CN102212108A

CN102212108A - Determination and application of human RACK1 (receptor for activated C kinase 1) epitope interacting with MKK7 (mitogen-activated protein kinase kinase 7)

Info

Publication number: CN102212108A
Application number: CN2011100718371A
Authority: CN
Inventors: 郭媛媛; 王晶; 王庆阳; 张纪岩; 冯健男; 沈倍奋
Original assignee: Institute of Basic Medical Sciences of AMMS
Current assignee: Institute of Basic Medical Sciences of AMMS
Priority date: 2011-03-24
Filing date: 2011-03-24
Publication date: 2011-10-12
Anticipated expiration: 2031-03-24
Also published as: CN102212108B

Abstract

The invention utilizes a related theoretical method of bioinformatics and calculation biology to predict that the functional epitope of an RACK1 (receptor for activated C kinase 1) molecule interacting with MKK7 (mitogen-activated protein kinase kinase 7) is 275Ile276Ser277Thr278Ser279Ser280Lys amino acid residue of the RACK1 molecule, which is verified by biological experiments; and further mutation of the functional epitope can be applied in inhibiting tumor growth.

Description

Determining and application of people RACK1 and MKK7 interaction epi-position

Technical field

The present invention relates to rationally determining and application of proteinic functional epitope.Based on the space structure information of discerning mutually between biomacromolecule, by the computer-aided analysis technology, the functional epitope of discerning mutually between the reasonable prediction biomacromolecule; By biological experiment the functional epitope of prediction is verified; Further this epi-position is applied to suppress tumor growth.

Background technology

JNK is the important member of MAPK family.Stress, multiple born of the same parents' external stimulus factor such as somatomedin, inflammatory stimulus can activate with JNK is the JNK signal transduction pathway at center.Classical MAPK protein sequence phosphorylation pattern is followed in the activation of JNK path.Its upstream kinases is MKK7.MKK7 makes JNK obtain protein kinase activity by Thr183, the Tyr185 site of bis phosphoric acid JNK.After the JNK activation, can regulate and control the functionally active of multiple substrate, these substrates had both comprised plasmosins such as bcl-2 family member, comprised transcription factor in the nuclears such as c-jun, c-myc again.In addition, JNK also regulates proteic stability such as p53 by the mode of the non-dependence of phosphorylation.By above these mechanism, JNK has participated in the adjusting of multiple vital movement, the generation of for example cell proliferation, apoptosis, cytodifferentiation, inflammatory factor, migration etc.And the active disorder of JNK has promoted the generation and the development of multiple disease.The MKK7-JNK approach is brought into play the important regulating and controlling effect under multiple physiological and pathological condition, the regulation mechanism of studying this path is significant, and novel targets, New Policy may be provided for the intervention of relative disease.

The molecular weight that RACK1 (acceptor of activated protein kinase C) is made up of the sequence of 7 Trp-Asp40 (WD40) high conservative is the albumen of 36KD.And RACK1 albumen is all guarded from the chlamydomonas to people.Because itself and Mammals G albumen heterotrimer β subunit have homology, so the GNB2L1 that is otherwise known as, promptly the fast nucleotide binding protein β of bird 2-sample 1.RACK1 can only interact with activated protein kinase C (PKC), both can be used as the anchorin of PKC simultaneously, can also recruit PKC or other albumen such as PDE4D5, Src and exercise the function of scaffolding protein.And the space structure that seven β spirals of RACK1 are formed regulates albumen for it and protein-interacting provides the polyprotein interactive surfaces.RACK1 is wide expression in the higher mammal and the mankind's tissue, comprises brain, liver and kidney or the like, and plays a significant role aspect physiology, for example: the growth of cells whose development, cell, the function of brain and immune response.In addition, bibliographical information RACK1 up-regulated in human cancer, liver cancer, melanoma, nonsmall-cell lung cancer, colorectal carcinoma or the like (Pablo Lopez-Bergami for example, Conway Huang, Conway Huang, et al.Rewired ERK-JNKSignalingPathwaysinMelanoma.Cancer Cell 11,447-460, May 2007).Therefore, RACK1 probably is found as new tumor markers and uses, and this will provide a new approach for the early diagnosis of tumour.

