CN105602910A

CN105602910A - Application of ELL as E3 ubiquitin ligase

Info

Publication number: CN105602910A
Application number: CN201610003565.4A
Authority: CN
Inventors: 肖武汉; 陈瑜; 姬伟; 周迟
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2016-01-05
Filing date: 2016-01-05
Publication date: 2016-05-25
Anticipated expiration: 2036-01-05
Also published as: CN105602910B

Abstract

The invention discloses application of ELL as an E3 ubiquitin ligase. The application of ELL protein or truncated protein of the ELL protein to at least one of 1-6: 1, a protein product for degrading c-Myc is prepared; 2, the ELL protein or the truncated protein of the ELL protein is used as the E3 ubiquitin ligase; 3, a product for inhibiting tumor cell proliferation is prepared; 4, a product for inhibiting tumorigenesis is prepared; 5, a product for identifying or assisting in identifying whether tissue to be detected is tumor tissue or not is prepared; 6, a product for identifying or assisting in identifying whether a patient to be detected is a tumor patient or not is prepared. The experiment proves that ELL is combined with c-Myc and induces c-Myc to be degraded through proteasome, in-vivo and in-vitro studies both indicate that the ELL has the characteristic of the E3 ubiquitin ligase, can inhibit tumor cell proliferation and tumorigenesis, can be used as a molecular marker for tumor clinical diagnosis, and can be used for preparing the medicine or a kit for inhibiting tumors and a kit for diagnosing the tumors as the novel E3 ubiquitin ligase of c-Myc.

Description

ELL is as the application of E3 ubiquitin ligase

Technical field

The present invention relates to biological technical field, relate in particular to the application of a kind of ELL as E3 ubiquitin ligase.

Background technology

The gene outcome of ELL first identified out be at acute myeloid leukaemia (AML) (Thirman with mll geneDeng people 1994) transposition merge part (MLL-ELL). Further Biochemical Research is found: ELL is a transcriptional elongation factor. ?In experiment in vitro (Invitro), it can increase transcribing of rna plymerase ii and extend active. Afterwards at body research (InVivo) disclose its be associated with Gene Transcription in vitro site (people 2002 such as Eissenberg, people such as Shilatifard1996). ELL is a part for two different extension complexs, i.e. super extension complex (SEC) and little extension complex(LEC) (people 2013 such as Hu, the people such as Luo 2012, Smith and Shilatifard2013). As positive transcriptional elongation factor (P-TEFb) one of most active form in, super extension complex (SEC) is carried out several critical functions, for example: HSP70 induces heatShock, development gene response environment are coerced people 2010 such as (, the people such as Lin 2011) Lin, the HOX base in MLL-AFF1-transformantBecause of imbalance people 2010 such as () Lin, and the transcriptional activation of HIV people 2010 such as (, the people such as Sobhian 2010) He. Little extension is compoundBody (LEC) snRNA genetic transcription open begin and the extension stage regulate small nuclear rna (smallnuclearRNA) transcribing andFunction (people 2013 such as Hu, the people such as Smith 2011).

ELL is essential in mammiferous embryonic development, because embryonic death appears in the mouse that ELL target knocks out(people 2000 such as Mitani). In addition, ELL is as steroid receptor, hypoxic inducing factor-1-α (HIF-1 α), and E2F1 and TFIIH are multipleFit combination albumen, it can regulate their protein-bonded activity (Pascual-Le in the mode of a kind of forward or negative senseThe people such as Tallec 2005) (the people 2010a such as Liu) (people 2013 such as Mourgues, the people such as Zhang 2014). In sum, ELL hasVery diversified function; But its mechanism of action definite under physiological condition is still unknown.

Proto-oncogene c-Myc is identified by being identified in similar v-Myc oncogene in host cell gene group at firstOut (people 1982 such as Vennstrom). Research subsequently shows, mankind c-Myc is frequent transposition (Shou in Huppert's diseaseDeng people 2000), and high expressed or sudden change (people 2010 such as Beroukhim) in many different human cancers, thereby it confirmedIt is a human carcinomas gene (Dang2012). C-Myc gene code is a kind of many what play an important role aspect regulatory gene expressionFunction transcription factor, its function involves many bioprocess, and these processes contribute to tumour generation, tumor development and tumourShift (Dang2012).

