Protein degradation product and application of non-structural protein NSs of rift valley fever virus or coding gene of non-structural protein NSs in preparation of protein degradation product

Abstract

The invention discloses a protein degradation product and application of non-structural protein NSs of rift valley fever virus or a coding gene of the non-structural protein NSs in preparation of the protein degradation product, and relates to the technical field of protein degradation. On the basis of the property that NSs and recombinant protein of the NSs can autonomously form a fiber-shaped high polymer structure, a degradation machine capable of degrading target protein is self-assembled, so that the specific and efficient degradation of the target protein is realized. Therefore, the new application of the non-structural protein NSs of the rift valley fever virus in targeted degradation of the protein is found, specific efficient degradation of the disease-related protein can be achieved, and the effect of treating diseases is expected to be achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein degradation, in particular, to a protein degradation product and application of a non-structural protein NSs of a Rift Valley fever virus or a coding gene thereof in preparation of the protein degradation product. BACKGROUND

[0002] Currently, due to the lack of available binding pockets and suitable chemicals, 80-85% of human proteins cannot become drug targets. The biggest advantage of targeted protein degradation (TPD) is different from traditional small molecule inhibitors, which only requires a binding agent to recruit target proteins for degradation, rather than a high-affinity inhibitor, which greatly solves the problem of protein undruggable. At present, through two natural protein degradation systems in cells, namely ubiquitin proteasome system (UPS) and lysosome degradation pathway, specific and efficient degradation of disease-related proteins can be achieved, thereby achieving the effect of treating diseases.

[0003] Among them, the most widely used is the PROTAC bifunctional small molecule: protein degradation targeting chimera (PROTACs) includes three parts, namely an E3 ubiquitin ligase ligand, a target protein ligand and a specially designed linker, and two active ligands are connected together through the linker structure. After the ligand binds the E3 ubiquitin ligase and the target protein to shorten the distance between them, the protein is tagged with ubiquitination and degraded through the ubiquitination process. Due to the high dependence of the ubiquitin protein system, PROTACs technology is mainly used to degrade cytoplasmic and nuclear protein targets. Subsequently, many technologies for degrading proteins using lysosome pathways, including endocytosis-lysosome pathway and autophagy-lysosome pathway, etc., such as LYTACs, ATTECs, AUTACs, etc., have emerged, expanding the range of degradable targets.

[0004] However, so far the targeted degradation of protein targets in organelles has not been achieved. Protein abnormalities in organelles are related to many diseases, the most common of which is protein abnormalities in mitochondria. Abnormalities in protein homeostasis in mitochondria are important characteristics and driving factors for a series of human diseases, such as Leber's hereditary optic neuropathy, maternal genetic deafness, type II diabetes, Parkinson's disease and various cancers. Although proteins located in the outer membrane of mitochondria can still be effectively degraded through the traditional ubiquitin-proteasome pathway, the interior of mitochondria cannot be degraded through the ubiquitin-proteasome pathway due to the absence of ubiquitination system proteins.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The application aims to provide a protein degradation product and application of non-structural protein NSs of a Rift Valley fever virus or a coding gene thereof in preparation of the protein degradation product to solve the above technical problems.

[0007] The application is implemented in the following manner:

[0008] In a first aspect, the application provides application of non-structural protein NSs of a Rift Valley fever virus or a coding gene thereof in preparation of a protein degradation product.

[0009] In a second aspect, the application further provides a protein degradation product, which comprises: a coding gene coding non-structural protein NSs of a Rift Valley fever virus and a coding gene of a polypeptide interacting with a target protein; and / or, contains a plurality of non-structural protein NSs of a Rift Valley fever virus and a polypeptide interacting with a target protein; each non-structural protein NSs is directly connected to the polypeptide capable of interacting with the target protein, and the plurality of non-structural protein NSs self-assembles into a filamentous high polymer structure.

[0010] In a third aspect, the application further provides a protein degradation method, which comprises the following steps:

[0011] Transferring an expression vector expressing non-structural protein NSs and a polypeptide interacting with a target protein into cells of the protein to be degraded, or introducing a coding gene coding non-structural protein NSs and a polypeptide interacting with a target protein into cells of the protein to be degraded, and co-culturing;

[0012] Preferably, the cells are cultured in a 37℃±0.5℃, 5%±0.5% CO2 incubator for 18-24 hours.

[0013] In a fourth aspect, the application further provides use of the protein degradation product in preparation of an anti-tumor drug.

[0014] In a fifth aspect, the application further provides a pharmaceutical composition comprising the above protein degradation product.

[0015] The application has the following beneficial effects:

[0016] The inventor found that the flexible structure at the C-terminal of non-structural protein NSs of a Rift Valley fever virus facilitates self-adapted design of a peptide segment (i.e. a motif), and through coupling of the NSs protein with the motif capable of interacting with a target protein in a cell, the E3 ubiquitin ligase property of the NSs protein can realize specific degradation of the target protein in the cell interacting with the motif. Based on the property of multiple NSs capable of self-assembly into a filamentous high polymer structure, the NSs are self-assembled into a degradation machine capable of degrading the target protein, thereby realizing high polymer assembly of the NSs recombinant protein degradation machine, and further realizing specific and efficient degradation of the target protein.

