CN112961236B

CN112961236B - Methylation modified KRAS4B protein and application thereof

Info

Publication number: CN112961236B
Application number: CN202110171830.0A
Authority: CN
Inventors: 肖田; 江承尧; 郑多
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2022-10-28
Anticipated expiration: 2041-02-08
Also published as: CN112961236A

Abstract

The invention belongs to the technical field of protein modification, and particularly relates to a methylation modified KRAS4B protein and application thereof. At least one amino acid in the methylation modified KRAS4B protein provided by the invention is a methylation modified amino acid. The protein degradation rate after modification can be promoted and the stability of the protein can be reduced through methylation modification. The invention also provides an application of the SETD7 in KRAS4B protein methylation modification and a corresponding action site. The methylation modified KRAS4B protein provided by the invention can be used for preparing various functional preparations, and has important significance in the aspects of regulation and control of KRAS4B protein, disease treatment and prognosis of tumor patients and the like.

Description

Methylation modified KRAS4B protein and application thereof

Technical Field

The invention belongs to the technical field of protein modification, and particularly relates to methylation modified KRAS4B protein and application thereof, application of SETD7 in preparation of methylation modified KRAS4B protein, and a preparation containing methylation modified KRAS4B protein.

Background

KRAS gene, named Kirsten rat sarcoma viral oncogene homolog, is a guanine nucleotide binding protein with GTPase enzymatic activity, and is named as "Kirsten rat sarcoma viral oncogene homolog" in the Chinese, belonging to the RAS super protein family. The KRAS gene may be transcribed into two splice variants, namely KRAS4A and KRAS4B. The KRAS4B protein has 188 amino acids and 21.6kDa molecular weight, plays a key important role in the occurrence and development of various tumors, and is a gene with high mutation frequency in tumors.

Researches show that the activating mutation of KRAS4B not only has a promoting effect on the proliferation of tumor cells, but also can affect the tumor microenvironment to further promote the occurrence and the development of cancers. Therefore, the research of a drug or a means capable of inhibiting the mutant KRAS4B protein is one of the current research directions of KRAS.

Disclosure of Invention

The invention aims to provide a methylation modified KRAS4B protein and application thereof, and provides application of Lysine Methyltransferase SETD7 (SET Domain control 7, histone Lysine Methyltransferase) in preparation of the methylation modified KRAS4B protein and a preparation comprising the methylation modified KRAS4B protein, aiming at solving the promotion effect of the existing KRAS4B mutation on the growth of tumor cells.

In order to achieve the above object, in one aspect of the present invention, a methylation modified KRAS4B protein is provided, wherein at least one amino acid in the methylation modified KRAS4B protein is a methylation modified amino acid.

According to the invention, at least one amino acid in the KRAS4B protein is subjected to methylation modification, so that the degradation rate of the obtained methylation-modified KRAS4B protein is obviously accelerated, the ubiquitination degree is obviously increased, the stability is lower, the inhibition of the cancer promotion activity of the KRAS4B protein is facilitated, the negative influence brought by the KRAS4B is reduced or inhibited, and the application prospect is good.

In another aspect of the invention, the application of lysine methyltransferase SETD7 in preparing methylation modified KRAS4B protein is provided.

The inventors of the present invention found that the methyltransferase SETD7 interacts with KRAS4B protein. A series of experiments prove that the site of the lysine methyltransferase SETD7 acting on KRAS4B is the 182 th lysine and the 184 th lysine in KRAS4B protein. Meanwhile, the SETD7 acts on the KRAS4B protein, so that at least one site of the 182 th lysine and the 184 th lysine of the KRAS4B protein is methylated, and the KRAS4B protein subjected to methylation modification is obtained, and has the effect of reducing the stability of the KRAS4B protein.

In a further aspect of the invention, the use of SETD7 to promote the degradation of KRAS4B protein and/or to reduce the stability of KRAS4B protein is provided.

Since SETD7 can act on KRAS4B protein by methylating at least one of the lysine at position 182 and the lysine at position 184, resulting in a methylated KRAS4B protein with lower stability, SETD7 can be used to promote KRAS4B protein degradation and/or reduce KRAS4B protein stability.