Bibliographical information, (Liver) compares with normal liver cell, JNK increased activity among the HCC (hepatocellular carcinoma), promptly the P-JNK level raises.And when knocking out JNK1, the Mouse Liver mean tumour volume reduces, illustrate that JNK is bringing into play short tumor activity (Lijian Hui in liver cancer forms, Kurt Zatloukal, Harald Scheuch, et al.Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation.The Journal of Clinical Investigation, Volume 118 Number 12 December2008).Simultaneously, RACK1 also presents rise trend (Katia Bourd-Boittin in the expression of hepatocarcinoma patient, He ' Ie`ne Le Pabic, Dominique Bonnier, et al.RACK1, a New ADAM12 Interacting Protein.THE JOURNAL OF BIOLOGICAL CHEMISTRYVOL.283, NO.38, pp.26000-26009, september 19,2008).Our previous work shows that RACK1 and P-JNK have dependency, and bibliographical information, PKC regulates WD1-4 structural domain (the Pablo L ó pez-Bergami that the JNK activity depends on RACK1, Hasem Habelhah, Anindita Bhoumik, et al.Receptor for RACK1 Mediates Activation of JNK by Protein Kinase C.Molecular Cell, Vol.19,309-320, August 5,2005).Simultaneously, we sift out the interaction protein that RACK1 is MKK7 by the yeast two-hybrid mode, and MKK7 is the upstream kinases of JNK.Therefore, we propose to provide new target position based on studying the research and development for the novel liver cancer medicine of RACK1 and MKK7 interaction sites.

The present invention is promptly based on top research background, utilize information biology, calculation biology correlation theory method to make up the interactional composite structure of RACK1-MKK7, by means such as distance geometries the structural region that RACK1 and MKK7 discern is mutually judged, by biological experiment theoretical model is verified, determined the functional epitope that RACK1 and MKK7 specificity are discerned mutually; Further the disappearance with this epi-position can suppress growth of tumor.

Summary of the invention

The invention discloses the RACK1 and the MKK7 space conformation that obtain based on computer homology simulation technique, and RACK1 and the interactional composite structure of MKK7 by the acquisition of molecular docking technology.

The 275-280 amino acids site that the invention discloses the interactional functional epitope of RACK1 and MKK7 and be RACK1 is ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰Lys.

The invention discloses the novel method based on RACK1 molecules in inhibiting tumor growth, i.e. sudden change at the 275-280 amino acids of RACK1 can suppress tumor growth.

The invention discloses the concrete mutation method that suppresses tumor growth at the sudden change of the 275-280 amino acids of RACK1: I275A/S276A/T277A/S278A/S279A/K280A.

The invention also discloses the specific implementation process of determining and using of people RACK1 and MKK7 interaction epi-position:

1) RACK1 and the simulation of MKK7 interaction composite structure and the theoretical prediction of functional target site

2) functional epitope of biological experiment proof theory prediction

3 based on RACK1 functional epitope inhibition tumor growth

By following specific embodiment, at length set forth the invention process process.It should be appreciated by those skilled in the art that the present invention not merely is defined in following embodiment, any variant that is equal to embodiments of the invention is also included among the present invention.

Description of drawings

Fig. 1, RACK1, the theoretical space conformation of MKK7.A represents the MKK7 space conformation; B represents the RACK1 space conformation.

The interactional mixture space conformation of Fig. 2, RACK1 and MKK7.

Co-immunoprecipitation MKK7 is removed in Fig. 3, RACK1 and sudden change thereof, and after the 275-280 amino acids residue sudden change of RACK1, RACK1 and MKK7 interact and obviously weaken.

