suppression LUC Photinus pyralis LUC Photinus pyralis FL catalysis luciferin, makes subsequent detection
Only it is measured to the activity of renilla luciferase, it is achieved the detection of luciferase reporter gene.LUC Photinus pyralis LUC Photinus pyralis FL is permissible
Catalysis luciferin is oxidized to oxyluciferin, during luciferin aoxidizes, can send bioluminescence.Sea pansy is glimmering
Light element enzyme can be catalyzed coelenteron fluorescein and be oxidized to coelenteramide, also can send life during coelenteron fluorescein aoxidizes
Thing fluorescence.May then pass through fluor tester to be also referred to as Chemiluminescence Apparatus or liquid flashing determining instrument to measure above-mentioned fluorescein enzymatic
The bioluminescence of release in oxidizing process.The strongest emission wavelength of LUC Photinus pyralis LUC Photinus pyralis FL catalysis luciferin luminescence is 560nm.
The strongest emission wavelength of renilla luciferase catalysis coelenteron fluorescein luminescence is 465nm.By luciferase and its substrate this
Bioluminescence system, can the expression of detection gene the sensitiveest, efficient.Generally the regulation and control of genetic transcription interested
Element is cloned in upstream or other suitable place of LUC Photinus pyralis LUC Photinus pyralis FL, is built into reporter plasmid, and then transfection is thin
Born of the same parents, cell lysis after suitably stimulating or processing, measure uciferase activity.Judge to stimulate by the height of uciferase activity
Front and back or the different stimulated impact on controlling element interested.Renilla luciferase is more used as the internal reference of transfection efficiency,
To eliminate cell quantity and the difference of transfection efficiency.
Experimental procedure: cultivate HEK293FT cell (or other purpose cell), and be inoculated in 24 orifice plates, grows 12-24
Hour (80% degree of converging).Luciferase reporter plasmid and sea pansy with lipo2000 transfection expression firefly luciferin are glimmering
Light element enzyme internal reference reporter plasmid, and required destination gene expression plasmid.After 24-48 hour, add cell cracking
Liquid (5X lysis buffer is diluted with water to 1X, purchased from promega): the 24 every holes of orifice plate add 120 μ l, the 48 every holes of orifice plate add
60μl.This plate is put-80 DEG C and freezes 1h.By plate from-80 DEG C of taking-ups, after room temperature is melted, by cell pyrolysis liquid sucking-off, transfer to
In EP pipe.Often pipe lysate takes 5 μ l and joins in 384 orifice plates.Add the reaction substrate of 10 μ l LUC Photinus pyralis LUC Photinus pyralis FL,
Survey the fluorescent value of reporter gene.Add the reaction substrate of 10 μ l, survey the fluorescent value of renilla luciferase.Reporter gene and sea pansy
The ratio of fluorescein fluorescence value is the relative fluorescence signal value of each sample.
In the experiment that intracellular reporter gene expression is affected by detection compound, HEK293FT cell transfects Flag label
RIG-I (residues 1-238), and IFN β or the reporter gene of ISRE, luciferase reporter gene.After 12 hours,
Peptic cell, is seeded in 384 orifice plates with the density in 10000 cells/every hole, adds the cell culture medium of 50 μ l.7 is little
Shi Hou, adds the compound that AlphaScreen experiment screening obtains, final concentration of 10 μMs of compound.Cultivate 15h for 37 DEG C
After, detect uciferase activity.
7. immunoblotting: prepare protein sample according to requirement of experiment, 100 DEG C of degeneration 10 minutes, 13200rpm is centrifuged 2 points
Clock, the supernatant taking equivalent is added in the loading hole of SDS-PAGE glue.Protein sample voltage when concentrating in glue is 80V,
Time in separation gel, voltage is 120V.After electrophoresis terminates, take off gel, membrane-transferring device is installed in the following order: (negative pole), filter
Paper, gel, nitrocellulose filter, filter paper, (positive pole).Membrane-transferring device is put into Cool Room 4 DEG C, 100V constant voltage transfer 1h.
After transferring film completes, take out nitrocellulose filter, film be immersed in the skim milk of use TBST buffer preparation 5%,
On shaking table, 1h is hatched under room temperature.Film, 5min × 3 time are washed with TBST buffer solution.
Add and resist with the one of the dilution of 5%BSA solution to scale, overnight incubation on the shaking table of Cool Room 4 DEG C.Delay with TBST
Dissolved liquid washes film, 10min × 3 time.Add and resist with the two of 5% milk dilution to scale, hatch on shaking table under room temperature
40-60min.Film, 10min × 3 time are washed with TBST buffer solution.Chromogenic substrate is covered on nitrocellulose filter, room
Temperature colour developing 2 minutes.Shoot with LAS4000 cold light/bioluminescence image analyzers.
The preparation of required reagent: 5%BSA solution: weigh 5g BSA powder and be dissolved in 100ml 1 × PBS solution, then
Add the sodium azide of 0.02%, be stored in 4 DEG C.10 × Western blot transferring film buffer: weigh 30.3g Tris alkali and 144
G glycine, adds 300ml methanol, then with deionized water constant volume to 1L.
The extracting of 8.RNA: the cell after transfection is discarded culture medium, and with the PBS of pre-cooling, cell is cleaned one time.Six orifice plates
Each hole add 500 μ l Trizol Reagent, room temperature treatment 5 minutes.Cell pyrolysis liquid is transferred to 1.5ml Eppendorf
Guan Zhong.Every part of sample adds the chloroform of 100 μ l, stands 5 minutes after the mixing of vortex low speed.4 DEG C, 12000g is centrifuged 15
Minute, in the upper strata aqueous phase of transferase 12 40 μ l to new Eppendorf pipe.Every part of sample adds the isopropanol of 240 μ l, reverse
Mixing, room temperature is placed 10 minutes.4 DEG C, 12000g is centrifuged 15 minutes, supernatant discarded, leaves the RNA precipitate of white.
Every part of sample adds 75% ethanol of 500 μ l, reverse mixing.4 DEG C, 12000g is centrifuged 5 minutes, abandons most supernatant.Room temperature
Dry.Add the deionized water that appropriate DEPC processed, fully dissolve.Measure with NanoDrop ND1000 and extracted
The concentration of RNA, the ratio of the light absorption value of 260nm and the light absorption value of 280nm should maintain between 1.9-2.0.To be extracted
RNA for reverse transcription or directly freeze to-80 DEG C of preservations.