In another aspect of the invention, a plurality of applications of the methylation modified KRAS4B protein in preparing medicines for inhibiting or treating tumor diseases, preparations for inhibiting lung adenocarcinoma cell proliferation, preparations for inhibiting an ERK1/2 signal pathway and/or preparations for inhibiting an AKT signal pathway are provided.

Experiments prove that the methylation modified KRAS4B protein provided by the invention has low stability, can inhibit or treat tumor diseases, inhibit cell proliferation, inhibit ERK1/2 and AKT signal pathways and other purposes, can be used for preparing preparations with various corresponding functions, and has important significance in the aspects of regulation and control of KRAS4B protein, disease treatment and prognosis of tumor patients and the like.

In a final aspect of the invention, there is provided a formulation comprising a methylated KRAS4B protein.

Because the methylation modified KRAS4B protein has multiple purposes of inhibiting or treating tumor diseases, inhibiting cell proliferation, inhibiting ERK1/2 and AKT signal pathways and the like, the preparation containing the methylation modified KRAS4B protein also has corresponding functions, and can be used for regulating and controlling the KRAS4B protein, preventing or treating tumor diseases and other fields.

Drawings

FIG. 1 is a flow chart of the method for confirming the action of the lysine methyltransferase SETD7 on KRAS4B by using the non-standard quantitative protein mass spectrometry technology in example 1 of the present invention;

FIG. 2 is a graph showing the results of confirming that lysine methyltransferase SETD7 is significantly enriched by KRAS4B using a non-standard quantitative protein mass spectrometry technique in example 1 of the present invention;

FIG. 3 is a graph showing the results of detecting the interaction between SETD7, lysine methyltransferase, and KRAS4B protein by co-immunoprecipitation in example 2 of the present invention;

FIG. 4 shows the amino acid sequence alignment of KRAS4B and other known substrates of lysine methyltransferase SETD 7. Wherein, K (black and white) at

positions

0 and 2 in KRAS4B are methylation sites for predicting two SETD 7;

FIG. 5 is a graph showing the results of predicting the region of interaction of KRAS4B with lysine methyltransferase SETD7 using computer simulation in example 3 of the present invention;

FIG. 6 is a graph showing the results of protein mass spectrometry of KRAS4B in which lysine 182 was methylated by lysine methyltransferase SETD 7;

FIG. 7 is a graph showing the results of measuring the stability of a methylated KRAS4B protein in example 5 of the present invention;

FIG. 8 is a graph showing the result of ubiquitination experiments performed on the methylated KRAS4B protein in example 6 of the present invention;

FIG. 9 is a graph showing the results of cell proliferation assay (left panel) and signal pathway assay (right panel) for methylated KRAS4B protein in example 7 and example 8;

FIG. 10 is a graph showing the results of the analysis of the correlation between the expression of the methyltransferase SETD7 and the survival of patients with lung cancer in example 9 of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

In the description of the present invention, the term "and/or" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of mass known in the chemical field such as μ g, mg, g, kg, etc.

In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, components, parts, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, components, parts, or combinations thereof.

It should be noted that the molecular biology experimental methods not specifically described in the examples of the present invention are performed by referring to the specific methods listed in the molecular cloning experimental manual (third edition) j. Sambrook, or according to the kit and the product specification; related reagents and biomaterials, if not specifically stated, are commercially available.

The embodiment of the invention provides a methylation modified KRAS4B protein, wherein at least one amino acid in the methylation modified KRAS4B protein is a methylation modified amino acid.

According to the embodiment of the invention, at least one amino acid in the KRAS4B protein is subjected to methylation modification, so that the degradation rate of the obtained methylation-modified KRAS4B protein is obviously accelerated, the ubiquitination degree is obviously increased, the higher instability is realized, the inhibition of the activity of the KRAS4B protein is facilitated, the negative influence brought by the KRAS4B is reduced or inhibited, and the good application prospect is realized.

In some embodiments, a typical methylatable site in a methylation modified KRAS4B protein is lysine 182 (K182).

