Application of aldolase A protein 330 amino acid modified posttranslational protein in tumor resistance and mutant

Abstract

The invention relates to the technical field of biology, in particular to application of aldolase A protein 330 amino acid modified posttranslational protein in tumor resistance and a mutant. The aldolase A protein mutant is obtained by carrying out substitution on an amino acid sequence as shown in SEQ ID No. 1; and lysine at the 330 position is substituted by arginine. Experiments prove that the aldolase A protein mutant can significantly reduce the growth speed of mouse malignant breast cancer cells 4T1 and mouse malignant black tumor cells B16-F10 under the skin of a mouse, and shows excellent antitumor activity. A brand-new tumor treatment method is created, and the technical blank in the field is filled.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to an anti-tumor application and mutant of a post-translational protein modification of the amino acid at position 330 of an aldolase A protein, and in particular to an aldolase A protein mutant and its application in the preparation of anti-tumor drugs, and specifically studies the effect of a mutant obtained by replacing the lysine at position 330 of the aldolase A protein on tumor growth. Background Art

[0002] Cancer is a disease that seriously threatens human health. It continues to threaten human quality of life and life expectancy, and has already jumped to the forefront of causes of death. Faced with this grim situation, the medical community continues to explore and innovate, striving to break through the limitations of traditional treatment methods and bring more effective treatment options to patients. At present, the treatment strategies for tumors cover a variety of approaches such as surgical resection, radiotherapy, chemotherapy, and emerging immunotherapy. However, although these therapies have prolonged the survival of patients to a certain extent, they are inevitably accompanied by challenges such as drug resistance, significant toxic side effects, and individual differences in treatment response, forcing researchers to constantly seek new treatment ideas and targets.

[0003] In recent years, with the continuous development of biotechnology, especially the in-depth development of molecular biology, genetics and bioinformatics, a series of cutting-edge anti-tumor therapies have emerged, injecting new vitality into the field of tumor treatment. Among them, gene therapy directly corrects or replaces pathogenic genes to achieve treatment at the root of the disease; immunotherapy uses the patient's own immune system to stimulate its ability to recognize and eliminate tumor cells; targeted therapy focuses on specific tumor-related molecules or signal pathways to accurately attack tumors without affecting normal tissues; in addition, the identification of new tumor markers also provides the possibility for early diagnosis and personalized treatment.

[0004] Metabolic reprogramming of tumor cells is a significant feature that distinguishes them from normal cells. The most striking of these is the shift from oxidative phosphorylation to aerobic glycolysis, the Warburg effect. This process not only meets the energy needs of rapid proliferation of tumor cells, but also promotes their ability to adapt to the hypoxic microenvironment. The metabolic reprogramming that occurs in tumor cells involves a shift from ATP production mainly through oxidative phosphorylation to aerobic glycolysis, which is the so-called Warburg effect. Human aldolase proteins are essential for glycolysis and need to catalyze the reversible conversion of fructose-1,6-bisphosphate to glyceraldehyde-3-phosphate (G3P) and dihydroacetone phosphate (DHAP). There are three isoenzymes in the human body, called aldolase 1, 2, and 3, which are expressed at different levels in specific tissues during development. The aldolase protein family, including the three isoenzymes of aldolase A, B, and C, are expressed at specific levels in different tissues of the human body and jointly regulate the smooth progress of the glycolysis pathway. Aldolase A (ALDOA), as one of the key enzymes in the glycolysis pathway, is responsible for catalyzing the cleavage of fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This reaction is essential for maintaining cellular energy metabolism balance. Previous high-sensitivity post-translational modification histoneomics experiments have shown that the post-translational level of aldolase A at position 330 is higher in tumor tissues of lung cancer patients than in adjacent tissues. Although aldolase A is most abundantly expressed in adult muscle, it is widely distributed in a variety of tissues and upregulated in a variety of tumor types, suggesting that aldolase A may be involved in the process of tumor metabolic reprogramming and play a key role in the growth and survival of tumor cells.

[0005] It is worth noting that although the important role of aldolase A in tumors has been gradually recognized, research on its post-translational modification mechanism in regulating tumor cell growth is still relatively scarce. Summary of the invention

[0006] In view of the fact that no important post-translational modified amino acids on aldolase A that regulate the growth of tumor cells have been found so far, the main purpose of the present invention is to provide an application of post-translational protein modification of the amino acid at position 330 of aldolase A protein to inhibit tumor growth and a protein mutant with post-translational modification of lysine at position 330 of aldolase A protein, which not only achieves the purpose of successfully preparing the protein mutant with post-translational modification of lysine at position 330 of aldolase A protein for the first time, but also achieves the purpose of proposing a new method for the diagnosis and treatment of future diseases.