The preparation of required reagent: phosphate buffered saline(PBS) (PBS): 800ml distilled water dissolves 0.2g KCl, 8g NaCl,
0.24g KH2PO4 and 1.44g Na2HPO4.With the pH value of HCl regulation solution to 7.4, it is settled to 1L.High steam
Sterilizing or filtration sterilization.
9. co-immunoprecipitation: the cell after transfection is discarded culture medium, and with the PBS of pre-cooling, cell is cleaned a time.Add suitable
Cell lysis buffer solution RIPA (containing protease inhibitor) of amount, cracks 30min on ice, by cell pyrolysis liquid in 4 DEG C,
Maximum (top) speed takes supernatant after being centrifuged 30min.Take the desired amount of protein A/G sepharose 4B, wash 3 with appropriate lysis buffer
Secondary.Take a small amount of lysate in case Western blot analyzes, residue lysate adds 20 protein A/G pretreated for μ l
Sepharose 4B and the 1 corresponding antibody of μ g, slowly rotate overnight incubation on the rotator of Cool Room 4 DEG C.Immunoprecipitation is complete
Cheng Hou, at 4 DEG C with the centrifugation 1min of 7000rpm, is centrifuged sepharose 4B at the bottom of EP pipe.Supernatant is carefully sucked,
Sepharose 4B is washed 2 times with 300 μ l lysis buffers, then washes 2 times with 300 μ l PBS.It is eventually adding the 2 × SDS of 20 μ l
Sample-loading buffer, 100 DEG C are boiled 10 minutes.SDS-PAGE and Western blot is utilized to analyze the albumen with antibodies
The preparation of required reagent: RIPA buffer:150mM NaCl, 100mM Tris pH 8.0,1%TritonX-100,5
mM EDTA,10mM NaF.
10. cell is cultivated: HEK293, HEK293T, MEF cell is incubated in DMEM (Hyclone) culture fluid, training
Nutrient solution is added 10% serum, 100ug/ml penicillin, 100 μ g/ml streptomycins.Cell is in 37 DEG C of cultivations, titanium dioxide
Concentration of carbon is 5%.
11. real-time quantitative fluorescence PCRs: real-time quantitative fluorescence PCR uses the Applied Biosystems company real-time PCR of two-step method
(Realtime-PCR) system, detects relative CT value.(Toyobo company provides to use quantitative fluorescence PCR premixed liquidGreen Realtime PCR reagent) configure reaction system detection the expression of quantitative objective gene,
GAPDH is as internal reference.
12. data analysiss: use SAS data software analysis bag (9.1.3) that data are analyzed, the average of statistical data ±
Standard deviation.Single-factor analysis of variance (ANOVA) and Student ' s t-test are used for analyzing continuous variable.Confidence interval
For P < 0.05.
Embodiment 1 p97 negative regulation I type interferon signal path depends on its ATP enzyme and lives
1. experiment purpose: determine whether p97 participates in I type interferon response and the signal path of antiviral immunity.
2. experimental technique:
(1) cell infection experiment: people HEK293, HEK293T, mice MEF cell is purchased from Chinese Academy of Sciences Shanghai
Life science institute cell bank.Cell culture processes is as described in basic experiment method 10.Sendai virus Sendai virus (SeV)
And GFP-Vesicular stomatitis virus is big by Wuhan with the vesicular stomatitis virus (GFP-VSV) of green fluorescence
Learning professor Shu Hongbing to grant, SeV with VSV-GFP is with commercially available, and VSV-GFP remodeling method is with Construction and
expression of high titer pseudotyped GFP-retroviruses by cotransfection of VSV-G geneJournal of
Shanghai Jiaotong University(Medical Science)》2007-07.Poly (I:C), poly (dA:dT) are purchased from Sigma
(St.Louis, MO), poly (I:C) uses rotaring transfecting mode to process cell, and poly (dA:dT) is directly added into cell culture medium
Middle process cell.
(2) IFN β, IFN β control domain PRDIII and PRDI (PRDIII-I), the reporter gene of ISRE is by China
Academy of science's Shanghai biochemistry is granted with cell institute professor Wang Chen, and above-mentioned Reporter gene vector is inserted corresponding reporter gene by commercial vector
Element is transformed and is obtained, and carrier related information refers to TRIM21 Is Essential to Sustain IFN Regulatory Factor 3
Activation during Antiviral Response2009vol.182no.63782-3792。
(3) IFNB, RANTES and ISG56 are in the detection of cell transcription level: Cell extraction total serum IgE method is such as
Described in basic experiment method 8, AMV reverse transcriptase synthesizes the first chain, and with it as template, under the effect of DNA taq enzyme
PCR amplification is carried out with corresponding primer;Reverse transcription system is shown in Table 1, and reverse transcription reaction program is shown in Table 2, and cell sample expands
PCR primer is shown in Table 3, and Realtime-PCR reaction system is shown in Table 4, and Realtime-PCR amplification program is shown in Table 5.
Table 1 reverse transcription reaction system
Table 2 reverse transcription program
37℃ | 15min | 50℃ | 5min | 95℃ | 5min | 4℃ | 20min |
Table 3 primer sequence
Table 4 Realtime-PCR reaction system
Table 5 PCR reaction system program setting
(4) siRNA knocks out intracellular p97: for p97 coding region and the siRNA of noncoding region and comparison siRNA by
Shanghai JiMa pharmacy Technology Co., Ltd synthesizes, and siRNA sequence is as shown in table 6.
Table 6 siRNA sequence
(5) Rescue experimental verification p97 function: p97 wild type and p97QQ mutant be biochemical and cell by Chinese Academy of Sciences Shanghai
Institute professor Zhao Yun grants, and above-mentioned carrier is inserted p97 wild type or p97QQ mutant fragment by commercial vector and obtains, and specifically carries
Body information refers to Ter94 ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial
degradation.Dev Cell.2013Jun 24;25(6):636-44.
3. experimental result and analysis:
Transfection IFN β in HEK293 cell, the Regulatory domain PRDIII of IFN β, the Regulatory domain PRDI of IFN β,
The reporter gene of ISRE, and the siRNA for p97 coding region.Use SeV infection cell, inducing type I interferon afterwards
Signal path.After siRNA knocks out p97, IFN β, the activity of PRDIII-I and ISRE reporter gene substantially raises (Figure 1A).
Equally, the mRNA level in-site of IFNB, RANTES and ISG56, along with the increase of SeV infection time, constantly raises (figure
1B).In people's 293T cell and mice MEF cell, lure with poly (I:C), poly (dA:dT), VSV-GFP
Lead I type interferon signal path, it is thus achieved that similar result (Fig. 1 C-1D).These results suggest that, knocking out p97 can increase
The signal path of strong I type interferon.