In other embodiments, a typical methylatable site in a methylation modified KRAS4B protein is lysine 184 (K184).

The embodiment of the invention also provides application of lysine methyltransferase SETD7 in preparation of methylation modified KRAS4B protein.

The inventors of the present invention found that lysine methyltransferase SETD7 acts on KRAS4B protein. A series of experiments prove that the site of the lysine methyltransferase SETD7 acting on KRAS4B is the 182 th lysine and the 184 th lysine in KRAS4B protein. Meanwhile, lysine methyltransferase SETD7 acts on KRAS4B protein, so that at least one site of the 182 th lysine and the 184 th lysine is methylated, and then the methylation modified KRAS4B protein is obtained, and the KRAS4B protein has the effect of promoting the degradation of KRAS4B protein.

Specifically, when the lysine methyltransferase SETD7 acts on KRAS4B and acts at the 182 th lysine in KRAS4B protein, a methylation modified KRAS4B protein can be obtained, and the methylation modified KRAS4B protein is methylation modified at least at the 182 th lysine.

When the acting site is lysine 184 of KRAS4B protein, methylation modified KRAS4B protein can be obtained, and the methylation modified KRAS4B protein is methylation modified at least at the lysine 184.

When the acting sites are the 182 th lysine and the 184 th lysine in the KRAS4B protein, the methylation modified KRAS4B protein can be obtained, and the methylation modified KRAS4B protein is subjected to methylation modification at least at the 182 th lysine and the 184 th lysine simultaneously.

It was found through experiments that in the present example, if lysine in KRAS4B protein is to be methylated by lysine methyltransferase SETD7, the amino acid adjacent to the lysine site needs to have amino acid sequence of [ lysine or arginine ] - [ serine, tyrosine or alanine ].

Correspondingly, the embodiment of the invention also provides application of the lysine methyltransferase SETD7 in promoting the degradation of the KRAS4B protein and/or reducing the stability of the KRAS4B protein.

Since SETD7 can act on KRAS4B protein to methylate at least one of the 182 th lysine and 184 th lysine, resulting in a methylated KRAS4B protein with lower stability, SETD7 can be used to promote KRAS4B protein degradation and/or reduce KRAS4B protein stability to obtain a methylated KRAS4B protein with lower stability.

In the embodiment of the present invention, when SETD7 is used to promote degradation of KRAS4B protein and/or reduce stability of KRAS4B protein, the specific method includes, but is not limited to, preparing SETD7 into a corresponding preparation to act on KRAS4B protein to allow methylation of KRAS4B protein, thereby promoting degradation of KRAS4B protein and/or reducing stability of KRAS4B protein.

The inventor of the invention also finds that in practical research, SETD7 acts on KRAS4B protein, at least one of the 182 th lysine and the 184 th lysine is methylated to obtain methylated KRAS4B protein, and RabGEF1 acts on the methylated KRAS4B protein to promote ubiquitination of the KRAS4B protein and further promote degradation of the methylated KRAS4B. Thus, acting in combination with SETD7 and RabGEF1, will further promote degradation of KRAS4B and/or reduce protein stability of KRAS4B.

The embodiment of the invention also provides multiple applications of the methylation modified KRAS4B protein in preparing medicines for inhibiting or treating tumor diseases, preparations for inhibiting cell proliferation, preparations for inhibiting an ERK1/2 signal pathway and/or preparations for inhibiting an AKT signal pathway.

Experiments prove that the methylation modified KRAS4B protein provided by the embodiment of the invention has low stability, and can inhibit or treat tumor diseases, inhibit cell proliferation, inhibit ERK1/2 and AKT signal pathways and other purposes, so that the methylation modified KRAS4B protein can be used for preparing preparations with various corresponding functions, and has important significance in the aspects of regulation and control of KRAS4B protein, disease treatment and prognosis of tumor patients and the like.

Accordingly, the embodiment of the invention also provides a preparation, and the preparation comprises methylation modified KRAS4B protein.

In order to make the above implementation details and operation of the present invention clearly understood by those skilled in the art, and to make the progress of the methylation modified KRAS4B protein and its application obvious in the present invention, the above technical solution is illustrated by a plurality of examples below.