The 275-280 amino acids residue sudden change of Fig. 4, RACK1 does not influence the JNK phosphorylation level.

The evaluation of the cell strain of Fig. 5, stably express RACK1 and mutant thereof; 1 expression PCDNA3.1 control cells strain, 2 expression RACK1 wild-type expression cell lines, 3 expression RACK1 (275-280) Mut mutant expression cell lines.

Fig. 6, RACK1 and mutant thereof are to the influence (* represents P＜0.05) of tumor growth.

The simulation of embodiment one, RACK1, MKK7 space conformation and the theoretical prediction of functional target site

1. material

Insightll 2005 routine packages (MSI molecular simulation, San Diego), this routine package comprises:

Homology homology mould is built

The Discover mechanics optimization

Discover_3 normal temperature dynamics simulation

The Docking molecular docking

The apparent static potential energy of Delphi is analyzed

Ludi molecular designing program (1995);

The IBM graphics workstation;

PDB 2008 databases (network www.rcsb.org download);

SwissProt database (2008, network download).

2. method and result

1) RACK1 and MKK7 space conformation simulation

The RACK1 crystalline structure (PDB code:1mpx), the MKK7 crystalline structure (PDB code:2dyl) that provide based on protein structure database PDB, under CVFF, the Amber field of force, select steepest descent (convergence criterion 0.05kCal/mol successively, 10000 steps of optimization step-length), conjugate gradient (convergence criterion 0.02kCal/mol, optimize 20000 steps of step-length), obtain the theoretical space conformation (Fig. 1) of RACK1, MKK7.By backbone c atoms root-mean-square displacement judgment method, RACK1, MKK7 structure and its crystalline structure that theory is obtained carry out structure superposition, calculate RMSD (the Root Mean Square Distance that obtains, root-mean-square displacement) value is 0.018nm, 0.023nm, and the structure that prompting optimization obtains is rational.

2) structural simulation of RACK1 and MKK7 interaction mixture

On the basis that obtains RACK1 and MKK7 space conformation, its epi-position structure is reasonably analyzed by the calculation biology correlation theory.On this basis, utilize molecular docking and dynamics simulation, under the situation of considering solvent effect, action scope is between the setting atom

Build the interactional mixture space conformation of RACK1 and MKK7 (Fig. 2).

3, the theory in RACK1 and MKK7 interaction recognition function territory is judged

Form theory, Van der Waals interaction pattern by intermolecular hydrogen bonding, learn a skill by computer graphics techniques, geometric distance RACK1 and MKK7 interaction pattern have been carried out theoretical analysis, the interactional functional epitope of results suggest RACK1 and MKK7 is the 275-280 amino acids residue of RACK1: ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰Lys.

The biometric authentication of embodiment two, RACK1 and MKK7 interactional function epi-position

1. experiment material:

Plasmid extraction kit Plasmid Midi Prep is available from Promega company; Eukaryotic cell transfection reagent Lopofectamin2000 is available from the Aprotinin of Invitrogen company, PMSF and Na ₃VO ₄All available from Sigma company; SDS-PAGE system and transferring system are available from Bio-RAD company; Quote with nitrocellulose filter Hybond-C available from Amersham company; The ECL of horseradish enzyme substrates is available from GE company; RACK1 antibody is available from BD Biosicences company; MKK7 antibody is available from Cell Signaling company; Anti-FLAG tag antibody is available from Sigma company; JNK antibody is available from Cell Signaling company; The goat anti-mouse (GAM) two of the anti-and horseradish enzyme labelling of the goat antirabbit of horseradish enzyme labelling (GAR) two are anti-available from company of middle China fir Golden Bridge.

Co-immunoprecipitation (Co-IP) lysis buffer prescription is as follows:

Tris-HCl(pH：7.5)20mM，NaCl?120mM，Glycerol?10％，EDTA?1mM，Triton?X-100?1mM。Add proteinase inhibitor before using: PNPP 10mM, PMSF 1mM, Na ₃VO ₄1mM, Aprotinin 10 μ g/ml.