Infecting MEF cell with the VSV-GFP that infection multiplicity is 2, the ability of the cell anti-virus infection knocking out p97 is higher,
GFP is positive, the cell fewer than matched group (Fig. 1 E) that i.e. virus infects.By cell flow cytometer detection, p97 knock out thin
In born of the same parents, GFP is positive, and the cell proportion that i.e. virus infects only accounts for 9%, and in the cell of matched group, the cell ratio that virus infects
Example is up to 93% (Fig. 1 F).In having transfected the cell for p97 noncoding region siRNA, carry out rescue experimental verification
The function of p97.The activity of IFN β reporter gene of the p97 suppression Sev induction of process LAN wild type and transcribing of IFNB,
And make its p97QQ mutant lacking atpase activity there is no above-mentioned functions (Fig. 1 G-1H).This shows, p97 negative regulation
Antiviral immunity, this function depends on its atpase activity.
Embodiment 2 Npl4 works in coordination with p97 and regulates and controls Immune responses of the antivirus
1. experiment purpose: p97 combines to participate in the regulation and control of different signal paths from different cofactors, and the present embodiment purpose exists
In determining in the regulation and control of Immune responses of the antivirus, the cofactor that p97 combines.
2. experimental technique:
(1) siRNA knocks out intracellular p97, Npl4, Ufd1, p47, FAF1: above-mentioned siRNA is by Shanghai Ji agate pharmacy skill
Art company limited synthesizes, and p97siRNA sequence is as shown in table 6, and Npl4, Ufd1, p47, FAF1 are as shown in table 7.
Table 7 siRNA sequence
(2) detection of the transcriptional level of the activity of reporter gene and gene is with embodiment 1.
(3) the Npl4 vector construction of process LAN in cell
Extract cell total rna with the method in embodiment 1 and system, and reverse transcription is cDNA, in Takara high-fidelity
Archaeal dna polymerase PrimeSTAREffect lower correspondence primer carry out PCR amplification;PCR primer is shown in Table 8, PCR
Reaction system is shown in Table 9, and PCR amplification program is shown in Table 10.
Table 8 primer sequence
Table 9 PCR reaction system
Table 10 PCR amplification program
The fragment that amplification obtains, by 1% agarose gel electrophoresis, confirms that size is identical with purpose band, cuts glue, use
Agarose gel reclaims test kit and reclaims (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP209).Npl4 fragment makes
Carrying out double digestion with restricted enzyme NcoI and SalI (thermo scientific), endonuclease reaction system is as shown in table 11,
Reaction carries out 2 hours at 37 degree.
Table 11 fragment endonuclease reaction system
After enzyme action, by common products purification kit (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP204),
Purification obtains the fragment after enzyme action.Fragment (HindIII/NheI double digestion) and carrier pCDNA3 (the double enzyme of HindIII/NheI
Cut), it is attached, shown in linked system table 12, coupled reaction carries out 2 hours in room temperature (25 DEG C).Carrier double digestion
Reaction system is as shown in table 13, and reaction carries out 4 hours at 37 DEG C.Sky root common products purification kit is used after having reacted
Purification obtains the carrier after enzyme action.Product after connection converts DH5 α competent cell, passes through resistance screening, it is thus achieved that sun
Sex clone, and sequence verification.
Table 12 coupled reaction system
Table 13 carrier endonuclease reaction system
3. experimental result and analysis:
Different p97 cofactors is knocked out in HEK293T cell, after Npl4, Ufd1, p47 and FAF1,
Detect activity and the transcriptional level of IFNB of IFN β reporter gene after SeV infects.After knocking out Npl4, transcribing of IFN β
The transcriptional level of activity and IFNB substantially raises, and the Ufd1 forming complex with Npl4 also can raise SeV infection mediation
The signal path of I type interferon.Thus, in antiviral immunity signal path, Npl4-Ufd1 is the cofactor of p97
(Fig. 2 A-2B).We transfect the Npl4 of various dose in the cell infected SeV, and result shows that Npl4 can suppress
The activation of IFN β of SeV induction and the activity of ISRE reporter gene, and its inhibitory action presents metering and relies on effect (figure
2C).Equally, after process LAN Npl4, after SeV stimulation cell, the mRNA level in-site of IFNB and ISG56 substantially lowers (figure
2D).Knock out endogenous Npl4 with siRNA, can significantly activate IFN β, the reporter gene (figure of PRDIII-I and ISRE
2E).Equally, in the cell that Npl4 knocks out, the transcriptional level of IFNB, RANTES and ISG56 stimulates along with SeV
Time constantly raises (Fig. 2 F).Rescue experiment shows, knocks out endogenous Npl4 with the siRNA for Npl4 noncoding region
The ratio of cell infection VSV, then process LAN Npl4 can be reduced, cell more easy infection VSV (Fig. 2 G) can be made.Will
After p97 in cell knocks out, p97 or Npl4 of cotransfection Npl4 and wild type and saltant type p97QQ, only cotransfection
The cell of Npl4 Yu p97 wild type, the activity of IFN β reporter gene jin and the transcribing of IFNB of SeV induction be suppressed,
Individually the corotation of Npl4 or Npl4 and p97QQ mutant all can not suppress the activity of IFN β reporter gene and IFNB
Transcribe (Fig. 2 H).Result above shows that Npl4 participates in I type interferon signal path and antiviral as the cofactor of p97
Immunoreactive negative regulation.
The structure elucidation of embodiment 3 p97-Npl4 complex
1. experiment purpose: by the method for structure biology, assemble complex with p97, Npl4 albumen that purification obtains, solve
Analyse its structure, study protein-interacting mode.
2. experimental technique:
(1) expression vector establishment that composite structure resolves:
HT-represents pET28a-HisTEV carrier, the carrier built voluntarily for laboratory, with commercial vectors pET28a (Merck)
Transform and obtain.Plasmid information sees (Shi, Z., et al.Structure of the MST4 in complex with MO25 provides
Insights into its activation mechanism.Structure 21,449-461,2013)
Fruit bat HT-Ter94-N (20-186), fruit bat HT-Npl4-UBD (1-77) is respectively with fruit bat Ter94 total length, and cell is inverse
The cDNA transcribed is template, at Takara high-fidelity DNA polymerase PrimeSTAREffect lower correspondence draw
Thing carries out PCR amplification;PCR primer is shown in Table 14, and carrier construction method and system are with embodiment 2.The carrier connected is above-mentioned
PET28a-HisTEV, restriction enzyme site is NcoI/XholI.Fruit bat Ter94 total length is biochemical and cell by Chinese Academy of Sciences Shanghai
Institute professor Zhao Yun grants, and above-mentioned carrier is inserted Ter94 fragment by commercial vector and obtains, and concrete carrier information refers to Ter94
ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial degradation.Dev
Cell.2013 24;25(6):636-44.The preparation method of the cDNA of cell reversal record and system are with embodiment 1.