Example 1

This example uses the non-standard quantitative protein mass spectrometry technique to confirm that the methyltransferase SETD7 can act on KRAS4B as follows, and the flow chart is shown in fig. 1:

(11) The pBOB-CMV-3 XHA plasmid and KRAS4B DNA fragment are cut by BamHI and Xho I for 16 hours at 37 ℃, the target fragment is purified and recovered by agarose gel electrophoresis, and is connected by DNA ligase for 16 hours at 16 ℃, then escherichia coli DH5 alpha is transformed, positive clones are picked, and the plasmid sequence is identified by DNA sequencing, so that the target plasmid pBOB-CMV-KARS4B-3 XHA is obtained.

(12) pBOB-CMV-KRAS4B-3 XHA (3 HA tags connected in series to the C-terminus of the KRAS4B gene) and the empty vector control plasmid were respectively overexpressed in 293T cells, and after 48 hours, the total protein was recovered in a lysate containing a protease inhibitor.

(13) After quantification of the proteins, 4mg of total protein was incubated with magnetic beads coupled with HA antibody at 4 ℃ and slowly rotated overnight.

(14) Washed three times with ice PBS buffer (containing protease inhibitors).

(15) A reaction solution containing sodium deoxycholate SDC (sodium deoxycholate), tris (2-carboxyethyl) phosphine Hydrochloride TCEP (Tris- (2-carboxyethyl) phosphine, hydrochloride) and Chloroacetamide CAA (Chloroacetamide) was added to the magnetic beads to perform one-step reduction, alkylation and elution. The steps are repeated for 2 times, the eluent is merged, and pancreatin is added for enzymolysis overnight after water is added for dilution. Desalting the peptide fragment solution after enzymolysis by a desalting column. After being dried by a centrifugal concentrator, the product is frozen at the temperature of minus 20 ℃ and waits for being tested on a machine.

(16) Mass spectrometry was performed using a TripleTOF 5600+ LC-MS system from SCIEX. The samples were separated by a liquid phase eksiogen microLC 415 system with microliter flow rate. The peptide fragment sample was dissolved in loading buffer, aspirated by an autosampler, bound to a C18 capture column (5 μm,

300 μm × 5 mm), and then eluted to an analytical column (3 μm,

300 μm × 15 mm). Using two mobile phases (mobile phase A:3% DMSO; 0.1% for formic acid; 97% ₂ 3 percent of O and the mobile phase BDMSO, 0.1%) formic acid,97% ACN) to establish an analytical gradient. The flow rate of the liquid phase was set to 5. Mu.L/min. For mass-spectrometric DDA mode analysis, each scan cycle consists of one MS full scan (scan range350-1500m/z, ion acquisition time 250 MS), followed by 40 MS/MS scans (scan range 100-1500m/z, ion acquisition time 50 MS). The signal of the peptide fragment ions with the signal of more than 120cps (+ 2- + 5) triggers MS/MS scanning. The exclusion time for MS/MS duplicate acquisitions was set at 18s.

(17) The mass spectra data generated by TripleTOF 5600+ were retrieved by ProteinPilot (V4.5) using the database retrieval algorithm, paragon. The database used for the search was the proteome reference database of Human in UniProt. The search parameters are as follows: the Sample Type selects Identification; cys Alkylation selects Iodoacetamide; digestion selects Trypsin; the Search efficiency is set to Rapid ID. And screening the retrieval result by taking Ununsed not less than 1.3 as a standard, deleting the retrieved items and the pollution protein in the anti-library, and using the remaining identification information for subsequent analysis. And screening proteins with obvious differences in different samples based on the number of the spectrogram of each protein. In order to facilitate statistical analysis and reduce false positive results caused by low-abundance protein identification, the data with the spectrogram number of 0 is artificially filled with 1. The ratio of the number of spectra (KRAS 4B/HA ratio) and the mean number of spectra (MeanSP) were calculated for each protein in different samples, with x = log2 (KRAS 4B/HA ratio) and y = log2 (MeanSP). The quantitative difference and the abundance of each protein in different samples are integrated, a difference protein screening boundary line y = c/(x-x 0) is set, and the parameters related to the cut-off line of the protein which is obviously adjusted up and down are shown in the following table 1:

TABLE 1 differential protein screening borderline parameters

Boundary line	Constant c	Constant x0	Marking
				y1
	1	log ₂ 1.5	+
				y2	log ₂ 2.5	1	++
y3	-1	-log ₂ 1.5	-
				y4	-log ₂ 2.5	-1	--

In Table 1, "+" indicates an up-regulated protein and "-" indicates a down-regulated protein. More labels indicates more significant differences. That is, in the screening, the larger the ratio of the number of spectra of the protein, the larger the average number of spectra, and the more significantly the protein is different among different samples. The boundaries of the different significance differences are shown in fig. 2. Proteins that are distributed outside the line are labeled as differential proteins of corresponding significance. According to the screening criteria, proteins with more than 1.5-fold difference in spectra (spectra number approaching. Infinity) to 3-fold difference in spectra number approaching 1) will be labeled as differential proteins ("+/-"), and proteins with more than 2-fold difference in spectra number (spectra number approaching. Infinity) to 4-fold difference in spectra number approaching 1 will be labeled as significantly differential proteins ("+/-").

As can be seen from FIG. 2, the signal of lysine methyltransferase SETD7 (point indicated by arrow)Log of the number of spectra ₂ Value of about 8, mean log of number of spectra ₂ A value of about 7 indicates significant enrichment of SETD7 by KRAS4B. Thus, this data suggests that the lysine methyltransferase SETD7 is an interacting protein of KRAS4B.

Example 2

In this example, a co-immunoprecipitation experiment was used to confirm that lysine methyltransferase SETD7 acts on KRAS4B as follows:

(21) The pcDNA3.0-CMV-HA plasmid and the SETD7 DNA fragment are digested by XbaI and Xho I at 37 ℃ for 16 hours, the target fragment is purified and recovered by agarose gel electrophoresis, and is connected by DNA ligase at 16 ℃ for 16 hours, then escherichia coli DH5 alpha is transformed, positive clone is selected, and the plasmid sequence is identified by DNA sequencing, so that the target plasmid pcDNA3.0-CMV-SETD7-HA is obtained.

(22) KRAS4B was overexpressed in 293T cells along with pcDNA3.0-CMV-SETD7-HA plasmid and after 48 hours total protein was recovered in lysates containing protease inhibitors.

(23) After protein quantification, 1mg of total protein was incubated with 2. Mu.g of anti-SETD7 antibody at 4 ℃ and slowly spun overnight.

(24) Binding to the antibody was performed by adding protein A/G coupled magnetic beads, and the mixture was slowly rotated at 4 ℃ for 4 hours to wash 5 times with ice PBS buffer (containing protease inhibitor).

(25) Using the denatured cell lysate, the reaction was carried out at 95 ℃ for 10 minutes.

(26) After polyacrylamide gel electrophoresis, the HA-tag was incubated overnight at 4 ℃ using an antibody specific for RAS protein (dilution 1.

(27) After 3 washes with PBST buffer, incubation with secondary antibody (anti-rabbit antibody conjugated peroxidase, 1.

(28) After 3 washes with PBST buffer, protein signals were detected with Enhanced Chemiluminiscence (ECL) substrate and a chemiluminescent detector, and the results are shown in fig. 3.

As can be seen from FIG. 3, KRAS4B was enriched in the "anti-SETD7" group after the co-immunoprecipitation with anti-SETD7 antibody, indicating that SETD7 and KRAS4B have a protein interaction in 293T cells.

Example 3

This example utilizes SETD7 action amino acid domain analysis and protein structure simulation analysis to determine the site of action of SETD7 and KRAS4B by the following steps:

(31) The crystal structures determined by SETD7 (PDB ID:1O 9S) and KRAS4B (PDB ID:6 CCX) via X-ray diffraction were obtained from the Protein Database (PDB).

(32) The G117-K366 amino acid sequence of SETD7 and the M1-C185 amino acid sequence of KRAS4B were selected as the initial docking structure.