2. experimental technique

1) RACK1 and mutant expression vector establishment thereof

RACK1 and the MKK7 interactional critical sites definite according to theoretical prediction ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰Lys sports L-Ala (Ala) with each amino-acid residue, is specially: I275A/S276A/T277A/S278A/S279A/K280A; Utilize conventional genetic engineering technique, wild-type RACK1 gene and mutant RACK1 (275-280) Mut gene clone are arrived on pCDNA3.1 (-) carrier,, introduce the FLAG label at the gene 5 ' end for ease of detecting.

2) transfection 293T cell

The 293T cell of taking the logarithm vegetative period becomes single cell suspension with trysinization, with contain the DMEM substratum of 10%FBS resuspended after, according to 4 * 10 ⁵The density in/hole is inoculated in 6 orifice plates; About 24h, cell rate in blocks is carried out transfection about 70%-80% with Lipofectamin 2000 reagent, and the transfection step is followed specification sheets.

Be used for detecting RACK1 molecule and the interactional critical sites of MKK7, during transfection, 2 holes of every sample, every hole is transfection 1 μ g wild-type RACK1 respectively, perhaps mutant RACK1 (275-280) Mut expression vector, cotransfection wild-type GFP-MKK7 1 μ g;

Be used to detect RACK1 and mutant thereof and JNK and interact, during transfection, 2 holes of every sample, every hole is transfection 1 μ g wild-type RACK1 respectively, perhaps mutant RACK1 (275-280) Mut expression vector, cotransfection wild-type GFP-JNK 1 μ g;

3) lysing cell

Behind the transfection 24h, prepare the Co-IP buffering night of precooling, the cell in cracking 6 orifice plates, each sample uses cell to scrape and collects cell with 400 μ l lysates; Cracking, scrape the process of getting and collect lysate and all on trash ice, carry out; Lysate closes in the 1.5ml Eppendorf tube, be inserted in place 10min on ice after, move in 4 ℃ of miniature refrigerated centrifuges the centrifugal 15min of 12000rpm.

4) co-immunoprecipitation

Get supernatant after centrifugal and move in the clean Eppendorf tube of another, take out 5 μ l as confidential reference items; All the other every pipe supernatants add Protein A/G beads 20 μ l successively, anti-FLAG tag antibody (M2) 1 μ l, 4 ℃ of overnight incubation; Wash beads night with the Co-IP buffering, each 1ml, washing 5min, centrifugal 30 seconds of 3000rpm abandons supernatant, repeats 3 times; Washing and centrifugally all carry out at 4 ℃.Last washing supernatant adds 2 * albumen sample-loading buffer after inhaling and abandoning, and 100 ℃ of boiling water boil 10min, and centrifugal 30 seconds of 12000rpm gets supernatant and carries out western blot analysis.

5) Western blot analyzes

Working concentration is 12% polyacrylamide gel electrophoresis, uses 15mA/ piece glue to carry out the constant current electrophoresis, the about 120min of electrophoresis time; Behind the electrophoresis, the albumen in the polyacrylamide gel is transferred on the nitrocellulose filter, the transfer printing condition is 60V, 180min; The nitrocellulose filter that transfer printing finishes is put in the TBST solution that contains 5% skim-milk and seals 2h; One anti-4 ℃ of (MKK7 or JNK or M2 antibody) overnight incubation; Wash each 10min in the TBST washing lotion 3 times; Nitrocellulose filter is hatched corresponding two resist room temperature 1h; Wash in the TBST washing lotion 3 times, each 10min washes the ECL luminescent solution that the back drips the horseradish enzyme substrates, the analysis of developing in the darkroom.