Table 14 primer sequence
(2) purification of Ter94-N/Npl4-UBD complex proteins:
It is purified by basic experiment method 3, has obtained the purity destination protein higher than 95%, then by a step gel filtration
Chromatograph homogeneous albumen.
By HT-Ter94-N and HT-Npl4-UBD of the purification ratio mixing with mol ratio as 1:1, hatch 2h for 4 DEG C, then
It is purified by the method for gel permeation chromatography, occurs in that single ultraviolet absorption peak, the position at this peak and independent purifying protein
Uv absorption peak position compare, there occurs significantly skew to the left, illustrate Ter94 and Npl4 can stoichiometrically than for
The mode of 1:1 combines, and forms stable binary complex.
(3) structure elucidation of HT-Ter94-N/HT-Npl4-UBD complex:
HT-Ter94-N/HT-Npl4-UBD complex proteins is concentrated to 25mg/ml, uses sitting-drop methods to carry out crystal growth bar
The Preliminary screening of part.After three days, in No. 69 condition of Index kit (Hampton Research), there is irregular lamellar
Crystal produces, and constituent is 0.1M Tris pH 8.5, and 25%PEG 3350,0.2M ammonium sulfate, by crystal
Take Shanghai synchrotron radiation light source biological macromolecule crystal light beam line station BL17U-MX to and carry out the collection of diffraction data.
(4) composite structure resolves:
In order to resolve the phase place of Ter94-N/Npl4-UBD complex, we are in the M9 culture medium containing selenomethionine
Abduction delivering HT-Ter94-N and HT-Npl4-UBD selenoprotein, then according to the purification process identical with parent protein enters
The purification of row selenoprotein.The Ter94-N/Npl4-UBD complex proteins utilizing selenomethionine labelling repeats this complex
The growth conditions of parent crystal is Index-69 and Index-75 (Hampton Research),.By changing precipitant PEG
Kind and concentration respectively the seleno crystal grown in the two condition is optimized, these seleno crystal are taken to Beijing with
Step radiating light source (BSRF) carries out the collection of diffraction data, (peak under three different wavelengthinflectionAnd remote) have collected resolution respectively and beWithDiffraction data,
Space group is P212121.Utilize the diffraction data of peak, by the method pair of Single wavelength anomalous scattering (SAD)
Ter94-N/Npl4-UBD complex has carried out the parsing of phase place and the foundation of model, has finally given the crystal knot of this complex
Structure.The structure of complex shows: the UBD domain of Npl4 has been combined in dredging of two subdomains formation of Ter94N end
In water groove, the combination ratio in asymmetric cell is 1:1.
(5) detection that IFN β activates and IFNB transcribes is with embodiment 1.
3. experimental result and analysis
Npl4 has four domains: the UBD domain (1-80) of N end is combined with p97, zf-Npl4 domain (104-246),
Npl4 domain (248-557), and the NZF domain (580-608) of C end is combined (Fig. 3 A) with ubiquitin.Disappearance
The IFN β that the Npl4 of UBD domain can not suppress SeV to induce activates and IFNB transcribes, and this proves Npl4 further
Collaborative p97 suppression antiviral immunity reaction.
We by resolve fruit bat Ter94-N/Npl4-UBD complex crystal structure respectively with people source p97N terminal domains
The nuclear-magnetism structure (PDB numbering 2PJH) of crystal structure (PDB numbering 3QQ7) and Mus source Npl4UBD domain is carried out
Relatively, it has been found that the people source single crystal structure of p97-N can be good at being superimposed upon one with the Ter94-N in fruit bat complex
Rising, after superposition, the root-mean-square-deviation (rmsd) of C alpha atom isThe sequence homology (78%) of this and they height
Consistent.On the other hand still there is notable difference between fruit bat Npl4-UBD and Mus source Npl4-UBD the two structure.
By Cdc48/Ter94/p97-N and Npl4-UBD of different plant species being carried out with structure for the sequence alignment guided, Wo Menfa
Key residues Q47 interacted with fruit bat Npl4-UBD on existing Ter94-N, F49, R50, D52, K106, Y107
With Y140 is the most conservative between different plant species.The crystalline substance of the fruit bat Ter94-N/Npl4-UBD complex that we are resolved
Body structure may describe universal way and the feature that p97 Yu Npl4 interacts more accurately.
The Ter94/Npl4 composite structure that we resolve and single Ter94, Npl4 is similar, the UBD domain knot of Npl4
Being combined in the hydrophobic groove of two subdomains formation of Ter94N end, the combination ratio in asymmetric cell is 1:1 (figure
3B).The heterodimer that Ter94/Npl4 complex is formed has two sites interacted.First site interacted
L7, R9 and R18 on first and second β lamella of Npl4 form hydrophobic phase interaction with the F49 of Ter94N end
With, R50, the D52 and Q47 of R9, R18 and the H69 of Npl4 and Ter94 forms hydrogen bond and ionic bond simultaneously.The
Y107 and Y140 of A13 and R49 and the Ter94 N end of two interaction sites Npl4 is by hydrophobic phase interaction
With connection, A13, Q11 and the R49 of Npl4 are interacted by hydrogen bond and ionic bond with the Y107 of Ter94.
Interact additionally, the Y140 of R49 Yu Ter94 of Npl4 forms cation-π.Above-mentioned these interact residual
Base is all conservative in fruit bat and people, thus the composite structure resolved with the Ter94-Npl4 of fruit bat, equally applicable and people
The structural analysis (Fig. 3 C) of p97-Npl4 complex.
The mutation research of embodiment 4 p97-Npl4 complex
1. experiment purpose: it is made in antiviral signal path by the interaction of mutation analysis research p97-Npl4 complex
Impact.
