CN109810959B - Protein polypeptide combined with KEAP1 and used for regulating stability of NRF2 protein - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to a protein polypeptide which is combined with KEAP1 and regulates the stability of NRF2 protein. The regulation of oxidative stress plays an important role in the process of tumorigenesis and development, NRF2 is used as a key molecule for cellular antioxidation, and factors influencing the expression of NRF2and the stability of protein play an important role in regulating the oxidative stress tolerance of cells and the resistance of chemotherapeutic drugs. The invention provides a protein polypeptide competitively combined with KEAP1 on the basis of RMP (RNA polymerase II subustit 5-mediating protein), which improves the antioxidant capacity of cells by enhancing the stability of NRF2 protein. The invention provides a useful target for regulating the antioxidant capacity of cells.

Description

Protein polypeptide combined with KEAP1 and used for regulating stability of NRF2 protein

Technical Field

The invention relates to the field of biomedicine, in particular to a protein polypeptide which is combined with KEAP1 and regulates the stability of NRF2 protein, wherein the protein polypeptide is combined with KEAP1 in a competitive way, so that the ubiquitination degradation of cell antioxidant regulatory key protein NRF2 is reduced, and the tumor antioxidant capacity is enhanced.

Background

RMP (RNA polymerase II subbunit 5-mediating protein, also known as URI, Uncinstantial preproldin RPB5 Interactor) was first discovered in 1998 by Japanese researchers during the search for proteins involved in the regulation of HBx transcription: the fifth subunit of RNA polymerase II (RNA polymerase II subbunit 5, RPB5) is directly combined with HBx, and simultaneously the two are combined with TF IIB through respective combination sites, and the complex formed by the three components promotes the transcription of HBx; however, they found that RMP competitively binds to RBP5 at low HBx levels, covering the binding sites with HBx and TF IIB in the latter protein structure, thereby inhibiting transcription of HBx. In 2011 swiss scientists reported that URI as an oncogene plays a tumor promoting role in ovarian cancer: URI binds to PP1 gamma to form a complex and inhibits its phosphatase activity, thereby disrupting the negative feedback pathway composed of the latter and S6K1-BAD, leading to increased BAD phosphorylation, decreased apoptosis, and increased survival (J.P.Theurilat, S.C.Metzler, N.Henzi, et al.URI is an on gene amplified in overhead Cell and is required for the same survivor [ J ]. Cancer Cell,2011,19(3): 317-32.). Subsequently, more studies find that RMP plays a tumor promotion role in various malignant tumors such as prostate cancer, multiple myeloma, endometrial cancer, and the like. In addition, it has also been reported that RMP can facilitate the combination of p65 and IL-6 promoter region, promote its transcription, make hepatoma cell secrete more IL-6, and promote hepatoma metastasis and self-renewal through IL-6/STAT3 signal channel.

ROS (reactive oxygen species) play an important role in tumorigenesis and tumor development. Excessive intracellular ROS causes key gene mutations that promote cell proliferation and tumorigenesis. During the growth process of the tumor, the tumor needs to deal with various internal and external environmental factors. The large amount of ROS generated and released can cause the oxidation-reduction equilibrium state in cells to be broken, so that a series of phenomena such as DNA damage, cell membrane structure damage, organelle damage and the like can be caused, and finally, the apoptosis can be caused. Therefore, the method has very important significance for exploring the cell antioxidant regulation mechanism in the process of tumorigenesis and development.

The KEAP1-NRF2-ARE signal pathway plays an important role in the anti-oxidation process of cells. KEAP1(Kelch-like ECH-associated protein 1) was anchored to actin in the cytoplasm and was able to bind to NRF2 via the Kelch region. KEAP1 provides a scaffold for E3 ubiquitin ligase CUL3, which is capable of mediating degradation of NRF2 by ubiquitin-proteasome. Normally, NRF2 is maintained only at a low level by the above mechanism to ensure daily vital activity. Upon oxidative stress, the upstream related stimuli mediate the termination and accumulation of NRF2 degradation, eventually entering the nucleus. The NRF2 entering the nucleus is combined with an Antioxidant Response Element (ARE) to promote the transcription of antioxidant related genes and participate in various physiological activities related to the antioxidant of cells. (John P.Fruehauf, Frank L.Meyskens, Jr.reactive oxygen species: A break of life or death

The important molecules in the relevant pathways of the targeted intervention oxidation resistance play an important role in the occurrence and development of tumors and provide personalized medicine guidance for tumor treatment.