[0017] Therefore, the application finds a new use of the non-structural protein NSs of the Rift Valley fever virus in protein degradation, and can realize specific and efficient degradation of disease-related proteins, and is expected to achieve a disease treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some of the embodiments of the application, and therefore should not be considered as a limitation to the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 Principle diagram for degrading target proteins;

[0020] Figure 2 Structure diagram of NSs protein cryo-EM analysis;

[0021] Figure 3 Structure mode diagram of NSs monomer C-terminal fusion polypeptide fragment;

[0022] Figure 4 Structure mode diagram of NSs filament high polymer structure fusion polypeptide motif;

[0023] Figure 5 Western blot detection result diagram of NSs induced degradation of target proteins with C-terminal coupling motif;

[0024] Figure 6 Result diagram of using Andor confocal fluorescence microscope to observe cell crawling slices and result diagram of using Fiji software to analyze the co-localization of fluorescence pictures;

[0025] Figure 7 Western blot detection result diagram of NSs induced degradation of target proteins with C-terminal coupling motif after treatment with proteasome inhibitor MG132;

[0026] Figure 8 PRK-Strep-(RV)NSs vector schematic diagram (expression vector containing only non-structural protein NSs encoding gene without motif encoding gene). DETAILED DESCRIPTION

[0027] Reference will now be made in detail to embodiments of the application, one or more examples of which are described hereinbelow. Each example is provided as an explanation and not a limitation of the application. Indeed, it will be apparent to one of ordinary skill in the art that numerous modifications and variations of the present application are possible in light of the above teachings without departing from the scope or spirit of the application. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further embodiment.

[0028] Practicing the present application will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety.

[0029] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions not noted in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted by the manufacturers are all conventional products that can be obtained by market purchase.

[0030] In a first aspect, the present application provides a use of a non-structural protein NSs of Rift Valley fever virus or a coding gene thereof in the preparation of a protein degradation product.

[0031] The present application first discovers a brand-new property of the non-structural protein NSs of Rift Valley fever virus, which is a reprogrammable E3 ubiquitin ligase, can recruit the target proteins with interaction and target the proteasome for degradation. The reprogramming strategy of the non-structural protein NSs of Rift Valley fever virus is simple and easy to realize, does not need complex sequence design, and only uses the coupled polypeptide motif and the self-assembled NSs filamentous high polymer degradation property to realize the degradation of the target protein.

[0032] The C-terminal of the NSs protein is a flexible and disordered region, which is suitable for fusion of a polypeptide fragment without affecting the three-dimensional structure of the NSs protein. It is convenient for people to adaptively design a peptide segment (i.e. motif), which can interact with the intracellular target protein by coupling the motif that can interact with the intracellular target protein, and the specific degradation of the intracellular target protein that interacts with the motif can be realized by using the E3 ubiquitin ligase property of the NSs protein (the principle diagram of degrading the target protein is referred to Figure 1 Based on the property of multiple NSs that can autonomously form a filamentous high polymer structure, a degradation machine that can degrade the target protein is self-assembled, so as to realize the high polymer assembly of the NSs recombinant protein degradation machine, and then the specific and efficient degradation of the target protein is realized.

[0033] In a preferred embodiment of the present application, the amino acid sequence of the non-structural protein NSs of Rift Valley fever virus is shown as SEQ ID NO. 1, and the amino acid sequence is as follows:

[0034] MDYFPVISVDLQSGRRVVSVEYIRGDGPPRIPYSMVGPCCVFLMHHRPSHEVRLRFSDFYNVGEFPYRVGLGDFASNVAPPPAKPFQRLIDLIGHMTLSDFTRFPNLKEAISWPLGEPSLAFFDLSSTRVHRNDDIRRDQIATLAMRSCKITNDLEDSFVGLHRMIVTEAILRGIDLCLLPGFDLMYEVAHVQCVRLLQAAKEDISNAVVPNSALIALMEESLMLRSSLPSMMGRNNWIPVVPPIPDVEMESEEESDDDGFVEVD. In other embodiments, the amino acid sequence of the non-structural protein NSs of the Rift Valley fever virus has at least 90% identity to the sequence set forth in SEQ ID NO. 1.

[0035] In a preferred embodiment of the application, the protein degradation product degrades proteins by the following means:

[0036] The non-structural protein NSs recruits target proteins that interact with the non-structural protein NSs for protein degradation.

[0037] In the present application, the terms "target protein", "targeted protein", "targeted proteins", and "target proteins" are synonymous and refer to the target proteins to be degraded by the protein degradation product.

[0038] The protein degradation product comprises a plurality of recombinant non-structural proteins NSs, and the plurality of recombinant non-structural proteins NSs self-assemble into a filamentous high polymer structure.

[0039] The filamentous high polymer structure refers to the fact that NSs spontaneously form a filamentous high polymer structure in transfected cells.

[0040] In a preferred embodiment of the application, the C-terminus and / or N-terminus of the non-structural protein NSs is coupled to a polypeptide that interacts with the target protein. The C-terminus and / or N-terminus coupled polypeptide is a characteristic sequence that recognizes the target protein, and NSs protein that is not coupled to the target protein does not have degradation activity; after coupling to a polypeptide that interacts with the target protein and self-assembling into a filamentous high polymer structure, the protein degradation product has high and specific protein degradation activity.

[0041] Definition: Motif is a typical sequence or structure, usually refers to the basic structure that constitutes any characteristic sequence. In biology, motif usually refers to a conserved sequence with biological function, which may contain specific binding sites and common sequence segments involved in a specific biological process.

[0042] In a preferred embodiment of the application, the polypeptide comprises one or more motifs. In order to achieve the degradation of multiple proteins, it is also easy for those skilled in the art to design the polypeptide to comprise multiple motifs. In order to improve the degradation effect, those skilled in the art can set up a motif, and it is also easy to implement to set up multiple same motifs in series.