(33) The HADDOCK server version 2.447 is set as a default parameter value for simulating the docking of KRAS4B with SETD7 binding pockets, and the most common 5 of the simulated molecular clusters are dynamic simulation, 3 of which are dynamic simulation.

(34) The FF14SB force field was applied to the protein preparation simulation, and the two protein-bound complexes were placed into TIP3PBOX and counter ions were added to neutralize the system.

(35) First, the whole system is minimized until convergence, after which the system is gradually heated to 300k with all heavy atoms constrained, with the constraint force constant set to 100 kcal/(mol · rad 2), and then the system is relaxed in four stages in the NPT ensemble, with the force constants set to 50, 20, 10 and 5 kcal/(mol · rad 2), respectively.

(36) The entire simulation lasted 100 nanoseconds, taking a snapshot of each binding pattern, and finally performing cluster analysis on the 20 nanosecond trace. Selecting a connection method and setting the intercept point to

(37) Prediction of lysine methylation site reference is made to the summary in the literature (epitopes volume 6, issue 9,1059-1067,2011), which, in conjunction with the results of this example, identifies that lysine, if it is to be methylated by SETD7, needs to have an amino acid sequence [ lysine or arginine ] - [ serine, tyrosine or alanine ], i.e., [ K/R ] - [ S/T/A ] -K, where the last K is methylated. FIG. 4 shows the conserved amino acid sequence rules for KRAS4B and other known substrates of lysine methyltransferase SETD 7. The amino acid sequences for K (black and white) at

positions

0 and 2 of KRAS4B also follow this rule, corresponding to K182 and K184, respectively, of KRAS4B. Therefore, it is assumed that KRAS4B is highly likely to be methylated by SETD7, and two sites K182 and K184 are predicted to be two SETD7 methylation sites of KRAS4B protein.

In fig. 5, the SET enzyme activity region of SETD7 sterically approaches three amino acid residues K182, T183, and K184 of KRAS4B into the plausible region of predicted protein interaction, and thus SETD7 is predicted to have a high probability of interacting with KRAS4B.

Example 4

In this embodiment, a high-sensitivity protein mass spectrometry technology is used to determine that methylation modification exists on KRAS4B protein, and the steps are as follows:

(41) KRAS4B and HA-SETD7 recombinant protein or KRAS4B and empty plasmid were overexpressed in 293T cells and total protein was recovered in lysates containing protease inhibitors after 48 hours.

(42) After protein quantification, 4mg of total protein was incubated with magnetic beads coupled with HA antibody at 4 ℃ and slowly rotated overnight.

(43) After washing three times with ice PBS buffer (containing 0.5% Triton X-100), denatured cell lysate (containing 2% SDS) and an appropriate amount of beta-mercaptoethanol (. Beta. -ME) were added, and reaction was carried out at 95 ℃ for 10 minutes, followed by polyacrylamide gel electrophoresis.

(44) After silver staining, the band corresponding to the target protein was cleaved and subjected to in-gel digestion. Gel using 50%50mM NH ₄ HCO ₃ Decolorizing with 50% acetonitrile and dehydrating with 100% acetonitrile.

(45) After the reduction and alkylation of the protein, trypsin or chymotrypsin was added to digest overnight at 37 ℃ and the peptide fragment obtained was dissolved in 0.1% by weight of FA for further use.

(46) The peptides were analyzed by LC-MS technique by liquid chromatography on a reverse phase column (40cm. Times.75um i.d.) packed with Magic C18 AQ 3- μm at 50 deg.C

Resin with a pulled emitting end. Solution A in H ₂ 0.1% FA in O,0.1% FA in acetonitrile in B solution.

(47) The peptide fragments were separated in a linear gradient from 0% to 5%B solution over 5 minutes, then increased to 30% b solution over 105 minutes, further increased to 35% b solution over 5 minutes, then washed and re-equilibrated under 95% b solution.