[0007] Our research team discovered that lysine 330 of aldolase A (ALDOA) ​​is a highly promising new tumor therapeutic target, which is of great significance for tumor treatment. By using highly sensitive post-translational modification group quantitative proteomics technology (SCASP-PTM technology), we observed that the post-translational modification level of lysine 330 of aldolase A in tumor tissues of lung cancer patients was significantly higher than that in the surrounding non-cancerous tissues. This discovery provides new clues to the occurrence and development mechanism of tumors.

[0008] In order to achieve the above object, the technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides an aldolase A protein mutant, wherein the mutant is obtained by substitution of the amino acid sequence shown in SEQ ID No. 1; Lysine at position 330 was substituted with other amino acids.

[0009] Furthermore, the other amino acid is arginine.

[0010] Furthermore, the aldolase A protein has a GenBank accession number of: Ensembl: ENSG00000149925.

[0011] The aldolase A protein is UniProtKB-P04075, which is SEQ ID No. The amino acid sequence shown in .1 is specifically: MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYA SICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFVSNHAY.

[0012] Furthermore, the amino acid sequence of the aldolase A protein mutant is the amino acid sequence shown in SEQ ID No. 2. Specifically: MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNG IVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVRRALANSLACQGKYTPSGQAGAAASESLFVSNHAY.

[0013] Furthermore, the aldolase A protein mutant has more than 80% homology at sites other than the mutation position in the amino acid sequence shown in SEQ ID No. 2.

[0014] Furthermore, the aldolase A protein mutant undergoes substitution, deletion, insertion and / or addition of at least one amino acid at a site other than the mutation position in the amino acid sequence shown in SEQ ID No. 2.

[0015] Furthermore, the aldolase A protein mutant is further connected to a transcriptional regulatory factor protein or domain.

[0016] Furthermore, the transcription regulatory factor is selected from a transcription activator, a transcription silencer or a transcription repressor.

[0017] In a second aspect, the present invention provides a cell comprising a polynucleotide encoding the amino acid sequence of the aldolase A protein mutant as described in the first aspect.

[0018] The cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; fungal cells such as yeast; CHO, etc. Transformation of host cells with a recombinant expression vector can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with methods such as CaCl2, and the steps used are well known in the art. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. The embodiment of the present invention specifically constructs a wild-type 293T cell line and a K330R mutant 293T cell line, and constructs K330R knock-in A375, A549, 4T1 and B16F10 cells.

[0019] The polynucleotide encodes the amino acid sequence of the aldolase A protein mutant as described in the first aspect.

[0020] Further, the polynucleotide is in an expression vector. Methods well known to those skilled in the art can be used to construct a recombinant expression vector containing the coding sequence of the arginine deiminase mutant and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis. The recombinant expression vector can be a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vector well known in the art. In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells. The embodiment of the present invention specifically constructs 3 Flag-Aldolase A and K330R 3 Flag-Aldolase A plasmid.

[0021] In a third aspect, the present invention provides a method for preparing the aldolase A protein mutant, the method comprising: 1) transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide described in the second aspect; 2) Host cells cultured in an appropriate culture medium; 3) Isolation and purification of proteins from culture medium or cells.

[0022] In a fourth aspect, the present invention provides a use of a drug containing a targeted aldolase A protein mutant in the preparation of an anti-tumor drug. Further, the use may include: use in immune sensitization target drugs, use in anti-tumor active ingredients; use in anti-tumor inhibition.

[0023] Furthermore, the drug targeting the aldolase A protein mutant is an ALDOA K330 site inhibitor.

[0024] Further, the anti-tumor drug used for the anti-tumor active ingredient refers to a tumor prevention and treatment drug used to regulate the ubiquitination and acetylation modification function of the ALDOA K330 site, and / or downregulate the expression level of ubiquitination and acetylation modification of the ALDOA K330 site, and / or reduce the ubiquitination and acetylation of the ALDOA K330 site. Further preferred is a drug that can target and regulate the ubiquitination and acetylation of the ALDOA K330 site to treat tumors. In certain embodiments, specifically: drug treatment targeting the aldolase A protein mutant reduces the ubiquitination and acetylation level of the ALDOA K330 site, thereby inhibiting tumor cell growth. In certain embodiments, the ALDOA K330 site ubiquitination and acetylation modification is highly expressed in human lung cancer tissue samples.