[0043] In a preferred embodiment of the application, the motif comprises at least 3 amino acids; for example, 3-30 amino acids, or 3-20 amino acids, or 3-15 amino acids, or 3-10 amino acids, or 3-9 amino acids, or any one point value between the above-mentioned amino acid number ranges.

[0044] In a preferred embodiment of the application, the motif can interact with at least one protein selected from an anti-apoptotic protein, a protein involved in necroptosis or necrosome formation, and a protein involved in ferroptosis.

[0045] In a preferred embodiment of the application, the anti-apoptotic protein is selected from an anti-apoptotic protein of the Bcl-2 protein family, a BAG protein, XIAP, cIAP1, cIAP2, and cyclin D1; the protein involved in necroptosis or necrosome formation is selected from MLKL, RIPK1, RIPK3, DIABLO, and FADD.

[0046] In a preferred embodiment of the application, the anti-apoptotic protein of the Bcl-2 protein family is selected from BCL-2, BCL-xL, BCL-w, MCL-1, A1, and BCL-B protein.

[0047] In a preferred embodiment of the application, the BAG protein is selected from BAG3 protein.

[0048] In a preferred embodiment of the application, the protein involved in ferroptosis is selected from Glutathione Peroxidase (GPX). For example, selected from GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, or GPX8.

[0049] In a preferred embodiment of the application, the motif is selected from at least one of KFERQ, NGR, AVPI, VQVG, ATIG, and VQLG.

[0050] In a preferred embodiment of the application, the protein for protein degradation of the protein degradation product is an intracellular protein of a eukaryotic cell.

[0051] In a preferred embodiment of the application, the protein degradation product is selected from a cell, an expression vector, a protein degradation drug, a reagent, or a kit.

[0052] In a preferred embodiment of the present application, the cell or the expression vector contains a gene encoding the non-structural protein NSs of the Rift Valley fever virus, or the cell contains the non-structural protein NSs of the Rift Valley fever virus;

[0053] In a preferred embodiment of the present application, the cell or the expression vector contains a gene encoding the non-structural protein NSs of the Rift Valley fever virus and a gene encoding a polypeptide interacting with the target protein, or the cell contains the non-structural protein NSs of the Rift Valley fever virus and the polypeptide interacting with the target protein;

[0054] The cell further contains the target protein.

[0055] In a second aspect, the present application further provides a protein degradation product, which comprises: a gene encoding the non-structural protein NSs of the Rift Valley fever virus and a gene encoding a polypeptide interacting with the target protein; and / or, contains a plurality of the non-structural protein NSs of the Rift Valley fever virus and the polypeptide interacting with the target protein; each of the non-structural protein NSs is directly linked to the polypeptide capable of interacting with the target protein, and the plurality of the non-structural protein NSs self-assembles into a filamentous high polymer structure.

[0056] In a preferred embodiment of the present application, the protein degradation product is selected from a cell, an expression vector, a protein degradation drug, a reagent or a kit.

[0057] The cell is a mammalian cell; in a preferred embodiment of the present application, the mammalian cell is a human cell, a mouse cell, a rat cell;

[0058] In a preferred embodiment of the present application, the mammalian cell is selected from any one of 293 cell, 293T cell, 293FT cell, CHO cell, COS cell, mouse L cell, LNCaP cell, 633 cell, Vero, BHK cell, CV1 cell, Hela cell, MDCK cell, Hep-2 cell and Per6 cell;

[0059] In a preferred embodiment of the present application, the human cell is a primary human blood cell (PBMC);

[0060] In a preferred embodiment of the present application, the cell is an immune cell;

[0061] In a preferred embodiment of the present application, the immune cell is a T cell or a B cell.

[0062] In a preferred embodiment of the present application, the expression vector is a eukaryotic expression vector;

[0063] The nucleotide sequence of the gene encoding the non-structural protein NSs of the Rift Valley fever virus has at least 90% identity with the sequence shown in SEQ ID NO. 6, and the gene encoding the polypeptide interacting with the target protein has at least 90% identity with the sequence shown in any one of SEQ ID NOs. 7-10.

[0064] In a preferred embodiment of the application, the medicament further comprises a pharmaceutically acceptable excipient.

[0065] In a preferred embodiment of the application, the pharmaceutically acceptable excipient is selected from at least one of a filler, a disintegrant, a lubricant, a flavoring agent, a binder, a suspending agent, and a flavoring.

[0066] The pharmaceutically acceptable excipient includes, but is not limited to, a pharmaceutically acceptable carrier, an auxiliary substance, or a solvent. The pharmaceutically acceptable excipient includes various organic or inorganic carriers and / or auxiliary materials, as they are commonly used for pharmaceutical purposes, particularly for solid pharmaceutical formulations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, acacia, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, carboxymethylcellulose calcium salt, hydroxypropyl starch, glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavors such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, p-hydroxybenzoic acid esters (e.g., methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate); stabilizers such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats, and oils such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, and the like.

[0067] In a preferred embodiment of the application, the dosage form of the above-mentioned medicament is selected from any one of an injection, a tablet, a granule, a capsule, an emulsion, a suspension, an elixir, a syrup, a powder, and a tablet.

[0068] In an alternative embodiment, the aforementioned drug is a liquid pharmaceutical preparation (such as an injectable formulation), such as a solution, suspension, or gel, which typically contains a liquid carrier, such as water, and / or a pharmaceutically acceptable organic solvent. Furthermore, such liquid preparations may also contain pH adjusters, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (e.g., methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs may be isotonic, i.e., they may have the same osmotic pressure as blood. The isotonicity of the drug can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickener, such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the reagent selected.