2. experimental technique:
(1) interact detection carrier S trep-Npl4, the vector construction of His-Npl4, Strep-p97, p97-His:
Strep-Npl4, His-Npl4 are with HA-Npl4 carrier as template, at Takara high-fidelity DNA polymerase PrimeSTAREffect lower correspondence primer carry out PCR amplification;PCR primer is shown in Table 15, and carrier construction method and system are with real
Execute example 2.Strep-Npl4 uses SEQ ID NO:31 in table 15, and 32 amplifications, the carrier of connection is commercial vector
PET51b-Strep, His-Npl4 use SEQ ID NO:31 in table 15, and 33 amplifications, the carrier of connection is build voluntarily
PET28a-HisTev, restriction enzyme site is NcoI/SalI.
Table 15 primer sequence
P97-His, Strep-p97 are with V5-p97 carrier as template, at Takara high-fidelity DNA polymerase PrimeSTAREffect lower correspondence primer carry out PCR amplification;PCR primer is shown in Table 16, and carrier construction method and system are with real
Execute example 2.P97-His uses SEQ ID NO:34 in table 16,35 amplifications, and the carrier of connection is commercial vector pET21a,
Restriction enzyme site is NdeI/SalI.Strep-p97 uses SEQ ID NO:36 in table 16, and 37 amplifications, the carrier of connection is
Commercial vector pET51b-Strep, restriction enzyme site is BamHI/SalI.
Table 16 primer sequence
(2) structure of the mutational vector of p97 and Npl4:
P97 mutant F52A/D55A, Y110A, with p97-His as template, use SEQ ID NO:38-41 in table 17
After shown primer carries out PCR amplification, using DpnI demethylase to remove unmutated wild type p97 template, amplification obtains
With the carrier segments with p97 gene of specific locus mutation, PCR primer is converted DH5 α competent cell, passes through
Resistance screening, it is thus achieved that positive colony, and sequence verification.
P97 mutant 3A, with p97-His F52A/D55A as template, uses SEQ ID NO:40 in table 17,41 institutes
After showing that primer carries out PCR amplification, use DpnI demethylase to remove unmutated p97-His F52A/D55A template, expand
Increase and obtain the carrier segments with p97 gene with specific locus mutation, PCR primer is converted DH5 α competent cell,
Pass through resistance screening, it is thus achieved that positive colony, and sequence verification.
Table 17 primer sequence
Npl4 mutant I6A/R8A, R50A, with Npl4-His as template, SEQ ID NO:42 in employing table 18,43,
After primer shown in 46,47 carries out PCR amplification, DpnI demethylase is used to remove unmutated wild type Npl4 template,
Amplification obtains the carrier segments with Npl4 gene with specific locus mutation, PCR primer converts DH5 α competence thin
Born of the same parents, pass through resistance screening, it is thus achieved that positive colony, and sequence verification.
Npl4 mutant I6A/R8A/R17A, with Npl4-His I6A/R8A as template, uses SEQ ID NO in table 18:
After primer shown in 44,45 carries out PCR amplification, DpnI demethylase is used to remove unmutated Npl4-His I6A/R8A
Template, amplification obtains the carrier segments with Npl4 gene with specific locus mutation, and PCR primer converts DH5 α sense
By state cell, pass through resistance screening, it is thus achieved that positive colony, and sequence verification.
Npl4 mutant 4A, with Npl4-His I6A/R8A/R17A as template, uses SEQ ID NO:46 in table 18,
After primer shown in 47 carries out PCR amplification, DpnI demethylase is used to remove unmutated Npl4-His I6A/R8A/R17A
Template, amplification obtains the carrier segments with Npl4 gene with specific locus mutation, and PCR primer converts DH5 α sense
By state cell, pass through resistance screening, it is thus achieved that positive colony, and sequence verification.
Table 18 primer sequence
3. experimental result and analysis:
We construct the mutant of a series of people p97 and Npl4 according to structural information, are tested by pull down, I
Find that (F52, Y110) (D55, the Y110) of p97 has conclusive effect for the combination of Npl4.Npl4's
The multipoint mutation of R50 and I6, R8, R17, R50, can break the interaction (Fig. 3 C-3E) of Npl4 Yu p97,
Co-immunoprecipitation experiment also demonstrates this result (Fig. 3 F-3G).Thus, F52A/D55A/Y110p97 (3A) suddenlys change
And Npl4 (4A) sudden change of I6A/R8A/R17A/R50A is to affect the critical sites that p97 Yu Npl4 interacts.?
Knocking out in the HEK293T cell of endogenous p97, under the stimulation of SeV, the p97 of transfected wild-type can suppress turning of IFN β
Record activates and the transcribing of IFNB, and transfection p973A mutant can not (Fig. 3 H).Equally, the Npl4 of transfected wild-type
Can suppress the activation of IFN β promoter and transcribing of IFNB, and Npl44A mutant can not (Fig. 3 I).More than tie
Fruit shows, p97-Npl4 forms complex, plays a role in antiviral signal path.
The protein level of embodiment 5 p97-Npl4 complex negative regulation RIG-I
1. experiment purpose: p97 has AAA ATP enzyme and lives, can be with the stability of modulin.For research p97-Npl4 complex
Whether by the stability of the molecule in the antiviral signal path of regulation and control RLR mediation, we have studied p97-Npl4 multiple
The compound regulation and control to RIG-I protein level.
2. experimental technique:
(1) slow virus restructuring shRNA knocks out intracellular p97, Npl4, Ufd1, p47
The target sequence (shRNA) of one section of 21 base of design, this sequence can form the hair fastener shape structure of reverse complemental, heavy
Silent related gene expression.ShRNA and comparison shRNA (scramble) of corresponding gene are connected to slow virus carrier
pLKO.1-puro.ShRNA (scramble) is comparison.ShRNA sequence is as shown in table 19.
Table 19 shRNA sequence
The forward of 0.01M and the mixing of reverse DNA chain, be diluted to 50ul, through following reaction:
95℃ | 5min | 70℃ | 10min | 55℃ | 30min | 37℃ | 30min |
Annealing is connected as double-strand.Double-stranded DNA is connected with pLKO.1-puro (EcoRI/AgeI double digestion) carrier, converts DH5 α,
Identify that the positive colony obtained extracts plasmid after sequence verification.By plasmid rotaring redyeing 293 cell, with 2 μ g/ml puromycin
The cell of screening-gene group stable integration shRNA sequence, after 48-72 hour, uses Vivaspin 20ml (Sartorius
Stedim Biotech, Aubagne, France) with the centrifugal force of 3000g, centrifugal 50min, concentrating virus liquid.Infect virus
After, the cell knocking out corresponding gene can be obtained.PLKO.1-puro plasmid is biological from Shanghai life science institute of the Chinese Academy of Sciences
Obtain at chemistry and the refined researcher of Institute of Cell Biology JIHONG, for the common carrier of commercially available structure RNAi, can be from
Addgene company buys.