[0025] Furthermore, the anti-tumor drug is used for immune sensitization target drugs, including using the aldolase A protein mutant (ALDOA K330 site inhibitor) as an anti-tumor active ingredient, or using the ALDOA K330 site inhibitor in the preparation of anti-tumor drugs.

[0026] Furthermore, the application of the anti-tumor active ingredient refers to a tumor prevention and treatment drug for regulating the ubiquitination and acetylation modification function of the ALDOA K330 site, and / or downregulating the expression level of ubiquitination and acetylation modification of the ALDOA K330 site, and / or reducing the ubiquitination and acetylation of the ALDOA K330 site. Further preferred is a drug that can target and regulate the ubiquitination and acetylation of the ALDOA K330 site to treat tumors; specifically: drug treatment reduces the ubiquitination and acetylation level of the ALDOA K330 site, thereby inhibiting tumor cell growth.

[0027] Furthermore, the application for anti-tumor inhibition includes the application of the ALDOA K330 site ubiquitination and acetylation modification detection reagent to tumor screening / prognosis judgment reagents, or the application of the preparation of efficacy / prognosis judgment kits.

[0028] Furthermore, the tumor is selected from lung cancer, liver cancer, intestinal cancer, gastric cancer, breast cancer, malignant melanoma, nasopharyngeal cancer, ovarian cancer, prostate cancer, basal cell carcinoma, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, kidney cancer, etc.

[0029] Furthermore, the anti-tumor drugs further include alkylating agents (such as nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide and glyphosate mustard, etc.), antimetabolites (such as nucleotide analogs such as deoxyflurane, doxiflurane, fluorouracil, mercaptopurine, methotrexate, etc.), anti-tumor antibiotics (such as antibiotics such as actinomycin D, doxorubicin and daunorubicin), anti-tumor animal and plant component drugs (such as vinorelbine, paclitaxel, harringtonine, irinotecan, taxotere and vinblastine, etc.), anti-tumor hormone drugs (such as atamestane, anastrozole, aminoglutethimide, letrozole, formestane and tamoxifen, etc.) and commonly used chemotherapy drugs such as cisplatin, dacarbazine, oxaliplatin, oxadiazine, coplatin, mitoxantrone and procarbazine.

[0030] Further, the anti-tumor drug further includes targeted drugs. Targeted drugs include EGFR blockers such as gefitinib (Gefitinib, Iressa and Iressa) and erlotinib (Erlotinib, Tarceva), monoclonal antibodies for specific cell markers such as cetuximab (Cetuximab, Erbitux) and anti-HER-2 monoclonal antibodies (Herceptin, Trastuzumab, Herceptin), tyrosine kinase receptor inhibitors such as crizotinib (Crizotinib, Xalkori), anti-tumor angiogenesis drugs such as Bevacizumab, endostatin and Bevacizumab, Bcr-Abl tyrosine kinase inhibitors such as Imatinib and Dasatinib, anti-CD20 monoclonal antibodies such as Rituximab, IGFR-1 kinase inhibitors such as NVP-AEW541, mTOR kinase inhibitors such as CCI-779, ubiquitin-proteasome inhibitors such as Bortezomib, etc.

[0031] Furthermore, the anti-tumor drug is further combined with other tumor treatment methods, and the other tumor treatment methods can be selected from surgical resection, radiofrequency ablation, argon-helium superconducting surgical treatment, laser ablation treatment, high-intensity focused ultrasound and radiotherapy including one or more of X-knife, R-knife, 3D-CRT and IMRT.

[0032] In a fifth aspect, the present invention provides an application of a drug targeting an aldolase A protein mutant in the preparation of an anti-tumor therapeutic agent, wherein the drug targeting an aldolase A protein mutant is a drug targeting ubiquitination and acetylation modification of the ALDOA K330 site.

[0033] Furthermore, the application of the tumor therapeutic agent includes using the ALDOA K330 site ubiquitination and acetylation inhibitor as a component with tumor therapeutic function, or combining the ALDOA K330 ubiquitination and acetylation inhibitor with an immunotherapy drug for anti-tumor application.

[0034] The function of the tumor therapeutic agent is to target the ubiquitination and acetylation of the ALDOA K330 site to slow down the cell proliferation rate, thereby inhibiting tumor growth.

[0035] The application of the tumor therapeutic agent includes the application of the ALDOA K330 ubiquitination and acetylation post-translational modification content detection reagent to tumor screening / prognosis judgment.