[0069] In a preferred embodiment of the present invention, the drug is administered orally or by injection.

[0070] When protein degradation products are used as reagents, the reagents may be in the form of liquid, solid, or semi-solid.

[0071] Thirdly, the present invention also provides a method for protein degradation, comprising the following steps:

[0072] Expression vectors expressing non-structural proteins (NSs) and peptides that interact with target proteins are transferred into cells containing the proteins to be degraded, or genes encoding non-structural proteins (NSs) and peptides that interact with target proteins are introduced into cells containing the proteins to be degraded and co-cultured.

[0073] Preferably, the culture is carried out in an incubator at 37℃±0.5℃ and 5%±0.5%CO2 for 18-48 hours.

[0074] For example, incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0075] Thirdly, the present invention also provides the use of protein degradation products in the preparation of antitumor drugs;

[0076] In a preferred embodiment of the present invention, the tumor is a solid tumor or a hematologic tumor.

[0077] In a preferred embodiment of the present invention, the solid tumor is at least one of epithelial tumors and gliomas. Epithelial tumors include, but are not limited to, papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma, or squamous cell carcinoma.

[0078] Gastrointestinal cancers are selected from esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, pancreatic cancer, bile duct cancer, small bowel cancer, colon cancer, rectal cancer, colorectal cancer, and anal cancer.

[0079] In one alternative implementation, adenocarcinoma includes, but is not limited to, lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0080] In one alternative implementation, the adenoma includes, but is not limited to, cystic adenoma, fibroadenoma, pleomorphic adenoma, or polypoid adenoma.

[0081] In one alternative implementation, breast cancer includes, but is not limited to, breast epithelial carcinoma;

[0082] Preferably, the breast cancer is ductal carcinoma or lobular carcinoma of the breast.

[0083] Breast cancer, preferably stage II to IV and / or poorly differentiated invasive ductal carcinoma, comedo carcinoma and medullary carcinoma (preferably grade 2).

[0084] Ovarian cancer, serous and mucinous carcinoma (preferably stage Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0085] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0086] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0087] Lung cancer, preferably including small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma), and large cell lung cancer.

[0088] Squamous cell carcinoma includes one or more of the following: oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal squamous cell carcinoma, esophageal squamous cell carcinoma (e.g., esophageal squamous cell carcinoma), squamous cell carcinoma of the lip, uterine squamous cell carcinoma, vaginal squamous cell carcinoma, and skin squamous cell carcinoma.

[0089] Oral squamous cell carcinoma (OSCC) is also known as oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include, but are not limited to, tongue squamous cell carcinoma.

[0090] In other embodiments, the aforementioned squamous cell carcinoma can also be squamous cell carcinoma formed by squamous metaplasia of the bronchus, bladder, renal pelvis, etc.

[0091] Hematologic malignancies are selected from at least one of lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, mantle cell lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.

[0092] Fourthly, the present invention also provides a pharmaceutical composition comprising the above-described protein degradation product.

[0093] In a preferred embodiment of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0094] Pharmaceutically acceptable excipients include a variety of organic or inorganic carriers and / or auxiliary materials, as they are commonly used for pharmaceutical purposes, particularly for solid drug formulations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylene pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, hydrolyzed starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, talc, and sodium lauryl sulfate; and flavorings such as citric acid, menthol, and glycine. Orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (e.g., methylparaben, ethylparaben, propylparaben, butylparaben); stabilizers, such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0095] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0096] Example 1

[0097] This embodiment provides a protein degradation product (eukaryotic expression vector) comprising the coding sequence for the non-structural protein NSs encoding Rift Valley fever virus and the coding sequence for a motif (AVPI) linked to the C-terminus of NSs. A schematic diagram of the full-length structure of the non-structural protein NSs monomer is shown below. Figure 2 As shown, its C-terminus is a flexible and disordered region, suitable for fusing polypeptide fragments without affecting the three-dimensional structure of the NSs protein itself. A schematic diagram of the structure of the NSs monomer fused with a polypeptide motif at its C-terminus is shown below. Figure 3 As shown. A schematic diagram of the structure of the NSsfilament polymer fused with the polypeptide motif is referenced. Figure 4 As shown.

[0098] The recombinant protein expressed by the eukaryotic expression vector in this embodiment can target cIAP1 and XIAP. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.2. The tetrapeptide motif AVPI can mediate the interaction with the BIRC3 domain of the target proteins cIAP1 and XIAP [1-4].

[0099] The eukaryotic expression vector was constructed as follows: the coding sequences encoding the recombinant protein shown in SEQ ID NO.2, the coding sequences encoding the AVPI protein, and the terminator were constructed into a eukaryotic expression plasmid, and the nucleotide sequence of the inserted gene was shown in SEQ ID NO.11. The nucleotide sequence of the constructed eukaryotic expression vector was shown in SEQ ID NO.15.

[0100] First, the plasmid vector fragment is amplified using PCR, and then the fragment is ligated into a circular plasmid using homologous recombination.

[0101] Methods for constructing cloning vectors include: using Figure 8 The PRK-Strep-(RV)NSs plasmid shown was used as a template for the PCR reaction. Primers containing tetrapeptide motif nucleotides were synthesized, and a linearized plasmid fragment containing the tetrapeptide motif was amplified by reverse PCR. Finally, the linear plasmid fragment was assembled into a circular plasmid under the action of homologous recombinase. After transformation into E. coli cells, the plasmid was extracted and sequenced for identification. Successful sequencing indicates the completion of clone construction.