3. experimental result and conclusion

RACK1 and MKK7 bonded ability are weighed by the amount of the MKK7 that co-immunoprecipitation experiment RACK1 co-precipitation is got off, and the MKK7 amount that co-precipitation is got off is big more, and RACK1 then combines strong more with MKK7.The result shows: RACK1 compares with wild-type, and the amount of the MKK7 that mutant RACK1 (275-280) Mut co-precipitation is got off obviously reduces.After the 275-280 amino acids residue sudden change of prompting RACK1, RACK1 and MKK7 interact and obviously weaken; The 275-280 amino acids residue of proof RACK1 is the critical sites (Fig. 3) that itself and MKK7 do usefulness mutually.

RACK1 and JNK bonded ability are weighed by the amount of the JNK that RACK1 co-precipitation in the co-immunoprecipitation experiment is got off, and the JNK amount that co-precipitation is got off is big more, and then RACK1 combines strong more with JNK; The result shows that the amount of the JNK that wild-type RACK1, mutant RACK1 (275-280) Mut co-precipitation are got off does not have significant difference.The 275-280 amino acids residue of prompting RACK1 does not participate in RACK1 and JNK interaction (Fig. 4).

Prompting: the 275-280 amino acids residue of prompting RACK1 ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰Lys is the interactional specificity of RACK1 and MKK7 site.

Embodiment three, sudden change RACK1 and MKK7 interactional function epi-position suppress growth of tumor

1. experiment material

Plasmid extraction kit Plasmid Midi Prep is available from Promega company; Eukaryotic cell transfection reagent Lopofectamin2000 is available from Invitrogen company; G418 is available from invitrogen company; ScaI enzyme and reaction buffer thereof are all available from NEB company; Cut glue and reclaim test kit available from TianGen company; M2 antibody is available from Sigma company; P-JNK antibody is available from Cell Signaling company; SDS-PAGE system and transferring system are available from Bio-RAD company; Transfer printing uses nitrocellulose filter Hybond-C available from Amersham company; The ECL of horseradish enzyme substrates is available from GE company; The goat anti-mouse of horseradish enzyme labelling (GAM) two is anti-available from company of middle China fir Golden Bridge.

M2 lysis buffer prescription is as follows:

Tris-HCl (pH:7.5) 20mM, NaCl 250mM, EDTA 3mM, EGTA 3mM, 0.5%NP40; Add before using: PNPP 10mM, DTT 1M, Na ₃VO ₄1mM, Aprotinin 10 μ g/ml.

2. experimental technique

1) transfection HepG 2 cell

The HepG2 cell of taking the logarithm vegetative period becomes single cell suspension with trysinization, with contain the DMEM substratum of 10%FBS resuspended after, according to 4 * 10 ⁵The density in/hole is inoculated in 6 orifice plates; About 24h, cell degree of converging be about 70%-80%, and PCDNA3.1 empty carrier and the carrier that contains wild-type RACK1, mutant RACK1 (275-280) Mut gene are carried out transfection with Lipofectamin 2000 reagent respectively; The plasmid amount of every hole transfection is 2 μ g, and the transfection step is followed specification sheets.

2) pressurization screening

24h after the transfection becomes single cell suspension with trysinization, with contain the DMEM substratum of 10%FBS resuspended after, be inoculated in 96 orifice plates according to the density in 10/hole.Behind the 24h, add G418 and screen, concentration is 600 μ g/ml, and replacings in per 3～4 days contain the substratum of G418.Microscopically is observed the clone and is formed, with the mono-clonal amplification culture that forms, with Western blot methods analyst destination gene expression situation.

3) Western blot analyzes

Prepare the M2 buffering night of precooling, cracking purpose clone cell, lysate close in the 1.5mi Eppendorf tube, place 10min on ice after, 4 ℃, the centrifugal 15min of 12000rpm; Get supernatant and carry out Western blot analysis.Working concentration is 12% polyacrylamide gel protein isolate, uses 15mA/ piece glue to carry out the constant current electrophoresis, the about 120min of electrophoresis time; Behind the electrophoresis, the albumen in the polyacrylamide gel is transferred on the nitrocellulose filter, the transfer printing condition is 60V, 180min; The nitrocellulose filter that transfer printing finishes is put in the TBST solution that contains 5% skim-milk and seals 1h; One anti-4 ℃ of overnight incubation; Wash each 10min in the TBST washing lotion 3 times; Corresponding two anti-incubated at room 1h; The TBST washing lotion is put in two anti-film taking-ups of hatching later washed 3 times, each 10min washes the ECL luminescent solution that the back drips the horseradish enzyme substrates, the analysis of developing in the darkroom.