Disclosure of Invention

The invention aims to provide a protein polypeptide RMP-D2 which is capable of competitively binding KEAP1, stabilizing NRF2 protein and improving the antioxidant capacity of cells in RMP protein molecules. The invention also provides a coding gene of the protein polypeptide RMP-D2, a recombinant vector, a recombinant bacterium, a recombinant cell or an expression cassette containing the coding gene, and application of the coding gene in preparing a medicament for regulating the anti-oxidation capacity of tumors.

In order to achieve the above objects, according to a first aspect of the present invention, there is provided a RMP-D2 polypeptide having an amino acid sequence as shown in (I) or (II):

(I) an amino acid sequence shown as SEQ ID NO. 1;

(II) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1.

The RMP protein is mainly considered to form a complex by combining with PP1 gamma and inhibit the phosphatase activity of the complex, so that a negative feedback channel formed by the complex and S6K1-BAD is damaged, the BAD phosphorylation is increased, the apoptosis is reduced, and the survival is increased. This mechanism is used to explain why RMP promotes progression in a variety of tumors. However, the inventor firstly finds that the RMP protein competitively binds to the NRF2 binding site in the KEAP1 protein molecule through the region containing the E structure domain between the 164-containing and 288-amino acids of the protein molecule, thereby enhancing the stability of the NRF2 protein and further regulating the antioxidant capacity of cells in the long-term research process. The amino acid sequence and the nucleotide sequence of the peptide segment consisting of 164-288 amino acids of the RMP protein molecule are shown as SEQ ID NO. 1. It should be noted that the derived sequence obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 and having the cell antioxidant capacity also belongs to the protection scope of the present invention.

In a second aspect of the present invention, there is provided a gene encoding the RMP-D2 polypeptide as described above, which has the nucleotide sequence shown in (i) or (ii):

(i) a nucleotide sequence shown as SEQ ID NO. 2;

(ii) a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown as SEQ ID NO. 2.

The derivative sequence obtained by substituting and/or deleting and/or inserting one or more bases on the nucleotide sequence and the coded peptide segment has the same antioxidant capacity as the peptide segment of the amino acid sequence also belongs to the protection scope of the invention.

In a third aspect of the invention, there is provided a recombinant vector, a recombinant bacterium, a recombinant cell or an expression cassette comprising a gene encoding the RMP-D2 polypeptide as described above.

Further, the recombinant vector is one of a lentiviral vector, a retroviral vector or an adenoviral vector.

Further, the recombinant vector contains an enhancer (usually referred to as a constitutive specific enhancer such as CMV, SV40, PGK enhancer, etc.) which promotes the expression of the encoding gene upstream or downstream of the encoding gene of the polypeptide.

In a fourth aspect of the invention, the invention provides an application of the RMP-D2 polypeptide in preparing a medicine for regulating and controlling the antioxidant capacity of cells.

Furthermore, the RMP-D2 polypeptide can be competitively combined with KEAP1, so that the degradation of NRF2 is reduced, the stability of NRF2 protein is enhanced, and the antioxidant capacity of cells is improved.

The invention also provides application of the RMP-D2 polypeptide in preparing a medicament which competitively binds with KEAP1, reduces NRF2 degradation, enhances the stability of NRF2 protein and improves the oxidation resistance of cells.

In a fifth aspect of the invention, the invention provides an application of the encoding gene of the RMP-D2 polypeptide in preparing a medicine for regulating and controlling the antioxidant capacity of cells.

The sixth aspect of the invention provides an application of the recombinant vector, the recombinant bacterium, the recombinant cell or the expression cassette in preparation of a medicine for regulating and controlling the antioxidant capacity of cells.

The seventh aspect of the invention provides an application of the RMP-D2 polypeptide, the coding gene, the recombinant vector, the recombinant bacterium, the recombinant cell or the expression cassette in preparing an anti-tumor medicament.

The eighth aspect of the invention provides an application of the RMP-D2 polypeptide, the encoding gene, the recombinant vector, the recombinant bacterium, the recombinant cell or the expression cassette in preparing a medicament for assisting in relieving the resistance of tumor cells to other chemotherapeutic drugs.