C-Myc is a labile protein that can regulate multiple physiology courses. Ubiquitin-Proteasome Pathway (UPS) isOne of the most outstanding mechanism that c-Myc degrades in cell (FarrellandSears2014). In view of the vast as the open sea endThing needs corresponding selectively targeted E3 ubiquitin ligase, and therefore the estimated number of mankind E3 ligase is greater than 600 (BerndsenAnd Wolberger2014). E3 ligase can be divided into three extended familys: (RING)-FINGER family of latest find, HECT familyAnd RING-between-RING (RBR) family (Berndsen and Wolberger2014). RING-FINGERE3 ligase canTransfer to substrate with direct catalysis ubiquitin from E2. In contrast, HECT is connected enzymatic with RBRE3 be two-step reaction, whereinFirst ubiquitin transfers to an active cysteine site of E3 from E2, and then from E3 to substrate. As RING-FINGEROne of component of structure ubiquitin junctional complex (Farrell and Sears2014), FBW7 (presses down by mediation c-Myc degradedMake its activity) be to study to obtain the most sufficient E3 ubiquitin ligase; It identifies the phosphorylation of the 58th threonine of c-Myc (T58),This phosphorylation is by by glycogen synthase kinase 3 (GSK3) mediation people 2004 such as (, the people such as Yada 2004) Welcker. AnotherRING-FINGERE3 ligase Skp2, can be identified in the aminoterminal one section of conserved sequence element of c-Myc (MBII) andHLH-LZ motif (367-439 amino acid), promotes its many ubiquitinations and degraded, and causes the enhancing of c-Myc transcriptional activity(people 2003 such as Kim, the people such as vonderLehr 2003). The 3rd RING-FINGERE3 ligase β-TRCP, is combined in c-The amino terminal of Myc, and use UbcH5 ubiquitin binding enzyme (E2) to form the special-shaped ubiquitin chain on c-Myc; Thereby strengthen c-The stability (people 2010 such as Popov) of Myc. The HECTE3 ligase HectH9 of unique c-Myc being in the news, ubiquitination c-Myc forms poly ubiquitin chain people 2005 such as () Adhikary of 63 lysines, the c-Myc degraded that it does not cause, butBut be the multiple target genes of c-Myc trans-activation necessary (people 2005 such as Adhikary). Therefore, the stability of c-Myc and generalBetween elementization, exist complicated relevance, as the relation between transcriptional activity and the stability of same c-Myc.

As one of classical proto-oncogene, there is overexpression in c-Myc in approximately 70% human tumor; But, theseIn tumour, only have 20% c-Myc gene magnification or the transposition showing, this is enough to explain the high expressed of c-Myc at protein level(Farrell and Sears2014). Therefore excessively expressing of the c-Myc albumen of, observing in human tumor may be with accordinglyThe downward of E3 ubiquitin ligase is relevant. In fact, in tumour, reported the unconventionality expression of some E3 ligases of c-MycAnd/or sudden change. Point mutation that it should be noted that FBW7 in human cancer can cause its inactivation, and may not express(Farrell and Ears2014). There are some researches show recently, FBW7 is initial by regulating the stability of c-Myc to affect leukaemiaCytoactive, the adjusting of the ubiquitination of FBW7 to c-Myc can control that chronic granulocytic leukemia occurs and the development (heat such as KingThe people such as 2013, Reavie 2013). In contrast, the positive regulating gene Skp2 of the transcriptional activity of c-Myc and HectH9 demonstrate and causeCarcinous, (Farrell and Sears2014) verified in their expression of crossing in many human tumors.

Summary of the invention

An object of the present invention is to provide the purposes of ELL albumen or its truncate.

The invention provides ELL albumen or its truncate following 1)-6) application at least one:

1) preparation degraded c-Myc protein product;

2) as E3 ubiquitin enzyme ligase;

3) prepare inhibition tumor cell propagation product;

4) preparation suppresses tumour formation product;

5) as the molecular labeling of diagnosing tumor and/or qualification;

The amino acid sequence of described ELL albumen truncate is to comprise 583-614 bit amino in ELL Argine Monohydrochloride sequenceArbitrary section of acid residue.

The amino acid sequence of described ELL albumen is sequence 4 in sequence table;

The amino acid sequence of described c-Myc albumen is sequence 2.

In above-mentioned application, described ELL albumen truncate is following 1)-6) in any:

1) amino acid sequence of the ELL albumen truncate shown in is sequence 4 46-621 positions;

2) amino acid sequence of the ELL albumen truncate shown in is sequence 4 447-621 positions;

3) amino acid sequence of the ELL albumen truncate shown in is sequence 4 509-621 positions;

4) amino acid sequence of the ELL albumen truncate shown in is sequence 4 466-621 positions;

5) amino acid sequence of the ELL albumen truncate shown in is sequence 4 447-621 positions;

6) amino acid sequence of the albumen shown in is sequence 4 583-614 positions.

In above-mentioned application, described tumour is colon cancer; Described tumour cell is colon cancer cell; Described colon cancer cell toolBody is HCT116 cell.

In above-mentioned application, described product is kit or medicine.

Another object of the present invention is to provide a kind of following 1)-5) any.

1) degraded c-Myc protein product;

2) as E3 ubiquitin enzyme ligase;

3) inhibition tumor cell propagation product;

4) suppress tumour and form product;

5) as the molecular labeling of diagnosing tumor and/or qualification;

The amino acid sequence of described c-Myc albumen is sequence 2.

In the said goods, described ELL albumen truncate is following 1)-6) in any:

6) amino acid sequence of the albumen shown in is sequence 4 583-614 positions.

In the said goods, described tumour is colon cancer; Described tumour cell is colon cancer cell; Described colon cancer cell toolBody is HCT116 cell.

In the said goods, described product is kit or medicine.

The 3rd object of the present invention is to provide a kind of protein.