(3) vector construction of Flag label RIG-I, Flag label RNF125:
Extract cell total rna with the method in embodiment 1 and system, and reverse transcription is cDNA, in Takara high-fidelity
Archaeal dna polymerase PrimeSTAREffect lower correspondence primer carry out PCR amplification;PCR primer is shown in Table 20, carries
Body construction method and system are with embodiment 2.Flag-RIG-I uses SEQ ID NO:60 in table 20,61 amplifications, Flag-RNF125
Using SEQ ID NO:62 in table 20,63 amplifications, the carrier of connection is commercial vector pCDNA3.1-Flag, restriction enzyme site
It is HindIII/XholI.
Table 20 primer sequence
(4) RIG-I K181 mutation construction:
With Flag-RIG-I as template, after primer carries out PCR amplification shown in employing table 21, use DpnI demethylase
Removing unmutated template, amplification obtains the carrier segments with Npl4 gene with specific locus mutation, by PCR primer
Convert DH5 α competent cell, pass through resistance screening, it is thus achieved that positive colony, and sequence verification.
Table 21 primer sequence
(5) antibody detection protein expression: experimental technique is with basic experiment method 7.Detection Flag, Myc and β-actin's
Antibody is purchased from Sigma (St.Louis, MO).Detection p97, RIG-I, MAVS, p-TBK, TBK, IKK ε, p-IRF3,
The antibody of IRF3 and K48Ub is purchased from Cell Signaling (Danvers, MA).Detection HA, Ubiquitin, RNF125
Antibody purchased from Santa Cruz (Santa Cruz, CA).The antibody of detection Npl4 (IF) is purchased from Novus Biologicals
(Littleton, CO).Detection Npl4 (IP and WB) is purchased from Abcam (Cambridge, UK).
3. experimental result and analysis:
We have detected the protein level (Fig. 4 A) of RIG-I, MAVS and IRF3 in the cell having transfected shp97.Strike
Except p97 can dramatically increase the RIG-I expression that poly (I:C) induces, the protein level of same MAVS also increases, IRF3
Total protein levels be not changed in, but its phosphorylation level substantially increases.In turn, process LAN Npl4 significantly reduces poly
(I:C) expression of RIG-I and MAVS induced, the phosphorylation level of IRF3 is significantly reduced (Fig. 4 B).Additionally,
Knock out Npl4 and can dramatically increase the expression of what poly (I:C) induced RIG-I, MAVS, strengthen the phosphorylation of IRF3
Level, and the protein level of TBK1, IKK ε and IRF3 is not changed in (Fig. 4 C).Equally, knock out p47, p97's
Another cofactor, does not has regulating and controlling effect to RIG-I antiviral path.Thus p97 leads to for RIG-I antiviral signal
The regulating and controlling effect on road depends on Npl4.Process LAN Npl4, can't cause the mRNA of the RIG-I that poly (I:C) induces
Level changes, and the RIG-I protein level that after processing cell with proteasome inhibitor MG132, p97 and Npl4 mediates
Decline and be also suppressed (Fig. 4 D-4E).This explanation p97-Npl4 complex albumen by proteasomal degradation regulation and control RIG-I
Level.At intracellular p97 or Npl4 that knock out of HEK293, then transfect the K48 chain ubiquitin of Flag label RIG-I, RIG-I
Change level is decreased obviously (Fig. 4 F-4G).On the contrary, the HEK293 cell knock out p97 transfects Flag label RIG-I simultaneously
And the K48 chain ubiquitination level of wild type p97, RIG-I substantially increases, and transfect Flag-RIG-I and can not be with simultaneously
The p973A mutant that Npl4 combines, does not affect (Fig. 4 H) to the ubiquitination level of RIG-I.Equally, process LAN is wild
The Npl4 of raw type can increase the K48 ubiquitination level of RIG-I, and the Npl44A that process LAN can not be combined with p97 suddenlys change
The ubiquitination level of RIG-I is not affected (Fig. 4 I) by body.These results suggest that, p97-Npl4 complex is by promoting RIG-I
K48 chain ubiquitination so that it is by proteasomal degradation, suppress antiviral immunity signal path.P97-Npl4 complex leads to
Cross mediation RIG-I degrading and regulating antiviral signal path, and research before show E3 ubiquitin ligase c-Cbl and
RNF125 participates in the K48 chain ubiquitination of RIG-I, we have studied the RIG-I degraded participating in the regulation and control of p97-Npl4 complex
Ubiquitin ligase.We have separately designed the shRNA of RNF-125 and c-Cbl, demonstrate it by Q-PCR and knock out
Efficiency.Double luciferase element Reporter Gene Experiments show, knock out endogenous RNF125, and Npl4 cannot lower the IFN β of SeV induction
The activation of promoter, and knock out c-Cbl and do not act on (Fig. 5 A).Equally, in the cell knocking out RNF-125, Npl4
The transcriptional activation of the IFN β that the card domain of process LAN RIG-I mediates, in the cell knocking out c-Cbl, Npl4 can not be suppressed
Inhibitory action unaffected (Fig. 5 B).In the cell knocking out endogenous RNF125, Npl4 can not reduce RIG-I's
Protein level (Fig. 5 C).These results suggest that, RNF125 is that p97-Npl4 complex suppression RIG-I antiviral signal leads to
E3 ubiquitin ligase special in road.We have detected the ubiquitination water of total RIG-I in the cell of transfection Npl4 or RNF125
Flat, process LAN Npl4 significantly increases the K48 chain ubiquitination level of RIG-I, and process LAN Npl4 and RNF125 simultaneously
Can increase its ubiquitination level (Fig. 5 D) further, this shows Nlp4 Yu RNF125 synergism, promotes RIG-I
K48 chain ubiquitination.QPCR experiment also indicates that cotransfection Npl4 with RNF125 is remote for the transcripting suppressioning action of IFN β
Higher than individually transfection Npl4 or RNF125 (Fig. 5 E).We are process LAN RNF125 in cell, and thinks non-process LAN
The cell of RNF125 is comparison, and by the ubiquitination site of the RIG-I that mass spectral analysis RNF125 is catalyzed, result shows that it is general
Elementization occurs and 181 lysines (Fig. 5 F).Build 181 by point mutation means not suddenlyd change by the RIG-I of ubiquitination
Body K181, cotransfection wild type or RIG-I Yu Npl4 or RNF125 of mutant in cell, after SeV stimulates, pass through
The protein level of immune-blotting method RIG-I, result shows that Npl4 or RNF125 can significantly reduce the RIG-I of wild type
Protein level, but the protein level of mutant K181R is not affected (Fig. 5 G), this shows that 181 lysines are
P97-Npl4 complex is by the major site of antiviral path relevant for RNF125 regulation and control RIG-I.Additionally, Npl4 is permissible
Increase the K48 chain ubiquitination level of RIG-I, but the K48 chain ubiquitination level of K181R mutant is not affected (figure
5H).Equally, RNF125 and Npl4 can substantially suppress the activity of the IFN β promoter that wild type RIG-I mediates,
But K181R mutant is not affected (Fig. 5 I).After knocking out Npl4 or RNF125 in cell, transfected wild-type
RIG-I can suppress VSV to infect (infection multiplicity MOI=20), and transfection K181R mutant can not produce antivirus action
(Fig. 5 J).We analyze the architectural feature of the 181st lysine of RIG-I further, and this site is structurally close 172
Position lysine, i.e. RIG-I activates relevant crucial K63 chain ubiquitination site, and the phosphorus of negative regulation RIG-I signal path
The 170th threonine of polyadenylation sites, thus, the 181st lysine of RIG-I is the RIG-I of RNF125/p97-Npl4 mediation
The critical sites of ubiquitination.