Further, the tumor is various types of tumors which are primarily generated in the liver, in particular various types of bile duct cancer.

In a ninth aspect of the invention, a medicament for regulating and controlling the antioxidant capacity of cells is provided, wherein the medicament comprises the RMP-D2 polypeptide, a coding gene, a recombinant vector, a recombinant bacterium, a recombinant cell or an expression cassette. Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.

The tenth aspect of the invention provides an anti-tumor medicament, which comprises the RMP-D2 polypeptide, a coding gene, a recombinant vector, a recombinant bacterium, a recombinant cell or an expression cassette. Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.

The invention has the advantages that:

the polypeptide provided by the invention, namely RMP-D2 polypeptide, can be competitively combined with KEAP1, thereby reducing the degradation of NRF2, enhancing the stability of NRF2 protein and improving the oxidation resistance of cells. The polypeptide can be used as a target for regulating and controlling the oxidation resistance of cells, and can be applied to preparation of antitumor drugs or auxiliary relief of drug resistance of tumor cells to other chemotherapeutic drugs.

The regulation of oxidative stress plays an important role in the process of tumorigenesis and development, NRF2 is used as a key molecule for cellular antioxidation, and factors influencing the expression of NRF2and the stability of protein play an important role in regulating the oxidative stress tolerance of cells and the resistance of chemotherapeutic drugs. The invention provides a protein polypeptide competitively combined with KEAP1 on the basis of RMP, which improves the oxidation resistance of cells by enhancing the stability of NRF2 protein. The invention provides a useful target for regulating the antioxidant capacity of cells.

Drawings

FIG. 1 shows that the KEAP1 protein according to the example of the present invention is capable of binding to the RMP protein and NRF2 protein, respectively, but the RMP and NRF2 proteins have no interaction relationship.

FIG. 2 shows that the RMP protein of the present example binds to KEAP1 through its D2 domain.

FIG. 3 shows that the region of the RMP protein D2 of different species of the present invention is conserved in a molecule similar to the NRF2 protein and the KEAP1 binding site E.

FIG. 4 shows that the RMP protein molecules of the present invention are capable of binding to KEAP1 truncation plasmids containing Kelch repeat domains.

FIG. 5 shows that the RMP protein molecules of the present example are capable of competitively binding to the NRF2 protein molecule with KEAP1 protein.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

The bile duct cancer cell lines HuCCT1 and HEK-293T cells are purchased from Chinese academy cells. pCDNA3.1A-GFP, the wild-type pCDNA3.1A-RMP plasmid and the corresponding truncated plasmid were purchased from Shanghai inkstone Biotech. The KEAP1 truncated plasmid was provided by The University of Medicine and dentist (The University of Medicine and department of New Jersey, UMDNJ) Charbin (also prepared according to literature methods: J.Ma, H.Cai, T.Wu, et al. PALB2 intermediates with KEAP1 to promoter NRF2 nuclear amplification and function [ J ]. Mol Cell Biol,2012,32(8): 1506-1). The reagents or instruments used are conventional products which are not indicated by the manufacturer and are commercially available.

Example 1: the KEAP1 protein was able to bind to the RMP protein and NRF2 protein, respectively, but the RMP and NRF2 proteins had no interactive relationship.