Protein provided by the invention, it is following 1)-6) in any:

1) amino acid sequence of the albumen shown in is sequence 4 46-621 positions;

2) amino acid sequence of the albumen shown in is sequence 4 447-621 positions;

3) amino acid sequence of the albumen shown in is sequence 4 509-621 positions;

4) amino acid sequence of the albumen shown in is sequence 4 466-621 positions;

5) amino acid sequence of the albumen shown in is sequence 4 447-621 positions;

6) amino acid sequence of the albumen shown in is sequence 4 583-614 positions.

The ELL of experiment showed, of the present invention has the feature of E3 ubiquitin ligase, and it is in conjunction with c-Myc and induce it to pass through eggWhite enzyme body degraded. ELL can pass through degraded c-Myc albumen, thereby inhibition tumor cell propagation and tumour form. Therefore, thisA bright previous unknown function that has disclosed ELL,, as the E3 ubiquitin ligase of a kind of novel c-Myc, mediates c-MycEffective degraded. It can be developed as the labelled molecule of clinical tumor diagnosis and the target molecules for the treatment of tumour.

Brief description of the drawings

Fig. 1 expresses ELL and disturbs ELL to express the impact on c-Myc degraded.

Fig. 2 is ELL experimental results show that as an E3 ubiquitin ligase.

Fig. 3 is that experiment in vitro proves that ELL is E3 ubiquitin ligase.

Fig. 4 is the determining of active region of ELL degraded c-Myc albumen.

Fig. 5 is that ELL suppresses Colon Cancer Cells.

Fig. 6 is that ELL suppresses the tumor growth of Colon grafting knurl and ELL in the application as in tumor markers.

Detailed description of the invention

The experimental technique using in following embodiment if no special instructions, is conventional method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

Cell and cultivation in following embodiment:

HEK293, HEK293T, HCT116 and COS7 cell are from ATCC.

HEK293, HEK293T and COS7 cell use and add 10% hyclone (FBS; Hyclone company)Dulbecco ' smodifiedEagle ' smedium culture medium (DMEM; HyClone company) cultivate; HCT116 cell usesAdd 10% hyclone (FBS; Hyclone company) McCoy 5A culture medium (HyClone company) cultivate. Condition of culture is37℃，5％CO₂. VigoFect (VigorousBiotech) is for cell transfecting.

Antibody and reagent in following embodiment

The antibody using is as follows: anti-ELLantibody (A0668, ABclonal; 51044-1-AP,Proteintech；HPA046076，Sigma-Aldrich)，anti-c-Mycantibody(9E10，SantaCruz；A0309，ABclonal；D84C12，CellSignaling)，anti-Flagantibody(F1804，Sigma)；anti-HAantibody(Covance)，anti-Hisantibody(H15，SantaCruz)，anti-GAPDHantibody(SC-47724, SantaCruz), anti-α-tubulinantibody (EPR1333, Epitomics). Use reagent asUnder: chloroquinediphosphate (BioVison), AICAR (Cayman), MG132 (Calbiochem),cycloheximide(Sigma-Aldrich)。

In following embodiment, co-immunoprecipitation and Westernblot analyze:

Anti-c-Mycantibody, anti-HAantibody and anti-Flagantibody-conjugatedAgarosebeads is purchased from Sigma-Aldrich. ProteinA/GSepharosebeads is purchased from GECompany.GlutathioneS-transferase (GST)-BindResin is purchased from Novagen.

The experimental procedure of endogenous co-immunoprecipitation is described in people 2014 such as () Zhang. Divide at GSTpull-downIn analysing, by the c-Myc of E.coli (BL21) bacterial expression GST label and the ELL of His label and carry out purifying. Use GST-BindResin carries out after co-immunoprecipitation, by SDS-PAGE by Separation of Proteins. By Coomassie blue stain or transfer for glueTo pvdf membrane, by Westernblot analyzing and testing His-ELL. Use FujiFilmLAS4000miniLuminescentimageanalyzer carries out the shooting of Westernblot result, uses MultiGaugeV3.0 basisThe ribbon density quantitative analysis of protein level that Westernblot obtains.

In following embodiment, in body, ubiquitination experiment is as follows:

Cotransfection plasmid HA-c-Myc, plasmid Myc-ELL, plasmid Myc-ELL (C595A) in HEK293T cell, plasmidHis-ubiquitin or plasmid His-ubiquitin (K48R). Pass through Ni²⁺-NTAresin (Novagen) is to His-Ubiquitin or His-ubiquitin (K48R) carry out purifying, finally use the ubiquitination of HA antibody test c-Myc.

In-vitro cell growth experiment in following embodiment:

Obtain cell by slow-virus infection and be inoculated in 6 orifice plates, every porocyte amount is 1 × 10⁵, use cell counter(TC10; Bio-Rad) continuous counter 4 days.

Cell clonal formation experiment in following embodiment:

The HCT116 stable cell lines of excessively expressing ELL is inoculated in six orifice plates to every hole 2 × 10⁴Individual cell; Strike and fall ELLThe every hole of HCT116 stable cell lines inoculation 1.5 × 10⁴Individual cell. Inoculate after six days, use methyl alcohol fixed cell and use crystallizationPurple dyeing. Use microscope photographing and clone's number is counted.