[0102] The names and sequences of the PCR primers used to amplify the tetrapeptide motif are shown in the table below:

[0103]

[0104]

[0105] This embodiment uses the recombinant protein encoding plasmid SEQ ID NO.2 as an example to briefly describe the specific steps of clone construction:

[0106] 1. Prepare the following PCR system in a PCR tube: (using NEB Q5 polymerase kit, catalog number: M0491V).

[0107]

[0108] 2. Perform PCR amplification in a PCR instrument according to the following PCR program:

[0109]

[0110] 3. Agarose gel electrophoresis was used to identify and recover the linearized fragment (approximately 5500 bp);

[0111] 4. Prepare the following recombination reaction in a PCR tube: (Use Novitan source recombination kit, catalog number: C112).

[0112]

[0113] 5. In a PCR instrument, react at 37°C for 30 minutes; immediately after the reaction, place on ice to cool for 3-5 minutes.

[0114] 6. Heat shock transformation of competent E. coli cells, followed by plate culture;

[0115] 7. Select single clones for amplification by shaking, extract plasmids, and then sequence them for identification.

[0116] Example 2

[0117] This embodiment provides a protein degradation product (eukaryotic expression vector) comprising the coding sequence for the non-structural protein NSs encoding Rift Valley fever virus and the coding sequence for a motif (VQVG) linked to the C-terminus of NSs. The recombinant protein expressed by the eukaryotic expression vector in this embodiment is capable of mediating homology interaction with the RHIM domain of the target protein RIPK1 [5-6], and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.3.

[0118] The method for constructing the eukaryotic expression vector is the same as in Example 1, except that the inserted nucleotide sequence is different. The nucleic acid sequence inserted in Example 1 is shown in SEQ ID NO.11, while the nucleic acid sequence inserted in this example is shown in SEQ ID NO.12.

[0119] Example 3

[0120] This embodiment provides a protein degradation product (eukaryotic expression vector) comprising the coding sequence of a non-structural protein NSs encoding Rift Valley fever virus and a motif (VQLG) linked to the C-terminus of NSs. The recombinant protein expressed by the eukaryotic expression vector in this embodiment has a motif (VQLG) that mediates homology interaction with the RHIM domain of the target protein RIPK1 [5-6]. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.4.

[0121] The method for constructing the eukaryotic expression vector is the same as in Example 1, except that the inserted nucleotide sequence is different. The nucleic acid sequence inserted in this example is shown in SEQ ID NO.13.

[0122] Example 4

[0123] This embodiment provides a protein degradation product (eukaryotic expression vector) comprising the coding sequence of a non-structural protein NSs encoding Rift Valley fever virus and a motif (ATIG) linked to the C-terminus of NSs. The recombinant protein expressed by the eukaryotic expression vector in this embodiment has an ATIG motif that mediates homology interaction with the RHIM domain of the target protein RIPK1 [5-6]. The amino acid sequence of the recombinant protein is shown in SEQ ID NO. 5.

[0124] The method for constructing the eukaryotic expression vector is the same as in Example 1, except that the inserted nucleotide sequence is different. The nucleic acid sequence inserted in this example is shown in SEQ ID NO.14.

[0125] Experimental Example 1

[0126] This experimental example demonstrates the degradation of target proteins cIAP1, XIAP, and RIPK1 in the protein degradation products prepared in Examples 1-4.

[0127] Huh7 cells were transfected with NSs, NSs-AVPI, or the empty vector pRK, and cells were collected 24 hours after transfection. Huh7 cells were first transfected with Flag-RIPK1, and then transfected with NSs, NSs-VQVG / VQLG / ATIG, or the empty vector 6 hours after transfection. Cells were collected 24 hours after transfection, and the protein level of the target protein was detected by Western blot.

[0128] The specific steps for pRK-NSs, pRK-NSs-AVPI, or pRK transfection are as follows:

[0129] (1) Huh7 cells in good growth condition were seeded into 24-well plates at a cell density of 1×10⁶ cells / well. 5 Cells per well, cell culture volume of 0.5 mL / well, cell culture was carried out by cross-shaking to distribute the cells evenly on the bottom of the plate, and the cells were incubated statically in a 37°C, 5% CO2 incubator.

[0130] (2) Incubate overnight for 14-16 hours. When the cells adhere to the wall and the cell confluence density is 70-80%, perform transfection using Lipo3000 transfection reagent (Thermo Fisher Scientific). The transfection system per well is: 100 μL Opti-MEM medium, 1 μL lipo3000, 1 μL P3000, and 1 μg plasmid (pRK-NSs or pRK-NSs-AVPI or pRK). Specifically, the transfection procedure is as follows: Add 50 μL of Opti-MEM medium to each of two clean 1.5 mL centrifuge tubes. Add 1 μL of LP3000 and 1 μg of plasmid to one tube, and add 1 μL of lipo3000 to the other tube. Gently mix each tube. After standing for 5 min, add the diluted plasmid to the diluted lipo3000 and mix slowly and thoroughly. After standing for 15 min, add the plasmid-liposome complex evenly to the cells, gently shake well, and then incubate in an incubator.

[0131] (3) Cells were collected and lysed 24 h after transfection, and Western blot was used to detect the protein expression of the target protein.