[0036] The present invention has the following advantages: The present invention provides an innovative treatment strategy. The lysine at position 330 of aldolase A is mutated to arginine through the Prime-Editing technology. This gene mutation can significantly reduce the growth rate of mouse malignant breast cancer cells 4T1 and mouse malignant melanoma cells B16-F10 under the mouse skin, showing excellent anti-tumor activity. Experiments show that lysine at position 330 of aldolase A is a highly potential tumor therapeutic agent. It has created a new tumor treatment method and filled the technical gap in this field.

[0037] The invention has a significant anti-tumor effect. Experiments have shown that the gene mutation can significantly reduce the growth rate of mouse malignant breast cancer cells 4T1 and malignant melanoma cells B16-F10 in mice, showing excellent anti-tumor activity.

[0038] The protein mutant of the present invention is a potential tumor immunosensitizer. Lysine 330 of aldolase A is found to have the potential to enhance tumor immune response and can be used as an effective tumor immunosensitizer to improve the effect of immunotherapy.

[0039] The present invention utilizes Prime-Editing technology to achieve high-precision editing of target gene sites, reduce off-target risks, and improve the safety and effectiveness of treatment.

[0040] The present invention has broad application prospects and is expected to be applied to the treatment of various types of malignant tumors.

[0041] The present invention can reduce the impact on normal cells through precise gene editing, reduce the side effects of traditional treatment methods, and improve the quality of life of patients.

[0042] This application reveals for the first time that lysine 330 of aldolase A is a key target that affects tumor growth. This discovery not only deepens our understanding of the tumor metabolic regulation mechanism, but also provides a theoretical basis for the development of targeted therapies for aldolase A. In the future, drug design and clinical trials around this target are expected to bring revolutionary progress to the field of tumor treatment, provide patients with safer and more efficient treatment options, and further improve the effectiveness and survival rate of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0044] Figure 1 The mass spectrometry analysis diagram of the intensity of ALDOA K330 ubiquitinated peptide and acetylated peptide in lung cancer tissue and its corresponding adjacent tissue of the patient of the present invention; Figure 2 The lysine at position 330 of the wild-type aldolase A protein expressed and purified by 293T of the present invention is ubiquitinated; Figure 3 The lysine at position 330 of the wild-type aldolase A protein expressed and purified in 293T of the present invention is acetylated; Figure 4 The in vitro enzyme activity assay of the wild-type and K330R aldolase A proteins expressed and purified by 293T of the present invention; Figure 5 The genome sequencing results of the aldolase A K330R knock-in human melanoma cell A375 and lung cancer cell A549 of the present invention; Figure 6 The mass spectrometry analysis diagram for the identification of ubiquitination and acetylation modification of aldolase A K330 in A375 cells of the present invention; Figure 7 It is an enzyme activity curve diagram comparing the aldolase A activity in the wild-type A375 and the aldolase A K330R knock-in A375 cells of the present invention; Figure 8 It is an enzyme activity curve diagram comparing the aldolase A activity in the wild-type A549 and the aldolase A K330R knock-in A549 cells of the present invention; Fig. 9 This is a graph showing the proliferation rate of wild-type A375 and aldolase A K330R knock-in A375 cells of the present invention; Fig.10This is a graph of the migration speed of wild-type A375 and aldolase A K330R knock-in A375 cells of the present invention; Fig.11 This is a graph showing the proliferation rate of wild-type A549 and aldolase A K330R knock-in A549 cells of the present invention; Fig.12 This is a graph of the migration speed of wild-type A549 and aldolase A K330R knock-in A549 cells of the present invention; Fig.13 The genome sequencing results of the aldolase A K330R knock-in mouse breast cancer cell 4T1 of the present invention; Fig.14 The genome sequencing results of the aldolase A K330R knock-in mouse melanoma cell B16F10 of the present invention; Fig.15 This is a tumor size measurement diagram of the 4T1 subcutaneous tumor of the present invention; Fig.16 This is a diagram showing the measurement of tumor size of the B16F10 subcutaneous tumor of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0046] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0047] The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0048] The above-mentioned term "aldolase A protein mutant" also includes variant forms of sequences having the same functions as the aldolase A protein mutant. These variant forms include but are not limited to: the deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and more preferably 1-8, 1-5) amino acids, and the addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when replacing with amino acids with similar or similar properties, the function of the protein is usually not changed. For another example, the addition or deletion of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of aldolase A protein mutants.