Statistical analysis:

The data of growth in vitro experiment and colony formation independently repeat the mean value of testing from three times, are expressed as flatAverage+SEM; In body, experimental data is expressed as mean value+SEM. Use GraphPadPrism5 (GraphPadSoftwareInc.) carry out data analysis.

Embodiment 1, ELL promote c-Myc degraded

The amino acid sequence of albumen c-Myc is sequence 2, and the nucleotides sequence of its encoding gene is classified sequence 1 as;

The amino acid sequence of albumen ELL is sequence 4, and the nucleotides sequence of its encoding gene is classified sequence 3 as.

Artificial synthetic above-mentioned sequence.

1, ELL reduces external source Myc expression

C-Myc encoding gene is inserted pCMV-HA carrier (Clontech) by the plasmid HA-c-Myc (WT) that expresses c-MycXbaI and the plasmid that obtains of BamHI site;

The plasmid HA-ELL that expresses ELL obtains SalI and the KpnI site of the encoding gene insertion pCMV-HA carrier of ELLThe plasmid arriving;

Plasmid HA-empty is pCMV-HA carrier.

Be 1:1 cotransfection cell with HA-c-Myc (WT) according to mass ratio respectively by plasmid HA-ELL and HA-emptyHEK293. After transfection the 2nd day, to collect supernatant and carry out Westernblot detection, the antibody of employing is as figure.

As shown in Figure 1A, cotransfection plasmid HA-ELL and HA-c-Myc (WT) can reduce the albumen water of HA-c-Myc to resultFlat, and HA-empty empty carrier and HA-c-Myc (WT) cotransfection can not reduce the protein level of HA-c-Myc, proves that ELL canDegraded c-Myc albumen.

2, ELL reduces endogenous Myc expression

The plasmid pcDNA-ELL that expresses ELL inserts pcDNA3.0 (+) by the encoding gene of ELL (Invitrogen) to carryThe plasmid that the KpnI of body and XhoI site obtain;

Plasmid pcDNA-empty is pcDNA3.0 (+) carrier.

By plasmid pcDNA-ELL and pcDNA-empty respectively according to different concentration (be not transfection plasmid ,+be transfection0.5 microgram plasmid, ++ be transfection 1 microgram plasmid transfection cell HCT116 (containing endogenous c-Myc). After transfection the 2nd day, in collectionClear liquid carries out Westernblot detection, and the antibody of employing is as figure.

As shown in Figure 1B, ELL is in HCT116 cell for result, and gradient is crossed expression ELL and can be caused the gradient of endogenous c-Myc to be fallenSeparate.

3, disturb ELL to express the stability that increases endogenous c-Myc

According to ELL, ELL-shRNA-1 and the ELL-shRNA-2 of its expression disturbed in design:

The encoding gene of ELL-shRNA-1: 5 '-CAACACCAACTACAGCCAGGA-3 ';

The encoding gene of ELL-shRNA-2: 5 '-GCGAGTACCTGCACAGCAA-3 '.

The encoding gene of ELL-shRNA-1 is inserted pSuper carrier (Brummelkamp by pSuper-ELL-shRNA-1TR1,BernardsR,AgamiR.AsystemforstableexpressionofshortinterferingRNAsinmammaliancells.Science.2002Apr19; 296 (5567): 550-3.) plasmid obtaining;

The encoding gene of ELL-shRNA-2 is inserted the plasmid that pSuper carrier obtains by pSuper-ELL-shRNA-2;

(be not transfection according to different concentration respectively by pSuper-ELL-shRNA-1 and pSuper-ELL-shRNA-2Plasmid ,+be transfection 1 microgram plasmid, ++ be transfection 2 microgram plasmids) transfectional cell HCT116, collects supernatant for after transfection the 2nd dayCarry out Westernblot detection, the antibody of employing is as figure.

(be documented in as in Publication about Document: ChenZ, LiuX, MeiZ, Wang with pSuper-GFP (ControlshRNA)Z*,XiaoW*.EAF2suppressesHIF-1alphatranscriptionalactivitybydisruptingitsinteractionwiththeco-activatorCBP/p300.MolecularandCellularBiology.2014,34 (6): 1085-1099.) be contrast.

Result as shown in Figure 1 C, can find out, strikes to fall ELL and can increase the stability of c-Myc in HCT116 cell.

In sum, the ELL c-Myc that can degrade.

Embodiment 2, ELL are as an E3 ubiquitin ligase degraded c-Myc albumen

1, ELL can strengthen the ubiquitination of c-Myc

In order to verify the type of the PD that ELL causes, use inhibitor, comprise chloroquine (lysosomal protein hydrolysisInhibitor, Chlq) (Sigma, 87111906), ammonium chloride (lysosomal protein hydrolysis inhibitor, NH₄CL) (traditional Chinese medicines reagent,12125-02-9), AICAR (autophagy inhibitor) (Cayman, 2627-69-2) and MG132 (proteasome inhibitor) (Sigma,M8699), to determine whether they can block the degraded of the c-Myc of ELL mediation.