(48) timsTOF-pro runs in PASEF mode, ddaPASEF files were searched using Peaks software for Swissprot human (downloaded in 2018, 9 months) with common contaminants. The search parameter settings are as follows: the parent monoisotope tolerance is 15ppm, the product ion tolerance is 0.05Da, the modification comprises cysteine iodoacetylation, potential modified protein N-terminal acetylation, acetylation and methylation on lysine or arginine, oxidation on methionine, phosphorylation on serine, tyrosine or threonine, and the maximum number of enzyme cutting errors is set to be 2. The results are shown in FIG. 6.

In FIG. 6, "y" represents the right-to-left cleavage of the peptide fragment, which is MIV (y 3), cKT (y 6), k (y 7) and SK; and "b" represents the left-to-right cleavage of the peptide fragment, which is KSkTK (b 5), c (b 6), V (b 7) and IM. On lysine "k", one more molecular weight of 14.04Da, which is the methylation-modified molecular weight, demonstrates that lysine 182 is methylated.

Example 5

This example examined the stability of the methylation-modified KRAS4B protein by the following steps:

(51) 293T cells were co-transfected with SETD7, rabGEF1 and KRAS4B wild or mutant, and after 48 hours the cells were seeded in 6-well plates until the cells were adherent.

(52) Adding cycloheximide CHX at different time points, collecting protein from the cell with denatured cell lysate, quantifying the protein, taking appropriate amount of protein and beta-ME, and reacting at 95 deg.C for 10 min.

(53) After polyacrylamide gel electrophoresis, the cells were incubated overnight at 4 ℃ using HA Flag-tagged protein, RAS, tubulin-specific antibody (dilution 1.

(54) After 3 washes with PBST buffer, incubation with secondary antibody (anti-rabbit, anti-mouse antibody conjugated peroxidase, 1.

(55) After 3 washes with PBST buffer, protein signals were detected with Enhanced Chemiluminiscence (ECL) substrate and a chemiluminescent detector.

(56) The results are shown in FIG. 7, which is a graph of the results of analysis using GraphPad Prism v6.01, after normalization of the obtained values to 0 hour values by AlphaEaseFC software.

As can be seen from FIG. 7, either KRAS4B wild type or KRAS4B ^G12D Activation mutant, SETD7, was able to promote its proteolytic degradation. However, in the case of K182 and K184 mutations (K182/184M), the degradation of KRAS4B was significantly inhibited and the stability was significantly improved, indicating that the degradation of KRAS4B is dependent on the methylation of K182 and K184.

Example 6

In this example, the ubiquitination experiment is performed on the methylation modified KRAS4B protein by the following steps:

(61) 293T cells were cotransfected with SETD7, rabGEF1, KRAS4B wild or mutant and Ubi-myc for 48 hours before MG132 treatment for 6 hours.

(62) After the cells were treated with 100. Mu.L of denatured cell lysate (2% SDS) to collect proteins, DNA was disrupted and 900. Mu.L of dilution buffer was added.

(63) After the protein was quantified, an appropriate amount of the protein was reacted with beta-mercaptoethanol (beta-ME) at 95 ℃ for 10 minutes.

(64) After polyacrylamide gel electrophoresis, myc, HA, flag-tagged protein, RAS-specific antibody (dilution factor 1: 1000) were incubated overnight at 4 ℃.

(65) After 3 washes with PBST buffer, incubation with secondary antibody (anti-rabbit, anti-mouse antibody conjugated peroxidase, 1.

(66) After 3 washes with PBST buffer, protein signals were detected with Enhanced Chemiluminiscence (ECL) substrate and a chemiluminescent detector. The results are shown in FIG. 8, in the presence of SETD7, whether KRAS4B wild type or KRAS4B ^G12D Activating mutant type, capable of making its proteinThe degree of ubiquitination increases. However, in the case of K182 and K184 mutations (K182/184M), ubiquitination of KRAS4B was significantly inhibited, indicating that ubiquitination of KRAS4B is dependent on methylation of K182 and K184.

Example 7

In this example, a cell proliferation assay was performed on a methylation-modified KRAS4B protein, using the following steps:

(71) Overexpression of Idle plasmid, KRAS4B in H1437 cell line ^G12D Or KRAS4B ^{G12D/K182/184M} 。

(72) Three over-expressed cells were seeded into 1000 cells per well in 96-well plates, with five replicate wells per group.