[0049] The above term "aldolase A protein mutant" also includes proteins having a homology of 80% or more to the polypeptide sequence defined by the above GenBank accession number; more preferably a homology of 85% or more, such as 90%, 95%, 98% or 99% homology) and having the same function as the aldolase A protein mutant involved in the embodiments of the present invention are also included in the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids or polypeptides based on the percentage of identical positions.

[0050] Pharmaceutical composition The present invention also provides a pharmaceutical composition, which contains an effective amount (such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably 0.0001-10wt%) of the aldolase A protein mutant or an inhibitor of its encoding gene, and a pharmaceutically acceptable carrier.

[0051] As a preferred embodiment of the present invention, a composition for inhibiting tumors is provided, the composition containing an effective amount of an inhibitor of an aldolase A protein mutant or a gene encoding the same, and a pharmaceutically acceptable carrier. Preferably, the inhibitor is a gene editing agent that specifically knocks out the gene encoding the aldolase A protein mutant, the gene editing agent recognizes the aldolase A protein mutant or a gene encoding the same and knocks out the gene, or can express or form a construct of the gene editing agent.

[0052] As used herein, the "effective amount" refers to an amount that can produce functions or activities in humans and / or animals and can be accepted by humans and / or animals. The "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers that are not necessary active ingredients in themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the composition may contain a liquid, such as water, saline, or a buffer. In addition, these carriers may also contain auxiliary substances, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc. The carrier may also contain a cell transfection reagent.

[0053] After knowing the use of the inhibitor of the aldolase A protein mutant or its encoding gene, various methods well known in the art can be used to administer the down-regulator or its encoding gene, or its pharmaceutical composition to mammals or humans.

[0054] Preferably, gene therapy can be used. For example, the aldolase A protein mutant or the inhibitor of its encoding gene can be directly administered to the subject by methods such as injection; or, the expression unit (such as expression vector or virus, etc., or siRNA) carrying the aldolase A protein mutant or the inhibitor of its encoding gene can be delivered to the target site through a certain route, and the target site can express the active aldolase A protein mutant or the inhibitor of its encoding gene. The specific situation depends on the type of the inhibitor, which is well known to those skilled in the art.

[0055] The effective amount of the inhibitor of the aldolase A protein mutant or its encoding gene of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the inhibitor of the aldolase A protein mutant or its encoding gene, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc.

[0056] Materials and methods are as follows: Commonly used drugs and reagents: Most of the drugs and reagents used in this description were purchased from Sigma, Shanghai Sangon Bioengineering Co., Ltd., and MedChemExprss (MCE). Antibodies used for Western Blot analysis were purchased from Sigma, CellSignaling (CST), and Proteintech.

[0057] Example 1 High-sensitivity quantitative mass spectrometry analysis of ubiquitination and acetylation levels in lung cancer clinical samples and adjacent normal tissues of lung cancer (1) Experimental process First, clinical samples and adjacent normal tissues were collected from lung cancer patients as the material basis for the control experiment. To ensure the accuracy of the experimental data, a unified homogenization process was implemented for these two types of tissue samples, aiming to destroy the cell structure and release all the proteins in the cells.

[0058] Next, trypsin was selected as the main enzymatic hydrolysis reagent to cut proteins in normal tissues and cancer tissues, and the proteases were hydrolyzed into peptides. To ensure the efficiency and specificity of the enzymatic hydrolysis process, strict control conditions were set, including precise control of protein concentration, enzymatic hydrolysis temperature, pH value, and enzyme-substrate ratio. The implementation of this series of control measures is aimed at minimizing experimental errors and ensuring the consistency of the quantity and quality of peptides extracted from normal cells and cancer cells.

[0059] Subsequently, immunoaffinity chromatography is used to enrich ubiquitinated peptides and acetylated peptides, respectively. For the enrichment of ubiquitinated peptides, an antibody that specifically recognizes double alanine on lysine is used to form an antibody-peptide complex through immunoaffinity, and the unbound peptides are washed away using a chromatography column. Finally, the ubiquitinated peptides are eluted from the antibody using appropriate elution conditions and collected. Similarly, the enrichment of acetylated peptides uses acetylation-specific antibodies or chemical groups with affinity for acetylated groups, which are effectively enriched through affinity chromatography columns and eluted for subsequent mass spectrometry analysis.