By plasmid HA-ELL, plasmid HA-ELL and plasmid HA-c-Myc transfectional cell HEK293 respectively, after transfection the 2nd day,In cell culture fluid, add respectively Chlq and AICAR, and the final concentration of Chlq and AICAR all reaches 20mM, continue to cultivate 6 littleTime, to collect supernatant and carry out Westernblot detection, the antibody of employing is as figure. Not add inhibitor as contrast (con.).

Result, as Fig. 2 A-2C, can find out, the c-Myc that only has proteasome inhibitor MG132 can block ELL mediation fallsSeparate, this shows that ELL promotes the degraded of c-Myc via proteasome pathway.

In order to verify that ELL participates in the proteasome degraded of c-Myc really, next, at HEK293T cotransfection His-Ubiquitin or His-ubiquitin (K48R), the empty carrier of HA-c-Myc and Myc or Myc-ELL carry out ubiquitination in bodyExperiment, specific as follows:

Crossing the plasmid Myc-ELL that expresses ELL is that the encoding gene of ELL is inserted to CMV-Myc carrier (Clontech)The plasmid that SalI and KpnI site obtain;

His-Ub plasmid is by sequence 5 (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG) the ubiquitin encoding gene shown in inserts the SalI of pCI carrier (Promega)The plasmid obtaining with NotI site;

His-Ub (K48R) plasmid be by the K of the 48th of ubiquitin sport R obtain albumen encoding gene insertThe plasmid that the SalI of PIC carrier and NotI site obtain;

Plasmid Myc-empty is CMV-Myc empty carrier.

By His-Ub (K48R) or His-ub, HA-c-Myc, Myc-ELL or Myc-empty according to the combination of Fig. 2 D and 2ETransfection HEK293T cell, the 2nd day collecting cell lysate of transfection. The cell pyrolysis liquid of each combination is carried out to co-immunoprecipitation, logicalCross Westernblot and analyze, the antibody of employing is as figure.

Result is as follows, and Fig. 2 D showed the ubiquitination of expressing ELL and can strengthen c-Myc.

Consider the effect (Berndsen and Wolberger2014) of 48 lysines (K) in many ubiquitinations, useUb (K48R) mutant carries out ubiquitination experiment, and result shows, this mutant can not form the ubiquitin chain that K48 connects. As Fig. 2 EShown in, under the condition existing at wild type ubiquitin, ELL can induce the ubiquitination of c-Myc; In the time that ubiquitin K48R mutant existsCan not induce the ubiquitination of c-Myc.

This shows, the formation of the poly ubiquitin chain that 48 lysines of ELL promotion connect on c-Myc, thus further testDemonstrate,prove the effect of ELL in the proteasome degraded of c-Myc.

These data make to suppose that ELL may have the activity of E3 ubiquitin ligase, and which section (Fig. 4 A) of ELL promotes c-actuallyThe degraded of Myc, needs below further research.

2, ELL promotes the core fragment of c-Myc degraded

Plasmid HA-ELL (1-45) is by ELL (1-45) brachymemma shown in ELL amino acid sequence 1-45 amino acids residueThe encoding gene of body inserts the XbaI of CMV-HA carrier (Clontech) and the plasmid that BamHI site obtains;

Plasmid HA-ELL (1-379) cuts ELL (1-379) shown in ELL amino acid sequence 1-379 amino acids residueThe encoding gene of short body inserts the XbaI of CMV-HA carrier and the plasmid that BamHI site obtains;

Plasmid HA-ELL (1-508) cuts ELL (1-508) shown in ELL amino acid sequence 1-508 amino acids residueThe encoding gene of short body inserts the XbaI of CMV-HA carrier and the plasmid that BamHI site obtains;

Plasmid HA-ELL (46-621) is by ELL (46-shown in ELL amino acid sequence 46-621 amino acids residue621) encoding gene of truncate inserts the XbaI of CMV-HA carrier and the plasmid that BamHI site obtains;

Plasmid HA-ELL (447-621) is by ELL (447-shown in ELL amino acid sequence 447-621 amino acids residue621) encoding gene of truncate inserts the XbaI of CMV-HA carrier and the plasmid that BamHI site obtains;

Plasmid HA-ELL (509-621) is by ELL (509-shown in ELL amino acid sequence 509-621 amino acids residue621) encoding gene of truncate inserts the XbaI of CMV-HA carrier and the plasmid that BamHI site obtains;

Plasmid HA-ELL (466-621) is by ELL (466-shown in ELL amino acid sequence 466-621 amino acids residue621) encoding gene of truncate inserts the EcoRI of CMV-HA carrier and the plasmid that KpnI site obtains;

Plasmid HA-ELL (447-621) is by ELL (447-shown in ELL amino acid sequence 447-621 amino acids residue621) encoding gene of truncate inserts the EcoRI of CMV-HA carrier and the plasmid that KpnI site obtains;

By above-mentioned plasmid transfectional cell HEK293 respectively. After transfection the 2nd day, collect supernatant and carry out Westernblot inspectionSurvey, the antibody of employing is as figure.