Taking out Protein A/G agarose magnetic beads (MAG25K/Protein A/G, Ranui Cheng biochemicals, P28-002) and firstly carrying out magnetic bead pretreatment according to the specification: a pipettor blows or a vortex oscillator uniformly mixes the magnetic beads, 50 mu L of magnetic bead suspension (10 percent, v/v) is taken to be put into a centrifuge tube, and 1.5mL of magnetic rack is magnetically separated to remove the preservation solution; adding 100. mu.L PBS and mixing the magnetic beads (inverting for 30 seconds or mixing with a vortex shaker), and magnetically separating to remove the supernatant (repeating for 2 times); and then carrying out antibody binding: diluting 2-20 mu g of antibody sample by using 100 mu of LPBST (PBST, pH 7.4: PBS with 0.02% Tween-20), and adding the diluted antibody sample into a centrifuge tube with magnetic beads; putting the mixture into a shaking table or a rotary mixer, and uniformly mixing the mixture in a reverse mode at room temperature or 37 ℃ or uniformly mixing the mixture in a vortex mode for 10-15 minutes; putting the centrifugal tube filled with the magnetic beads and the antibodies into a magnetic frame, and removing supernatant after the magnetic beads are completely attached to the wall; the magnetic bead-antibody complexes were washed 3 times with 100 μ LPBST. Antigen precipitation reaction: antigen adsorption: adding 100-1000 mu L of prepared cell lysate into the centrifugal tube filled with the magnetic bead-antibody compound in the previous step, and blowing and uniformly mixing the cell lysate with a pipette gun; reversing and uniformly mixing at 37 ℃ or uniformly mixing by low-speed vortex for 15-20 minutes to combine the antigen and the magnetic beads combined with the antibody; putting the centrifuge tube and the mixture in the previous step into a magnetic frame, and sucking the supernatant (the supernatant can be discarded or stored for subsequent analysis); washing the magnetic bead-antibody-antigen complex for 3 times with 200 μ L PBS; after washing was complete, the beads were resuspended in 100. mu.L PBS for further processing. Antigen elution: placing the centrifuge tube filled with the magnetic bead-antibody-antigen compound into a magnetic frame, and sucking a supernatant after the magnetic beads are attached completely; add 25. mu.L of Elution Buffer (100mM Glycine, pH 2.8) and 5. mu.L of 5 XSDS-PAGE Loading Buffer to the centrifuge tube and resuspend the complex with a pipette; boiling for 5-10 minutes at 95-100 ℃; and putting the boiled centrifugal tube into a magnetic frame, and taking the supernatant for subsequent analysis.

Collecting and culturing in 10cm culture dish HuCCT1 cells, adding 1ml of cell lysate after being washed by normal saline, dividing the cell lysate into two parts with 1600ug of protein content after the BCA protein is quantified, adding a proper amount of protein lysate to fix the volume to 500ul, and preparing an Input group by the residual protein. Incubation was performed with rabbitigg and KEAP1 antibodies, respectively, according to the co-immunoprecipitation protocol described above. The immunoprecipitated products were separated by Western Blotting experiments using SDS-PAGE gels, transferred to NC (nitrocellulose) membranes and analyzed on the Odyssey CLx imaging System (LICOR) after incubation of primary and secondary antibodies. The KEAP1 antibody shown in fig. 1A can co-immunoprecipitate both NRF2and RMP protein molecules from cholangiocarcinoma HuCCT1 cell protein lysate. Fig. 1B is a co-immunoprecipitation of KEAP1 protein molecules from cholangiocarcinoma HuCCT1 cell protein lysates using RMP antibody. FIG. 1C shows the simultaneous transfection of pcDNA3.1A GFP and RMP plasmids based on the HuCCT1 extracellular KEAP1 plasmid, administered at 250. mu. M H after 36 hours₂O₂After 8 hours of treatment, the outturned RMP cells were able to observe an increase in binding of KEAP1 to RMP and a decrease in binding to NRF 2.

Example 2: the RMP protein binds to KEAP1 via its D2 region.

According to the previous reports of RMP protein functional regions, a truncation containing different domains was designed (D.Dorjsuren, Y.Lin, W.Wei, et al.RMP, alpha novel RNA polymerase II deletion 5-interacting protein, interactions transfer by biological genes B virus X protein [ J ]. Mol Cell Biol,1998,18(12): 7546-55; J.P.Thellulat, S.C.zler, N.Henzi, et al.URI is an on gene amplified in cells and is recovered for the same Cell, J.cancer [ J ]. Cancer, 2011,19 (3-317; step-32; antibody Cell, expression Cell, III. G.J.: 9. Cell, 9. C.2016. Cell 2016. 16. C.2016. friend, C.2016. Cell). Schematic representation of RMP truncations and mutant plasmids shown in fig. 2A. The preparation of the above-mentioned truncated plasmid is that firstly, according to the protein fragment the correspondent cDNA fragment is analyzed, effective primer is designed, and HindIII-ApaI is used as cloning site, and pcDNA3.1(+)/myc-His is used as vector, and the vector and fragment are connected, and the effective clone is selected and amplified. After the expression effect of the plasmids was identified, each of the above-described truncated plasmid, pcDNA3.1(+)/myc-His GFP and RMP plasmid was transfected into 293T cells together with the wild KEAP1 plasmid using PEI transfection reagent, respectively. After 36 hours of incubation, the proteins were collected, quantified, and co-immunoprecipitation experiments were performed as described in example 1. The wild-type KEAP1 plasmid shown in fig. 2B was only able to interact with RMP truncation (D2, D4, D5) protein molecules containing the D2 region.