[0060] (2) Data analysis Based on the quantitative evaluation of the peak intensity of modified peptides, the relative contents of all ubiquitination and acetylation modifications in lung cancer tissues and paracancerous tissues were compared. First, the mass information and sequence characteristics of a large number of ubiquitinated and acetylated peptides in these tissues were successfully obtained using advanced mass spectrometry technology. Subsequently, the peak intensity of the corresponding modified peptides in lung cancer tissues and paracancerous tissues was compared using DIA-NN software for in-depth quantitative comparison. The difference in peak intensity directly reflects the relative abundance of modified peptides in the two tissues, thereby revealing the difference in the degree of ubiquitination and acetylation modification at the K330 position of aldolase A protein between lung cancer tissues and paracancerous tissues.

[0061] (3) Experimental results The present invention uses high-sensitivity quantitative mass spectrometry to detect a large number of acetylation and ubiquitination modification levels in lung cancer tissues and adjacent tissues, and finds that the acetylation and ubiquitination levels of ALDOA K330 are higher in lung cancer tissues than in adjacent tissues of lung cancer. We detected a total of 6 acetylation sites and 5 ubiquitination sites on ALDOA, of which 5 acetylation sites and 4 ubiquitination sites did not change in tumor and adjacent tissues, and only the acetylation and ubiquitination levels at the K330 position were higher than those in adjacent tissues of lung cancer, such as Figure 1 shown.

[0062] Example 2 (1) Construction of plasmid First, the DNA sequence of the aldolase A gene and its K330R mutant is extracted from the organism by gene cloning technology, and amplified and purified by PCR and other technologies. Then, a suitable plasmid vector is selected, which must have the ability to replicate autonomously in the host cell, a selectable marker gene, and a cloning site for inserting foreign DNA. Next, the DNA sequence of the aldolase A gene or its K330R mutant is cloned and cloned into the 3 The Flag tag sequence was fused to form a fusion gene, which was inserted into the cloning site of the plasmid vector to construct a 3 Flag-Aldolase A and K330R 3 Flag-Aldolase A plasmid.

[0063] (2) Construction of K330R mutant cell line When 293T cells reached the logarithmic growth phase, the expression vectors encoding the wild-type and K330R mutant aldolase A genes were introduced into the cells using liposome-mediated transfection technology to construct wild-type 293T cell lines and K330R mutant 293T cell lines. Flag-Aldolase A and K330R 3 The plasmid of Flag-aldolase A was transfected into the recipient cells, inducing the cells to express 3 Flag-tagged wild-type aldolase A protein and K330R mutant aldolase A protein. Subsequently, anti-Flag-coupled agarose beads were added to the cell lysate using the specific binding principle between the Flag tag and the anti-Flag antibody, and the target protein was tightly bound to the beads through affinity. Afterwards, protein purification techniques such as affinity chromatography were used to separate and obtain highly pure wild-type aldolase A protein and K330R mutant aldolase A protein.

[0064] (3) Analysis of wild-type and K330R mutant aldolase A protease activity At room temperature, fructose diphosphate was used as a specific substrate to incubate with wild-type and K330R mutant aldolase A proteins. In order to accurately determine the activity of aldolase A, a reaction system and conditions that match the in vitro enzyme activity assessment were used to measure the activity of purified wild-type ALDOA and purified K330R mutant ALDOA. During the measurement, a UV-visible spectrophotometer was used to measure the OD at 5-minute intervals. 240nm The absorbance was measured at 100 nm. Subsequently, the collected data were statistically analyzed and an enzyme activity curve was plotted with time as the horizontal axis and OD as the horizontal axis. 240nm The absorbance value at the wavelength is used as the ordinate. Through this graph, the activity differences of aldolase A in six different cells can be intuitively evaluated and analyzed.

[0065] (4) Experimental results In 293T, aldolase A has ubiquitination and acetylation modifications at position K330, such as Figure 2 and Figure 3 Ubiquitination and acetylation of K330 inhibit enzyme activity in vitro, as shown in Figure 4 shown.

[0066] Example 3 Construction of K330R knock-in A375, A549, 4T1 and B16F10 cells (1) Experimental process First, PE-GFP and K330R-pegRNA plasmids were mixed, and then the mixed plasmids were introduced into wild-type A375, A549, 4T1 and B16F10 cell lines by electroporation technology to prepare mutant cell lines. After the electroporation operation was completed, the cells were placed in an incubator and cultured for 48 hours. After the end, the cells were sorted by green fluorescence and red fluorescence by flow cytometry to screen out the cell population that was successfully transfected and expressed PE-GFP. Next, monoclonal cells were isolated and cultured from the sorted cells to obtain purified monoclonal cell lines, and cell samples were collected and their genomic DNA was extracted. Next, PCR amplification was performed using specific primers for the K330 position of the aldolase A gene to obtain a DNA fragment containing the K330 position. The PCR amplification product was sequenced and analyzed, and the sequencing results were compared with the expected K330R sequence to obtain the complete sequence information of the DNA molecule.