Result as shown in Figure 4 B and 4C, can be found out, ELL (46-621) truncate, the ELL that contain 583-614 region(447-621) truncate, ELL (509-621) truncate, ELL (466-621) truncate and ELL (447-621) truncate canDegraded c-Myc albumen, proves that 583-614 region is essential for degraded c-Myc albumen.

Therefore 583-614 region is the active region of ELL degraded c-Myc albumen.

3、ELL(C595A)

Only have cysteine at the 595th in ELL (583-614) region, and this site is from zebra fish to the mankindAll (Fig. 2 F) guarding. Because HECT and RBR-domainE3 ligase all need to bring into play it by cysteine activity siteCatalytic activity (Berndsen and Wolberger2014), sports so studied the C595 of ELL the sudden change that alanine obtainsAfter body ELL (C595A), whether also there is the effect of E3 ligase.

The amino acid sequence of ELL (C595A) is to be alanine by the cysteine mutation of the 595th of ELL amino acid sequence 4The albumen obtaining, the nucleotides sequence of its encoding gene is classified as ELL coding gene sequence 3 the TGC is sported to GCC.

The plasmid HA-ELL (C595A) that expresses ELL (C595A) carries for the encoding gene of ELL (C595A) is inserted to CMV-HAThe plasmid that the SalI of body and NotI site obtain;

By plasmid HA-ELL (C595A) and plasmid HA-c-Myc cotransfection cell HEK293. After transfection the 2nd day, in collectionClear liquid carries out Westernblot detection, and the antibody of employing is as figure. Taking plasmid HA-ELL as contrast.

As shown in Figure 2 G, ELL (C595A) mutant can not promote the degraded of c-Myc to result.

By plasmid HA-ELL (C595A) and plasmid HA-c-Myc according to different mass ratio (1:1) cotransfection cellHEK293. After transfection the 2nd day, to collect supernatant and carry out Westernblot detection, the antibody of employing is as figure. With plasmid HA-ELLFor contrast.

Result is as shown in Fig. 2 H, and compared with wild type HA-ELL, albumen HA-ELL (C595A) is stable to endogenous c-Myc'sProperty does not have a significant effect.

The encoding gene of ELL (C595A) is inserted CMV-Myc by the plasmid Myc-ELL (C595A) that expresses ELL (C595A)The plasmid that the SalI of carrier and NotI site obtain;

By plasmid His-Ub, plasmid HA-c-Myc and plasmid Myc-ELL (C595A) transfection HEK293T cell, transfection 2 daysCollecting cell lysate. The cell pyrolysis liquid of each combination is carried out to co-immunoprecipitation, analyze by Westernblot, employingAntibody is as figure. Taking plasmid Myc-ELL as contrast.

Result, as shown in Fig. 2 I, can be found out, compared with wild type ELL, crosses expression ELL (C595A) mutant and can weakenThe ubiquitination of c-Myc. These results show, it is that it is lived that ELL may have active and 595 cysteines of E3 ubiquitin ligaseProperty site.

4, the E3 ubiquitin ligase of ELL is active detects

For further proving that E3 is an E3 ubiquitin ligase, utilize a business-like kit (UW9920,BIOMOL), in vitro the E3 ubiquitin ligase activity of ELL is verified.

Clone 11 E2 plasmids, specific as follows:

Plasmid Myc-UbcH1 be by the encoding gene of UbcH1 insert CMV-Myc carrier (Clontech) EcoRI andThe plasmid that XhoI site obtains;

Plasmid Myc-UbcH2 be by the encoding gene of UbcH2 insert CMV-Myc carrier (Clontech) BglII andThe plasmid that XhoI site obtains;

Plasmid Myc-UbcH3 be by the encoding gene of UbcH3 insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH5a be by the encoding gene of UbcH5a insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH5b be by the encoding gene of UbcH5b insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH5c be by the encoding gene of UbcH5c insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH6 be by the encoding gene of UbcH6 insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH7 be by the encoding gene of UbcH7 insert CMV-Myc carrier (Clontech) EcoRI andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH8 be by the encoding gene of UbcH8 insert CMV-Myc carrier (Clontech) EcoRI andThe plasmid that XhoI site obtains;

Plasmid Myc-UbcH10 be by the encoding gene of UbcH10 insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains;

Plasmid Myc-UbcH13 be by the encoding gene of UbcH13 insert CMV-Myc carrier (Clontech) BglII andThe plasmid that NotI site obtains.

The encoding gene of ELL is inserted the plasmid that pET-32a (+) carrier (Novagen) obtains by plasmid His6-ELL;

The coding nucleotide sequence of ELL (C595A) mutant is inserted pET-32a (+) by plasmid His6-ELL (C595A)The plasmid that carrier obtains;

The encoding gene of c-Myc is inserted the matter that pET-32a (+) carrier (Novagen) obtains by plasmid His6-c-MycGrain.