[0132] The specific pRK-Flag-RIPK1 transfection steps are as follows:

[0133] (1) Huh7 cells in good growth condition were seeded into 24-well plates at a cell density of 1×10⁶ cells / well. 5 Cells per well, cell culture volume of 0.5 mL / well, cell culture was carried out by cross-shaking to distribute the cells evenly on the bottom of the plate, and the cells were incubated statically in a 37°C, 5% CO2 incubator.

[0134] (2) After overnight culture for 14–16 h, when the cells adhere to the wall and the cell confluence density reaches 70–80%, transfection is performed using Lipo3000 transfection reagent (Thermo Fisher Scientific). The transfection system per well is: 100 μL Opti-MEM medium, 1 μL lipo3000, 1 μL P3000, and 1 μg plasmid (pRK-Flag-RIPK1). Specifically, the transfection procedure is as follows: Add 50 μL Opti-MEM medium to each of two clean 1.5 mL centrifuge tubes. Add 1 μL P3000 and 1 μg plasmid to one tube, and add 1 μL lipo3000 to the other tube. Mix gently. After standing for 5 min, add the diluted plasmid to the diluted lipo3000 and mix slowly and thoroughly. After standing for 15 min, add the plasmid-liposome complex evenly to the cells, shake gently, and then incubate in an incubator.

[0135] (3) Transfection was performed again 6 hours after transfection: Lipo3000 transfection reagent (Thermo Fisher Scientific) was used for transfection. The transfection system per well was: 100 μL Opti-MEM medium, 1 μL lipo3000, 1 μL LP3000, 1 μg plasmid (pRK-NSs or pRK-NSs-VQVG or pRK-NSs-VQLG or pRK-NSs-ATIIG or pRK). Specifically, the transfection procedure is as follows: Add 50 μL of Opti-MEM medium to each of two clean 1.5 mL centrifuge tubes. Add 1 μL of P3000 and 1 μg of plasmid to one tube, and add 1 μL of lipo3000 to the other tube. Mix each tube gently. After standing for 5 min, add the diluted plasmid to the diluted lipo3000 and mix slowly and thoroughly. After standing for 15 min, add the plasmid-liposome complex evenly to the cells, shake gently, and then incubate in an incubator.

[0136] (4) Cells were collected 24 hours after transfection and lysed. Western blot was used to detect the protein expression of the target protein.

[0137] The steps for detecting protein expression levels using Western blot are as follows:

[0138] (1) Prepare protein gel: Prepare protein gel of appropriate concentration according to the size of the target protein;

[0139] (2) Protein sample preparation: Add protein loading buffer to the sample to be tested, mix well, and boil in a water bath for 15 minutes to fully denature the protein. After the water bath, centrifuge at 12000×g for 1 minute to centrifuge the water droplets on the tube wall to the bottom of the tube. Vortex for 10 seconds to mix the sample evenly before loading.

[0140] (3) Prepare the electrophoresis buffer, Running Buffer.

[0141] (4) Protein loading: Add the sample to be tested into the gel well;

[0142] (5) Electrophoresis: 80V constant voltage in stacking gel for 30 min, 120V constant voltage in separating gel. Adjust the electrophoresis time according to the size of the target protein. Generally, electrophoresis is stopped when bromophenol blue is observed to be close to the bottom edge of the gel.

[0143] (6) Preparation of transfer buffer, blocking buffer, and washing buffer TBS-T during electrophoresis: Weigh 144g of glycine and 30.3g of Tris, add ddH2O to 800mL, stir thoroughly to dissolve, adjust the pH to 8.3, and then bring the volume to 1L with ddH2O to obtain the 10× transfer buffer stock solution. For dilution, measure 100mL of the 10× stock solution + 700mL of ddH2O + 200mL of methanol to obtain the 1× transfer buffer. The transfer buffer is generally recycled, and pre-cooled transfer buffer is required for transfer; therefore, it is usually prepared in advance and stored at 4℃. Weigh 1g of skim milk powder and add 20mL of 1×TBS to obtain the blocking buffer (5% skim milk powder). Measure 100 mL of 10×TBS + 900 mL of ddH2O + 500 μL of Tween20 to obtain TBS-T washing buffer containing 0.05% Tween20.

[0144] (7) Transfer: Activate the PVDF membrane (5.5cm × 8.5cm) in methanol for 1 min. Assemble the "sandwich" in the following order: positive electrode (white side) - filter paper - PVDF membrane - protein gel - filter paper - negative electrode (black side). Align the white side with the red side of the transfer tank (positive electrode) and the black side with the black side (negative electrode) before inserting the "sandwich". After correctly inserting the electrodes into the electrophoresis apparatus, set the transfer current to 350mA. Adjust the transfer time according to the protein gel thickness, protein gel concentration, and target protein size. Generally, for a 1.0mm thick protein gel with a 12% separating gel concentration, the transfer time is set to 2 hours.

[0145] (8) Blocking: After the transfer is completed, the PVDF membrane is placed in the blocking solution and incubated on a shaker at room temperature for 1 hour;

[0146] (9) Primary antibody incubation: After blocking, wash the PVDF membrane with ddH2O to remove any remaining skim milk, then proceed with primary antibody incubation. Dilute the primary antibody according to the dilution ratio recommended in the antibody instructions. Incubate on a shaker at room temperature for 60 min or at 4°C overnight.

[0147] (10) Primary antibody recovery and membrane washing: The incubated primary antibody was recovered, the membrane was washed with TBS-T, and incubated on a shaker at room temperature for 20 min, with a new TBS-T every 8 min.

[0148] (11) Secondary antibody incubation: After washing the membrane, the secondary antibody was incubated. The secondary antibody was diluted in 5% skim milk powder at a ratio of 1:50000 and incubated on a shaker at room temperature for 1-2 hours.