4) tumor animal is observed tumor growth rate

With stably express wild-type RACK1 gene, mutant RACK1 (275-280) Mut gene that utilizes aforesaid method to obtain and the monoclonal cell strain amplification culture of expressing the PCDNA3.1 empty carrier, get the monoclonal cell strain that is in logarithmic phase, become single cell suspension with trysinization, the centrifugal 5min of 1200r/min, abandon supernatant, and wash cell one time with serum free medium, counting is with cell furnishing 2.5 * 107/mL.Adopt hypodermic method, the purpose Transplanted cells in the 3-4 male nude mouse oxter in age in week, is carried out the tumor animal experiment, the transplanting amount of each implantation site is 100 μ L, two points of every nude mice injection.Observe nude mice tumor growth situation, measure the knurl volume weekly one time.Knurl volume size calculation formula is 0.52 * major diameter * minor axis ²

3. experimental result and conclusion

1) evaluation of the monoclonal cell strain of stably express goal gene

By above-mentioned experimental technique, with purpose carrier transfection HepG 2 cell, process pressurization screening has obtained monoclonal cell strain PCDNA3.1-G10, the monoclonal cell strain RACK1-G3 that expresses wild-type RACK1 gene of expression empty carrier PCDNA3.1, monoclonal cell strain RACK1 (275-280) Mut-G6 of expression mutant RACK1 (275-280) Mut gene respectively.Compare with the PCDNA3.1-G10 cell strain, RACK1-G3, RACK1 (275-280) Mut-G6 cell strain has specific target protein band to express in the 36KD position.(Fig. 5)

2) tumor animal experiment

It is described that monoclonal cell strain RACK1 (275-280) Mut-G6 that expresses the monoclonal cell strain PCDNA3.1-G10 of empty carrier PCDNA3.1, the monoclonal cell strain RACK1-G3 that expresses wild-type RACK1 gene, expression mutant RACK1 (275-280) Mut gene is pressed experimental technique, the subcutaneous vaccination nude mice is observed the tumor growth situation.Express the knurl volume of the monoclonal cell strain PCDNA3.1-G10 formation of empty carrier PCDNA3.1 with inoculation and compare, tumor growth rate is obviously accelerated behind the monoclonal cell strain RACK1-G3 of inoculation expression wild-type RACK1 gene; And tumor growth rate significantly slows down similar to control group PCDNA3.1-G10 (Fig. 6) behind monoclonal cell strain RACK1 (275-280) Mut-G6 of inoculation expression mutant RACK1 (275-280) Mut gene.Prompting is at the 275-280 amino acids residue of RACK1 ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰Lys carries out transgenation, can significantly slow down the tumor growth that RACK1 causes.

Claims

1. the functional epitope of a RACK1 molecule is characterized in that the molecule by RACK1 ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰Lys forms.

2. the application of the described functional epitope of claim 1 in suppressing tumor growth.

3. carry out the application of transgenation in suppressing tumor growth at the described functional epitope of claim 1.

4. the described transgenation of claim 3 is specially: with the RACK1 molecule ²⁷⁵Ile ²⁷⁶Ser ²⁷⁷Thr ²⁷⁸Ser ²⁷⁹Ser ²⁸⁰The Lys amino-acid residue sports respectively ²⁷⁵Ala ²⁷⁶Ala ²⁷⁷Ala ²⁷⁸Ala ²⁷⁹Ala ²⁸⁰Ala.