Embodiment 6 p97-Npl4 complex is combined with RIG-I and RNF125
1. experiment purpose: whether detection p97-Npl4 complex is directly in conjunction with RIG-I and RNF125.
2. experimental technique:
(1) Flag label RIG-I 1-238, the vector construction of 200-803,804-925:
With Flag-RIG-I as template, at Takara high-fidelity DNA polymerase PrimeSTAREffect under with correspondence
Primer carry out PCR amplification;PCR primer is shown in Table 20, and 22, carrier construction method and system are with embodiment 2.The load connected
Body is commercial vector pCDNA3.1-Flag, and restriction enzyme site is HindIII/XholI.(" Flag label RIG-I 1-238 expands
Increasing primer is table 20 primer 60, table 22 primer 66;Flag label RIG-I 200-803 amplimer is table 22 primer 67,68;
Flag label RIG-I 804-925 amplimer is table 22 primer 69, table 20 primer 61 ")
Table 22 primer sequence
(2) HA-Npl4 total length, Δ NZF 1-579, Δ UBD (81-608), and individually UBD vector construction: with HA-Npl4
For template, at Takara high-fidelity DNA polymerase PrimeSTAREffect lower correspondence primer carry out PCR
Amplification;PCR primer is shown in Table 8, and 23, carrier construction method and system are with embodiment 2.The carrier connected is commercial vector
PCDNA3, restriction enzyme site is HindIII/NheI.(" HA-Npl4 Δ NZF 1-579 amplimer is table 8 primer 25,
Table 23 primer 70;HA-Npl4 Δ UBD (81-608) amplimer is table 23 primer 71, table 8 primer 26;HA-Npl4
UBD amplimer is table 8 primer 25, table 23 primer 72 ")
Table 23 primer sequence
(3) vector construction of MBP-RIG-I CARD, MBP-RNF125: MBP-RIG-I with Flag-RIG-I as template,
MBP-RNF125 is with Flag-RNF125 as template, at Takara high-fidelity DNA polymerase PrimeSTARWork
PCR amplification is carried out with lower corresponding primer;PCR primer is shown in Table 24, and carrier construction method and system are with embodiment 2.Even
The carrier connect is that (vector construction information sees Shi, Z., et al.Structure of for the carrier pET28a MBPTev that builds voluntarily
MST4 in complex with MO25 provides insights into its activation mechanism.Structure 21,
449-461,2013)), restriction enzyme site is NcoI/XholI.
Table 24 primer sequence
3. experimental result and analysis:
We, in the HEK293T cell that SeV stimulates, carry out co-immunoprecipitation experiment, find that endogenous RIG-I can be with
Endogenous Npl4 combines, it is also possible to be combined (Fig. 6 A) with RNF125.Meanwhile, laser confocal microscope detection also demonstrates that
, under SeV stimulates, there is location (Fig. 6 B) altogether in Npl4 Yu RIG-I.Npl4 Yu the RIG-I restructuring of purification
Albumen proves in pulldown tests further, and the two albumen can be with direct interaction (Fig. 6 C).We intercept
The domain of different RIG-I and Npl4, studies its concrete region interacted, different RIG-I fragments and total length
Npl4 cotransfection HEK293T cell, co-immunoprecipitation experiment shows, the CRAD domain (amino acid/11-238) of RIG-I
Interacting with Npl4, helicase domain and CTD domain are not involved in the interaction (Fig. 6 D) of itself and Npl4.
Equally, the deletion mutant of Npl4 and the RIG-I cotransfection cell of total length, co-immunoprecipitation experiment shows, disappearance Npl4's
The mutant of NZF domain completely loses the ability being combined with RIG-I, and lack the Npl4 of UBD domain still can be with
RIG-I combines (Fig. 6 E), i.e. Npl4 is combined with RIG-I by NZF domain.External pulldown experiment is further
Prove that Npl4 can directly be combined (Fig. 6 F) with the CARD domain of RIG-I.Endogenous co-immunoprecipitation experiment shows RNF125
Can not only be combined with RIG-I, also can be combined (Fig. 6 G) with p97.Utilize the albumen of purification, it has been found that p97 can
To be combined with RNF125, exist between the two and directly interact (Fig. 6 H).Additionally, RNF125 can directly tie
Closing RIG-I and p97-Npl4 complex, p97-Npl4 complex can promote between RNF125 and RIG-I further
Interact (Fig. 6 I).Result above shows, under the stimulation of SeV, p97-Npl4 complex can with RIG-I and
RNF125 combines, the K48 chain ubiquitination of RNF-125 mediation RIG-I, and then promotes the proteasomal degradation of RIG-I, presses down
The antiviral signal path of RIG-I processed.
The screening of embodiment 7 p97-Npl4 complex inhibitor and the effect in antiviral signal path thereof
1. experiment purpose: determine the micromolecular compound detection compound pair that can interact with specific inhibition p97-Npl4
The impact of the antiviral signal path of p97-Npl4 mediation.