[0067] (2) Experimental results We successfully obtained ALDOA K330R knock-in A375, A549, 4T1 and B16F10 cells, and the genome sequencing results are as follows Figure 5 , Fig.13 and Fig.14 shown.

[0068] Example 4 Proliferation of WT and K330R knock-in A375 and A549 cells In order to compare the proliferation rates of wild-type and K330R mutant A375 and A549 cells, these cells were first evenly spread into 96-well plates to ensure that the number of cells in each well was consistent. Subsequently, an appropriate amount of CCK8 reagent was added to each well. This reagent can react with the dehydrogenase in living cells to generate a colored formazan product, the amount of which is proportional to the number of living cells. Next, the 96-well plate was placed in a 37°C incubation for one hour to allow for sufficient reaction. Afterwards, the OD absorbance of each well was measured at a wavelength of 450nm using a microplate reader. This value reflects the number of living cells in each well. Repeat the above steps for several consecutive days and record the OD at each time point. 450nm The absorption values ​​of the wild-type and K330R mutant A375 and A549 cells can be plotted, where the horizontal axis represents the time point from the beginning to the end of the experiment, and the vertical axis represents the cell proliferation. This allows for an intuitive comparison of the proliferation capabilities of different cell lines.

[0069] Experimental results: K330R knock-in A375 and A549 cells were established by Prime Editing, and then cell proliferation experiments showed that K330R knock-in cells showed a significant slowing of growth, such as Fig. 9 and Fig.11 shown.

[0070] Example 5 Comparison of migration speed of WT and K330R knock-in A375 and A549 cells To compare the migration speed of wild-type and K330R mutant A375 and A549 cells, the two types of cells were first evenly spread into 96-well plates to ensure that the number of cells in each well was consistent. Subsequently, a sterile pipette tip was used to accurately draw a straight line in the middle of the cell layer to simulate the starting point of cell migration and create a cell-free gap area. Next, the well plate was placed in a cell culture incubator and cultured under standard culture conditions. Every 12 hours, the cells were photographed using a microscope, with particular attention paid to the cell gap area previously formed by the pipette tip. By comparing the photos taken at different time points, the migration of cells from the edge of the gap to the center can be observed, and the change in the width of the gap area in the photos at each time point can be measured and recorded. Using time as the abscissa or the distance of cell migration as the ordinate, the migration speed of wild-type and K330R mutant A375 and A549 cells was plotted. In this way, by comparing the migration curves of different cell lines, their migration ability can be clearly evaluated.

[0071] Experimental results: K330R knock-in A375 and A549 cells were established by Prime Editing, and then cell migration experiments showed that K330R knock-in cells showed a significant slowing of migration. K330R knock-in cells showed a significant slowing of migration, specifically, Fig.10 and Fig.12 As shown, WT cells migrated to the intercellular space faster than K330R cells.

[0072] Example 6 Subcutaneous tumor implantation of WT and K330R knock-in 4T1 and B16F10 cells In order to compare the growth rate of wild-type and K330R mutant 4T1 and B16F10 cells in mice, the two types of cells were first suspended in an appropriate amount of saline and implanted into mice of the corresponding strains by subcutaneous injection. The concentration of the cell suspension should be consistent during injection, and the injection volume of each mouse should be the same to ensure the uniformity of the experiment. Starting from the 10th day after cell implantation, the size of the subcutaneous tumor of the mouse was measured every 2 days using a vernier caliper. When measuring, the long diameter (L) and short diameter (W) of the tumor should be accurately recorded, and the volume of the tumor should be calculated according to the formula V = (L× W^2) / 2, where V represents the tumor volume. This step needs to be continued until the end of the experiment or the tumor reaches the predetermined volume limit. In order to intuitively display the growth rate of the tumor, the measured data were sorted, and the volume change diagram of the tumor formed by wild-type and K330R knock-in 4T1 and B16F10 cells in mice was drawn with time as the horizontal axis and tumor volume as the vertical axis. By comparing the tumor growth curves formed by different cell lines, their growth rates can be clearly assessed.

[0073] Experimental results: The growth rate of aldolase A K330R knock-in 4T1 and B16F10 in mice was significantly reduced compared with wild-type 4T1 and B16F10. Fig.15 and Fig.16 As shown, the average volume of WT 4T1 and B16F10 cells reached 1 cubic centimeter around 25 days and 16 days, while that of K330R 4T1 and B16F10 was less than 200 cubic millimeters.