By above-mentioned 11 E2 plasmids, plasmid His6-ELL, plasmid His6-ELL (C595A) and plasmid His6-c-Myc according toCo-immunoprecipitation experiment is carried out in combination shown in Fig. 3.

Result is as follows, and shown in Fig. 3 A-B, UbcH8 and ELL have the strongest combination.

Further external ubiquitination analysis shows: UbcH8 can mediate the E3 ubiquitin ligase (Fig. 3 C) of ELL, provesUbcH8 is the E2 of ELL. In addition, ELL (C595A) mutant can not cause the ubiquitination (Fig. 3 D) of c-Myc.

The above results shows, ELL is an E3 ubiquitin ligase, and targeting, in c-Myc, causes c-Myc albumen to pass throughProteasome degraded, amino acid fragment 583-614 is the active region of ELL albumen, the cysteine in 595 sites is ELL conductsThe avtive spot of E3 ubiquitin ligase.

ELL (C595A) mutant has been lost E3 ubiquitin ligase activity.

Embodiment 2, ELL suppress Colon Cancer Cells

In order to prove the c-Myc degraded that ELL mediates and the biological function that suppresses its transcriptional activity, use slow virus packagingThree HCT116 clones: contrast, ELL and ELL (C595A) carry out cell proliferation experiment.

PHAGE-control is pHAGE-CMV-MCS-IZsGreen carrier;

PHAGE-ELL is for to insert pHAGE-CMV-MCS-IZsGreen carrier (Addgene) by ELL coding gene sequence 3XbaI and the carrier that obtains of BamHI site;

ELL (C595A) encoding gene is inserted pHAGE-CMV-MCS-IZsGreen carrier by pHAGE-ELL (C595A)XbaI and the carrier that obtains of BamHI site;

ELL-shRNA inserts the coded sequence CAACACCAACTACAGCCAGGA of ELLshorthairpinRNAThe carrier obtaining on pLKO.1 carrier (Sigma-Aldrich);

By above-mentioned each carrier respectively with package carrier psPAX2 (Addgene), pMD2.G (Addgene) transfection HEK293TCell, after transfection 8 hours, is replaced by culture medium the fresh DMEM culture medium that contains 10%FBS. After 40 hours, from culture mediumMiddle collection virus, obtains expressing the slow virus of contrast slow virus, expression ELL, slow virus, the expression of expression ELL (C595A)The slow virus of the slow virus of scrambled and expression ELL – shRNA. Filter, and transfection HCT116 cell. Above-mentioned containing virulentIn culture medium, add Polybrene (8 μ g/mL) to improve efficiency of infection.

Result is as follows:

Compared with the cellular control unit of transfection pHAGE-control, the HCT116 cell proliferation rate of transfection pHAGE-ELLObvious decline from second day (Fig. 5 A). Expression with Westernbolt (Fig. 5 C) confirmation ELL in HCT116 cell. TherewithOn the contrary, the growth rate of the HCT116 clone of transfection pHAGE-ELL (C595A) similar to cellular control unit (Fig. 5 D). CloneForm experiment and also verified the above results (Fig. 5 E). The expression of the HCT116 clone of transfection pHAGE-ELL (C595A) is passed throughWesternblot analyzes (Fig. 5 F) and confirms. On the contrary, the stable clone (cell of transfection ELL – shRNA) of falling ELL of striking is with rightTake group cell (cell of transfection scrambled) picture specific growth rate and accelerate (Fig. 5 G, 5H). Striking of mediation to ELL-shRNAFall efficiency and analyze (Fig. 5 I) confirmation by Westernblot.

In addition, utilize Rat1 cell (containing wild type c-Myc) and HO15.19 (from Rat1 cell, but lacking c-Myc)Whether the impact of having verified ELL on cell proliferation depends on c-Myc. The Rat1 cell proliferation rate of transfection pHAGE-ELL is obviousLower than the Rat1 cell (Fig. 5 J) of pHAGEControl transfection. But, the HO15.19 cell proliferation rate of transfection pHAGE-ELLWith transfection the HO15.19 cell of pHAGEControl without marked difference (Fig. 5 K).

Therefore, ELL can suppress the propagation of cell, and this inhibitory action may be the degraded reality that is mediated c-Myc by itExisting. ELL (C595A) can not suppress the propagation of cell.

Above-mentioned Rat1 cell and HO15.19 cell are documented in as in Publication about Document:

MateyakMK,ObayaAJ,AdachiS,SedivyJM.Phenotypesofc-Myc-deficientratfibroblastsisolatedbytargetedhomologousrecombination.Cellgrowth&differentiation:themolecularbiologyjournaloftheAmericanAssociationforCancerResearch8,1039-1048 (1997); Or YuanJ, Minter-DykhouseK, LouZ.Ac-Myc-SIRT1feedbackloopregulatescellgrowthandtransformation.TheJournalofcellbiology185,203-211(2009)。

Embodiment 3, ELL suppress the tumor growth of Colon grafting knurl

By the HT116 cell of the HT116 cell of transfection pHAGE-control in embodiment 2, transfection pHAGE-ELL with turnThe HT116 cell that dyes pHAGE-ELL (C595A) carries out growth of transplanted human experiment: 15 male nude mouses are divided into 3 groups at random, and everyOrganize 5, three groups of nude mices above-mentioned three kinds of different clones of hypodermic injection respectively, the cell concentration of every nude mice injection is 2 × 10⁶Individual. From the 3rd week, measure weekly the length, width and height of tumour and use V=π .abc/6 people 2010 such as () Flatz to calculate tumour bodyLong-pending. After 6 weeks, put to death nude mice, measure the weight of tumour and corresponding gene expression is analyzed. Lung tissue is through HE dyeingAfter carry out histologic analysis.