[0149] (12) Washing the membrane: Wash the membrane with TBS-T, incubate on a shaker at room temperature for 20 min, and replace with a new TBS-T every 8 min;

[0150] (13) ECL color development: After washing the membrane, exposure and color development can be performed. Mix ECL color development solution A and B in a 1:1 ratio (prepare fresh before use), drop the mixed color development solution onto the PVDF membrane, and then image it in a protein chemiluminescence imaging system.

[0151] Test results as follows Figure 5 As shown, by coupling these motifs to the C-terminus of NSs, the degradation of the target proteins cIAP1, XIAP, and RIPK1 was achieved more significantly than that of NSs expression.

[0152] Experiment Example 2

[0153] Huh7 cells (from the Wuhan Institute of Virology, Chinese Academy of Sciences) were seeded on cell crawling slides in a 24-well plate system at 1×10⁵ cells / well. After cell adhesion, the cells were transfected with Flag-cIAP1, Flag-XIAP, and Flag-RIPK1 (these three expression plasmids were constructed in our laboratory using the following method: first, the plasmid vector fragments were amplified by PCR, and then the fragments were ligated into circular plasmids using homologous recombination). Six hours after transfection, the cells were transfected with NSs, NSs-AVPI / VQVG / VQLG / ATIG, or an empty vector plasmid. Twenty-four hours after transfection, the cells were fixed with 4% paraformaldehyde and treated with 0.5% Triton X-ray distillate. After permeabilization blocking with X-100 and 5% goat serum, the target protein (red) was labeled with anti-Flag antibody (purchased from Mitsubishi Pharmaceuticals, catalog number: 66008-4-Ig), NSs (green) was labeled with anti-NSs antibody (a polyclonal antibody prepared by immunizing rabbits with NSs protein), and the cell nucleus (blue) was labeled with DAPI (purchased from Beyotime Pharmaceuticals, catalog number: C1002). Cell slides were mounted and observed under an Andorra confocal fluorescence microscope. Colocalization analysis of the fluorescence images was performed using Fiji software.

[0154] The fluorescence co-localization detection method is as follows:

[0155] (1) Place a round glass slide into the 24-well plate;

[0156] (2) Huh7 cells in good growth condition were seeded onto cell spread sheets in 24-well plates at a cell density of 0.6 × 10⁶ cells / well. 5 The cell culture volume was 0.5 mL / well. The cells were evenly distributed on the bottom of the plate by the cross-shaking method and then incubated statically in a 37°C, 5% CO2 incubator.

[0157] (3) Incubate overnight for 14-16 hours. When the cells adhere to the wall and the cell confluence density is 70-80%, perform plasmid transfection. The transfection steps are the same as described above.

[0158] (4) 24 h after plasmid transfection, discard the culture supernatant and wash twice with 500 μL of PBS each time;

[0159] (5) Fix with 4% PFA for 30 min, 500 μL per well;

[0160] (6) Wash 3 times with 500 μL of PBS each time;

[0161] (7) Permeabilize with 0.5% Triton X-100 (prepared with PBS) for 10 min, 500 μL per well;

[0162] (8) Wash 3 times with 500 μL of PBS each time;

[0163] (9) Block with 3% BSA (prepared with PBS) at room temperature for 30 min, or block overnight at 4°C;

[0164] (10) Primary antibody incubation: Dilute the primary antibody with 0.5% BSA solution and incubate at room temperature for 1 h;

[0165] (11) Wash 3 times with 500 μL of PBS each time;

[0166] (12) Secondary antibody incubation: The secondary antibody was diluted 1:1000 in 0.5% BSA and incubated at room temperature for 1 hour in the dark;

[0167] (13) Wash 3 times with 500 μL of PBS each time;

[0168] (14) Incubation with nuclear staining reagent DAPI: DAPI was diluted 1:2000 in 0.5% BSA, and incubated at room temperature for 10 min in the dark;

[0169] (15) Wash 3 times with 500 μL of PBS each time;

[0170] (16) Mounting medium: Take 5 uL of Mountant medium (anti-quenching mounting medium) and drop it onto a glass slide. Use tweezers to pick up the cell smear. After absorbing the residual PBS with absorbent paper, place the cell side onto the Mountant medium. After the smear adheres, let it stand at room temperature in the dark for more than 6 hours until the mounting medium solidifies before observation.

[0171] (17) Andorra fluorescence confocal microscope observation, using a 63× oil immersion lens.

[0172] Results reference Figure 6 As shown, immunofluorescence observation results indicate that the NSs C-terminal coupled motif exhibits significant co-localization with the target protein, suggesting that the target protein was recruited and enriched.

[0173] Experimental Example 3

[0174] This experiment involved Western blot analysis of the target protein degradation induced by the proteasome inhibitor MG132 treatment inhibiting the C-terminal coupled motif NSs.

[0175] Huh7 cells were transfected with NSs, NSs-AVPI, or an empty vector plasmid. Six hours post-transfection, cells were treated with MG132 (10 μM) or an equal volume of DMSO (as a solvent). Cells were collected 18 hours after treatment. Alternatively, Huh7 cells were first transfected with Flag-RIPK1, followed by transfection with NSs, NSs-VQVG / VQLG / ATIG, or an empty vector plasmid 6 hours later. After 6 hours, cells were treated with MG132 (10 μM) or DMSO. Cells were collected 18 hours after treatment. Western blot analysis was performed to detect the target protein levels. The transfection and Western blot methods were the same as in Example 1.