2. experimental technique:
(1) AlphaScreen experiment: in 384 shallow bore hole plates (PerkinElmer), add the Npl4 of 2.5 μ l band His labels,
2.5 the biotin labeled p97 of μ l, add reaction buffer (20mM Hepes pH 7.5,100mM NaCl, 0.1%BSA),
Every pore volume is 11 μ l, and the final concentration of 125nM of the Npl4 of band His label, biotin labeled p97 is final concentration of
31.25nM, by reaction system in incubated at room 60 minutes.With dilution buffer (20mM Hepes, 100mM NaCl,
PH 7.5) compound (10mM) being dissolved in DMSO is diluted to 100 μMs, the compound after dilution is added 1 μ l
To reaction system, room temperature lucifuge hatches 60 minutes.2.5 μ l nickel chelating acceptor beads (PerkinElmer) are added
In reaction system, final concentration of 10 μ g/ml, after room temperature lucifuge hatches 60 minutes, add the streptavidin crosslinking of equivalent
Donor beads (PerkinElmer), room temperature lucifuge hatches 60 minutes.At instrument EnVision Multilabel Plate Reader
(PerkinElmer) reading numerical values, every piece of 384 orifice plates all have positive control and negative control.
(2) intracellular Reporter Gene Experiments: transfect the RIG-I CARD domain of Flag label in HEK293FT cell
, and IFN β or the reporter gene of ISRE, luciferase reporter gene (1-238).After 12 hours, peptic cell, with
The density in 10000 cell/every holes is seeded in 384 orifice plates, adds the cell culture medium of 50 μ l.After 7 hours, add
The compound that AlphaScreen experiment screening obtains, final concentration of 10 μMs of compound.After 37 DEG C are cultivated 15h, detect glimmering
Light element enzymatic activity.
3. experimental result:
The antiviral signal path for IFN β mediation that interacts of p97 with Npl4 is necessary, and we pass through
These screening means of AlphaScreen, have bioactive compound composition compound library with 8120 and carry out high flux sieve
Choosing, finds and can break the inhibitor that p97-Npl4 interacts.Preliminary experiment is optimized by a series of experiment flow,
The Z factor of experiment reaches 0.7, shows that experiment condition or measuring method may apply in the experiment of ensuing high flux.Right
After 8120 compounds screen, it is thus achieved that suppression ratio > compound 132 of 50%.By biotin labeling Npl4, screening
False-positive condition, we determined that 40 suppression ratio > 30%, the compound that p97-Npl4 complex is formed can be suppressed.
By intracellular Reporter Gene Experiments, we have detected this 40 compounds effect in antiviral signal path, Qi Zhongsan
Individual compound is for reporter gene IFN β and the activation effect of ISRE > 30%, i.e. Thonzonium Bromide (TB),
Epirubicin Hydrochloride (EH) and Purpurin.TB is 102.6% for the activation multiplying power of IFN β, to ISER
Activation multiplying power be 107.6%;EH is 89.5% for the activation multiplying power of IFN β, and the activation multiplying power to ISER is 207%,
Thus the two Compounds Against viral signal path has obvious activation.
It is experimental verification TB, EH impact for p97-Npl4 complex by pulldown further, uses embodiment 4
The pulldown system of middle p97, Npl4 interaction detection, is separately added into contrast solution and compound, detection in system
Interaction between the two, uses gray analysis to electrophoresis result, shows that both micromolecular compounds have slackened both significantly
Interaction (Fig. 7).Result shows, the micromolecular compound that above-mentioned screening obtains can be combined by destroying p97-Npl4
The formation of thing, promotes antiviral signal path relevant for RIG-I.
Claims (11)
1. epirubicin hydrochloride purposes in the inhibitor preparing the interaction that can block p97 Yu Npl4.
Purposes the most according to claim 1, it is characterised in that described inhibitor can suppress the RIG-I that RNF-125 mediates
K48 chain ubiquitination.
Purposes the most according to claim 1, it is characterised in that described inhibitor can suppress the proteasomal degradation of RIG-I.
Purposes the most according to claim 1, it is characterised in that described inhibitor can promote the IFN β letter that RIG-I mediates
Number path.
Purposes the most according to claim 1, it is characterised in that described inhibitor can play raising viral infection resisting ability and enter
And play the effect that treatment virus infects.
6. epirubicin hydrochloride purposes in the pharmaceutical composition that preparation treatment virus infects.
Purposes the most according to claim 6, it is characterised in that described epirubicin hydrochloride is by promoting that RIG-I mediates
IFN β signal path thus improve viral infection resisting ability and then play the effect that treatment virus infects.
Purposes the most according to claim 7, it is characterised in that described epirubicin hydrochloride is the albumen by suppressing RIG-I
Enzyme body is degraded thus is played the effect of the IFN β signal path promoting RIG-I mediation.
Purposes the most according to claim 8, it is characterised in that described epirubicin hydrochloride is to be situated between by suppression RNF-125
Lead the K48 chain ubiquitination of RIG-I thus play the effect of the proteasomal degradation of suppression RIG-I.
Purposes the most according to claim 9, it is characterised in that described epirubicin hydrochloride be by suppression RNF125 with
Interacting thus play the effect of the K48 chain ubiquitination of suppression RNF-125 mediation RIG-I between RIG-I.
11. purposes according to claim 10, it is characterised in that described epirubicin hydrochloride is by blocking p97 and Npl4
Interaction suppress the interaction between the formation of p97-Npl4 complex, and then suppression RNF125 and RIG-I
's.
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Citations (3)
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CN1554354A (en) * | 2003-12-23 | 2004-12-15 | 中国药科大学 | Epi-doxorubicine liposome and its preparing method |
WO2006121359A1 (en) * | 2005-05-12 | 2006-11-16 | Institut National De La Sante Et De La Recherche Medicale, Inserm | Epirubicine hydrochloride for treating hepatitis c virus |
WO2007075092A1 (en) * | 2005-12-29 | 2007-07-05 | Instytut Biochemii I Biofizyki | New derivatives of epirubicin, their medicinal application and pharmaceuticals acceptable forms of drugs |
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CN1554354A (en) * | 2003-12-23 | 2004-12-15 | 中国药科大学 | Epi-doxorubicine liposome and its preparing method |
WO2006121359A1 (en) * | 2005-05-12 | 2006-11-16 | Institut National De La Sante Et De La Recherche Medicale, Inserm | Epirubicine hydrochloride for treating hepatitis c virus |
WO2007075092A1 (en) * | 2005-12-29 | 2007-07-05 | Instytut Biochemii I Biofizyki | New derivatives of epirubicin, their medicinal application and pharmaceuticals acceptable forms of drugs |
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