Gross tumor volume result, as Fig. 6 A and 6B, is crossed HCT116 cell (the transfection pHAGE-ELL of expression ELL (C595A)(C595A) HT116 cell) tumor growth rate and almost phase of control group (the HT116 cell of transfection pHAGE-control)The HCT116 cell (the HT116 cell of transfection pHAGE-ELL) of together, and excessively expressing ELL only forms a very little tumour.

Tumor weight analysis also shows, control group (the HT116 cell of transfection pHAGE-control) and excessively expression ELL(C595A) between the tumour of the HCT116 cell of mutant (the HT116 cell of transfection pHAGE-ELL (C595A)), nothing is obviously poorDifferent (Fig. 6 C). Cross and express the HCT116 cell (the HT116 cell of transfection pHAGE-ELL) of ELL and cross expression ELL (C595A) and dash forwardThe tumour of the HCT116 cell (the HT116 cell of transfection pHAGE-ELL (C595A)) of variant is expressed and is divided by WesternblotAnalysing (Fig. 6 D) confirms.

These data show, ELL can suppress the growth of colon tumor transplantable tumor.

Embodiment 4, ELL are in the application as in tumor markers

90 colon cancer tissue chip (production numbers: HCo1-that utilize Zuo Cheng bio tech ltd, Shanghai to provideAde180Sur-04), 86 normal structures are contrast. Utilize ELL antibody and c-Myc antibody to carry out immunohistochemical stainingAfter, take pictures under the microscope, carry out quantitative analysis according to the depth of dyeing; Detect ELL albumen and c-Myc albumen in colon cancer groupKnit with normal structure in expression.

As shown in Figure 6, ELL is high expressed in normal structure for result, and expression in cancerous tissue significantly reduce (Fig. 6 E,6G). In contrast, the expression of c-Myc, high expressed in cancerous tissue, and in normal structure, expression very low (Fig. 6 E, 6F).

These data show, the expression of ELL may can be used as the molecular labeling of colon cancer clinical diagnosis.

Claims

1.ELL albumen or its truncate are following 1)-5) application at least one:

1) preparation degraded c-Myc protein product;

2) as E3 ubiquitin enzyme ligase;

3) prepare inhibition tumor cell propagation product;

4) preparation suppresses tumour formation product;

5) as the molecular labeling of diagnosing tumor and/or qualification;

The amino acid sequence of described ELL albumen truncate is that in ELL Argine Monohydrochloride sequence, to comprise 583-614 amino acids residualArbitrary section of base.

2. application according to claim 1, is characterized in that: the amino acid sequence of described ELL albumen is order in sequence tableRow 4;

The amino acid sequence of described c-Myc albumen is sequence 2;

Described ELL albumen truncate is following 1)-6) in any:

6) amino acid sequence of the albumen shown in is sequence 4 583-614 positions.

3. application according to claim 1 and 2, is characterized in that:

Described tumour is colon cancer; Described tumour cell is colon cancer cell.

4. according to arbitrary described application in claim 1-3, it is characterized in that: described product is kit or medicine.

5. following 1)-5) any product, it comprises ELL albumen or its truncate;

1) degraded c-Myc protein product;

2) as E3 ubiquitin enzyme ligase;

3) inhibition tumor cell propagation product;

4) suppress tumour and form product;

5) as the molecular labeling of diagnosing tumor and/or qualification;

6. product according to claim 5, is characterized in that:

The amino acid sequence of described c-Myc albumen is sequence 2;

Described ELL albumen truncate is following 1)-6) in any:

6) amino acid sequence of the albumen shown in is sequence 4 583-614 positions.

7. according to the product described in claim 5 or 6, it is characterized in that:

Described tumour is colon cancer; Described tumour cell is colon cancer cell.

8. according to arbitrary described product in claim 5-7, it is characterized in that: described product is kit or medicine.

9. a protein, it is following 1)-6) in any:

1) amino acid sequence of the albumen shown in is sequence 4 46-621 positions;

2) amino acid sequence of the albumen shown in is sequence 4 447-621 positions;

3) amino acid sequence of the albumen shown in is sequence 4 509-621 positions;

4) amino acid sequence of the albumen shown in is sequence 4 466-621 positions;

5) amino acid sequence of the albumen shown in is sequence 4 447-621 positions;

6) amino acid sequence of the albumen shown in is sequence 4 583-614 positions.