[0176] Figure 7 The results showed that treatment with the proteasome inhibitor MG132 significantly inhibited the degradation of target proteins induced by the C-terminal coupling motif of NSs, suggesting that the C-terminal coupling motif of NSs induces proteasome-dependent degradation of target proteins.

[0177] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0178] References:

[0179] 1. Structure-based design of potent, conformationally constrained Smacmimetics. J Am Chem Soc, 2004.126(51):16686-16687.

[0180] 2. The present and the future of motif-mediated protein-protein interactions. Curr Opin Struct Biol, 2018.50:162-170.

[0181] 3.Structural basis of IAP recognition by Smac / DIABLO.Nature,2000.408(6815):1008-1012.

[0182] 4.The structure of the BIR3 domain of cIAP1 in complex with the N-terminal peptides of SMAC and caspase-9.Acta Crystallogr D Biol Crystallogr,2009.65(Pt 1):58-66.

[0183] 5.The role of RHIM in necroptosis.Biochem Soc Trans,2022.50(4):1197-1205.

[0184] 6.Receptor-interacting protein kinase 1(RIPK1)as a therapeutictarget.Nat Rev Drug Discov,2020.19(8):553-571.

Claims

1. Application of non-structural proteins NSs of Rift Valley fever virus or their encoding genes in the preparation of protein degradation products.

2. The application according to claim 1, characterized in that, The amino acid sequence of the non-structural protein NSs of the Rift Valley fever virus is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The protein degradation product degrades proteins in the following manner: The non-structural protein NSs recruits target proteins that can interact with the non-structural protein NSs for protein degradation; Preferably, the protein degradation product comprises a plurality of recombinant non-structural proteins NSs, and the plurality of recombinant non-structural proteins NSs self-assemble into a fibrous polymeric structure.

4. The application according to claim 3, characterized in that, The non-structural protein NSs has a polypeptide coupled to its C-terminus and / or N-terminus that can interact with the target protein. Preferably, the polypeptide includes one or more motifs; Preferably, the motif comprises at least three amino acids; Preferably, the motif can interact with at least one protein selected from anti-apoptotic proteins, proteins involved in necroptosis or necrosome formation, and proteins involved in ferroptosis; Preferably, the anti-apoptotic protein is selected from anti-apoptotic proteins of the Bcl-2 protein family, BAG protein, XIAP, cIAP1, cIAP2 and cyclin D1; the protein involved in necroptosis or necrosome formation is selected from MLKL, RIPK1, RIPK3, DIABLO and FADD. Preferably, the anti-apoptotic proteins of the Bcl-2 protein family are selected from BCL-2, BCL-xL, BCL-w, MCL-1, A1 and BCL-B proteins; Preferably, the BAG protein is selected from BAG3 protein; Preferably, the protein involved in ferroptosis is selected from glutathione peroxidase (GPX); Preferably, the motif is selected from at least one of the following: KFERQ, NGR, AVPI, VQVG, ATIG, and VQLG.

5. The application according to claim 4, characterized in that, The protein degradation product is used to degrade proteins that are intracellular proteins of eukaryotic cells. Preferably, the protein degradation product is selected from cells, expression vectors, drugs, reagents or kits for protein degradation; Preferably, the cell or expression vector contains a gene encoding the non-structural protein NSs of the Rift Valley fever virus, or the cell contains the non-structural protein NSs of the Rift Valley fever virus. Preferably, the cell or expression vector contains a gene encoding the non-structural protein NSs of the Rift Valley fever virus and a gene encoding a polypeptide that interacts with the target protein, or the cell contains the non-structural protein NSs of the Rift Valley fever virus and a polypeptide that interacts with the target protein. The cells also contain target proteins.

6. A protein degradation product, characterized in that, It includes: encoding The genes encoding the non-structural proteins NSs of Rift Valley fever virus and the genes encoding the polypeptides that interact with the target proteins. And / or, containing a plurality of the non-structural proteins NSs of the Rift Valley fever virus and a polypeptide that interacts with the target protein; each of the non-structural proteins NSs is directly linked to the polypeptide, and the plurality of non-structural proteins NSs self-assemble into a fibrous polymeric structure. Preferably, the peptide is coupled to the C-terminus and / or N-terminus of the non-structural protein NSs; Preferably, the protein degradation product is selected from cells, expression vectors, drugs, reagents or kits for protein degradation; Preferably, the cell is a mammalian cell; Preferably, the expression vector is a eukaryotic expression vector; Preferably, the nucleotide sequence of the gene encoding the non-structural protein NSs of the Rift Valley fever virus has at least 90% identity with the sequence shown in SEQ ID NO. 6, and the gene encoding the polypeptide that interacts with the target protein has at least 90% identity with the sequence shown in any one of SEQ ID NO. 7-10.

7. A method for protein degradation, characterized in that, It includes the following steps: transferring an expression vector that expresses non-structural proteins NSs and peptides that interact with target proteins into cells containing proteins to be degraded, or introducing the encoding genes that encode non-structural proteins NSs and peptides that interact with target proteins into cells containing proteins to be degraded, and co-culturing them. Preferably, the culture is carried out in an incubator at 37℃±0.5℃ and 5%±0.5%CO2 for 18-48 hours.

8. Use of the protein degradation product as described in claim 6 in the preparation of antitumor drugs; Preferably, the tumor is a solid tumor or a hematologic tumor.

9. A pharmaceutical composition, characterized in that, It includes the protein degradation product as described in claim 6.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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