(73) CCK8 reagent is added into 0,24,48,72 hours respectively, after reaction for two hours at 37 ℃, OD value with the wavelength of 450nm is read by a microplate reader, and cell proliferation is detected. Cell proliferation was analyzed and plotted as GraphPad Prism v6.01 and p-values were counted, the results are shown in the left panel of fig. 9.

KRAS4B can be seen from FIG. 9 ^G12D KRAS4B, which is an activating mutation, and can promote cell proliferation; on the basis, if K182 and K184 (K182/184M) are mutated again, KRAS4B is made ^G12D Can not be methylated, and further improves the stability of KRA4S4B protein, so that the effect of promoting cell proliferation is more obvious.

Example 8

This example detects the effect of the methylation modified KRAS4B protein on the signaling pathway by the following steps:

(81) Overexpression of Idle plasmid, KRAS4B in H1437 cell line ^G12D Or KRAS4B ^{G12D/K182/184M} 。

(82) After quantifying the protein using the denatured cell lysate, an appropriate amount of protein was reacted with beta-ME at 95 ℃ for 10 minutes.

(83) After polyacrylamide gel electrophoresis, the cells were incubated overnight at 4C using antibodies specific for SETD7, KRAS4B, pAkt, akt, pErk, erk, tubulin (dilution factor 1.

(84) After 3 washes with PBST buffer, incubation with secondary antibody (anti-rabbit antibody conjugated peroxidase, 1.

(85) After 3 washes with PBST buffer, protein signals were detected with Enhanced Chemiluminiscence (ECL) substrate and a chemiluminescent detector. The results are shown in the right panel of FIG. 9.

KRAS4B can be seen from FIG. 9 ^G12D KRAS4B, which is an activating mutation, is capable of activating downstream ERK1/2 and AKT signaling pathways. On the basis, if K182 and K184 (K182/184M) are mutated again, KRAS4B is made ^G12D Can not be methylated, further improves the stability of KRA4S4B protein, and has more obvious effect of activating downstream ERK1/2 and AKT signal pathways.

Example 9

This example examines the correlation of the expression of the methyltransferase SETD7 with survival in lung cancer patients as follows:

(91) Enter http:// kmplot. Com/analysis/index. Phpp = service & cancer = lung website.

(92) The Affy id column is filled with the gene name SETD7, the corresponding probe number is 224928 at, the history column selects all or adenocarinoma, and the others are analyzed according to default values, thus obtaining the graph 10. In the left panel of fig. 10, the X-axis shows the survival time (month) of the lung cancer patients and the Y-axis shows the survival rate (%) of the lung cancer patients, and the survival rate of the patients with high expression of SETD7 is higher than that of the patients with low expression of SETD7 in terms of five years (60 months); the same phenomenon also occurs in patients with lung adenocarcinoma (fig. 10, right panel). This result demonstrates that SETD7 plays a role as a tumor suppressor gene in lung cancer, suggesting that expression of SETD7 can be used to predict survival and prognosis in lung cancer patients.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A methylation modified KRAS4B protein, wherein the methylation modified KRAS4B protein is a methylation modification of lysine at position 182 and lysine at position 184 of KRAS4B protein; wherein, the KRAS4B protein is the protein with the number PDB ID:6 CCX.

Use of setdh 7 for the preparation of the methylated KRAS4B protein of claim 1; wherein the SETD7 is protein shown in a number PDB ID:1O 9S.

3. Use of the SETD7 of claim 2 to promote degradation of KRAS4B protein and/or to reduce stability of KRAS4B protein.

4. Use of the methylated KRAS4B protein of claim 1 in the preparation of a medicament for inhibiting or treating a neoplastic disease; wherein the tumor is lung cancer.

5. Use of the methylated KRAS4B protein of claim 1 in the preparation of an agent for inhibiting cancer cell proliferation; wherein the cancer cell is a lung cancer cell.

6. A formulation comprising the methylated KRAS4B protein of claim 1.