Example 3: the region of the RMP protein D2 from different species is conserved and resembles the KEAP1 binding site E in the NRF2 protein molecule.

Numerous literature reports that ETGE (strong binding site) and DLG (weak binding site) are important KEAP1 binding sites in the NRF2Neh2 domain during the binding of KEAP1 to NRF2 (m.rojo de la Vega, e.chapman, d.d.zhang.nrf2and the Hallmarks of Cancer [ J].Cancer Cell,2018,34(1):21-43；Hanna M.Leinonen，Emilia Kansanen，Petri

et al.Role of the Keap1–Nrf2Pathway in Cancer[J].2014,122:281-320；P.Canning，F.J.Sorrell，A.N.Bullock.Structural basis of Keap1interactions with Nrf2[J]Free Radic Biol Med 2015,88(Pt B): 101-. The sequence of the human RMP protein was queried by NCBI, and only the D2 region presents a sequence similar to ETGE. The sequence of RMP protein in mouse, norway rat, zebrafish, orangutan, chicken, etc. was also queried and analyzed by alignment, as shown in fig. 3, the D2 region of these species is a relatively conserved region, and two of the D2 regions in these protein sequences contain KEAP1 binding site E similar to that in NRF2 molecule. The amino acid sequence of the RMP protein in FIG. 3 is shown in SEQ ID NO.3-SEQ ID NO. 8.

Example 4: the RMP protein molecule is capable of competing with the NRF2 protein molecule for binding to KEAP1 protein.

HEK-293T cells were transfected ex vivo with NRF2, KEAP1 plasmid and wild type RMP plasmid (2. mu.g, 6. mu.g, 12. mu.g) simultaneously, and after 36 hours of transfection, proteins were collected and co-immunoprecipitated as described in example 1 above, as shown in FIGS. 4A, 4B, and by analysis, increased competitive binding to KEAP1 and decreased binding to NRF2 was observed with increasing RMP expression.

Example 5: RMP protein molecules can affect NRF2 protein stability.

RMP overexpressing HuCCT1 cell lines were constructed by a lentiviral system, and control and overexpressing cells were treated with the protein synthesis inhibitor chx (cycloheximide) on a time gradient of 0, 15, 30, 60, 120, 180 min. Protein lysate is added to collect protein, BCA is quantified, a sample is prepared, and RMP overexpression is found to inhibit NRF2 protein degradation through Western Blotting experimental analysis as shown in FIGS. 5A and 5B.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Sequence listing

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Claims (6)

1. An application of RMP-D2 polypeptide with an amino acid sequence shown in SEQ ID NO.1 in preparing a medicine for regulating and controlling cell antioxidant capacity.

2. An application of the coding gene of RMP-D2 polypeptide with the nucleotide sequence shown in SEQ ID NO.2 in preparing the medicine for regulating and controlling the anti-oxidizing ability of cells.

3. The recombinant vector, the recombinant bacterium, the recombinant cell or the expression cassette contains an encoding gene of RMP-D2 polypeptide with a nucleotide sequence shown as SEQ ID NO. 2.

4. The use of claim 3, wherein the recombinant vector is one of a lentiviral vector, a retroviral vector, or an adenoviral vector.

5. The use of claim 4, wherein said recombinant vector comprises an enhancer upstream or downstream of said gene encoding said polypeptide to promote expression of said gene.

6. A medicine for regulating and controlling the oxidation resistance of cells comprises RMP-D2 polypeptide with an amino acid sequence shown as SEQ ID No.1, an encoding gene of RMP-D2 polypeptide with a nucleotide sequence shown as SEQ ID No.2, and a recombinant vector, a recombinant bacterium, a recombinant cell or an expression cassette containing the encoding gene of RMP-D2 polypeptide with a nucleotide sequence shown as SEQ ID No. 2.

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