[0074] The present invention embodiment verifies the potential of mutating the lysine at the K330 position of the aldolase A protein to arginine in tumor treatment through high-sensitivity quantitative mass spectrometry, in vitro enzyme activity determination, cell proliferation and migration experiments, and in vivo tumor implantation experiments in mice. The experimental results show that the ubiquitination and acetylation modifications at the K330 position of the aldolase A protein significantly inhibit the enzymatic activity of the aldolase A and cause the cell growth and migration speed to slow down. Therefore, the K330 position of the aldolase A protein is expected to become a new target for tumor treatment, providing new ideas and methods for tumor treatment.

[0075] The above content is only an explanation of the preferred embodiments of the present invention, which is intended to demonstrate its core concepts and application methods, but is not intended to limit the wide applicability of the present invention. Under the premise of following the basic spirit and core principles of the present invention, any form of modification, equivalent alternative, technical improvement or innovation shall be deemed to fall within the scope of protection claimed by the present invention.

Claims

1. An aldolase A protein mutant, characterized in that: The mutant is obtained by substitution of the amino acid sequence shown in SEQ ID No. 1; The lysine at position 330 was substituted with other amino acids; Furthermore, the other amino acid is arginine.

2. The aldolase A protein mutant according to claim 1, characterized in that The amino acid sequence of the aldolase A protein mutant is the amino acid sequence shown in SEQ ID No.

2.

3. The aldolase A protein mutant according to claim 1, characterized in that: The aldolase A protein mutant has more than 80% homology at sites other than the mutation position of the amino acid sequence shown in SEQ ID No. 2; or, the aldolase A protein mutant replaces, deletes, inserts and / or adds at least one amino acid at sites other than the mutation position of the amino acid sequence shown in SEQ ID No.

2.

4. A cell, characterized in that The cell comprises a polynucleotide encoding the amino acid sequence of the aldolase A protein mutant according to any one of claims 1 to 3.

5. A method for preparing the aldolase A protein mutant according to any one of claims 1 to 3, characterized in that: The method comprises: 1) transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide according to claim 4; 2) Host cells cultured in an appropriate culture medium; 3) Isolation and purification of proteins from culture medium or cells.

6. Use of a drug targeting an aldolase A protein mutant as claimed in any one of claims 1 to 3 in the preparation of an anti-tumor drug.

7. The use according to claim 6, characterized in that: The drug targeting the aldolase A protein mutant is an ALDOAK330 site inhibitor; a drug used for immunosensitization target, an anti-tumor active ingredient; and / or an anti-tumor inhibitor.

8. The use according to claim 7, characterized in that: The anti-tumor drug used for the anti-tumor active ingredient refers to a tumor prevention and treatment drug used to regulate the ubiquitination and acetylation modification function of the ALDOA K330 site, and / or downregulate the expression level of ubiquitination and acetylation modification of the ALDOA K330 site, and / or reduce the ubiquitination and acetylation of the ALDOA K330 site; Furthermore, the anti-tumor drug is used for immune sensitization target drugs, including the use of aldolase A protein mutants as anti-tumor active ingredients, or the use of ALDOA K330 site inhibitors in the preparation of anti-tumor drugs; Furthermore, the application of the anti-tumor inhibitor includes the application of the ALDOA K330 site ubiquitination and acetylation modification detection reagent to tumor screening / prognosis judgment reagents, or the application of the preparation of efficacy / prognosis judgment kits.

9. The use according to claim 6, characterized in that: The tumor is selected from lung cancer, liver cancer, intestinal cancer, gastric cancer, breast cancer, malignant melanoma, nasopharyngeal cancer, ovarian cancer, prostate cancer, basal cell carcinoma, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer or kidney cancer.

10. Use of a drug targeting an aldolase A protein mutant as claimed in any one of claims 1 to 3 in the preparation of an anti-tumor therapeutic agent, wherein the drug targeting an aldolase A protein mutant is a drug targeting ubiquitination and acetylation modification of the ALDOA K330 site; Furthermore, the application of the tumor therapeutic agent includes using the ALDOA K330 site ubiquitination and acetylation inhibitor as a component of tumor therapeutic function, or combining the ALDOA K330 ubiquitination and acetylation inhibitor with an immunotherapy drug for anti-tumor application; Furthermore, the function of the tumor therapeutic agent refers to targeting ubiquitination and acetylation of the ALDOA K330 site to slow down cell proliferation, thereby inhibiting tumor growth.

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