Post-translational protein modification of amino acid 330 of aldolase a protein and its mutant for anti-tumor application

Abstract

The present application relates to the field of biotechnology, in particular to an application of post-translational protein modification of aldehyde dehydrogenase A protein 330 amino acids and mutant. The aldehyde dehydrogenase A protein mutant is obtained by substitution of the amino acid sequence shown in SEQ ID No. 1, and the lysine at position 330 is substituted by arginine. The present application proves by experiment that the aldehyde dehydrogenase A protein mutant can significantly reduce the growth rate of mouse malignant breast cancer cells 4T1 and mouse malignant melanoma cells B16-F10 in the mouse subcutaneous, and exhibits excellent anti-tumor activity. A new tumor treatment method is created, and the technical gap in the field is filled.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and relates to an application of post-translational protein modification of an aldehyde dehydrogenase A protein at position 330 of amino acids and a mutant, in particular to an aldehyde dehydrogenase A protein mutant and an application thereof in preparing an anti-tumor drug, and specifically to an effect of a mutant obtained after substitution of lysine at position 330 of an aldehyde dehydrogenase A protein on growth of a tumor. BACKGROUND

[0002] Tumor is a disease that seriously threatens human health, continuously threatens the life quality and life expectancy of human beings, and has ranked in the front row of causes of death. In the face of this severe situation, the medical field continues to explore and innovate, and strives to break through the limitations of traditional treatment methods, so as to bring more effective treatment programs for patients. At present, the treatment strategies for tumors cover surgical resection, radiotherapy, chemotherapy, and emerging immunotherapy and other multiple approaches. However, these therapies, although to some extent, prolong the survival period of patients, inevitably also have 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, which have injected new vitality into the field of tumor treatment. Among them, gene therapy directly corrects or replaces the pathogenic gene to achieve treatment at the root of the disease; immunotherapy utilizes the patient's own immune system to stimulate its recognition and elimination ability of tumor cells; targeted therapy focuses on specific tumor-related molecules or signaling pathways, and precisely attacks 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 which is the shift from oxidative phosphorylation to aerobic glycolysis, the Warburg effect. This process not only meets the energy needs of the rapid proliferation of tumor cells, but also promotes their ability to adapt to a hypoxic microenvironment. The metabolic reprogramming that occurs in tumor cells involves a shift from ATP production primarily through oxidative phosphorylation to aerobic glycolysis, which is known as the Warburg effect. Human aldolase proteins are essential for glycolysis, catalyzing the reversible conversion of 1,6-fructose diphosphate to 3-phosphoglyceraldehyde (G3P) and dihydroxyacetone phosphate (DHAP). There are three isozymes in the human body, known as aldolase 1, 2 and 3, which are expressed at different levels in specific tissues during development. The aldolase protein family, including aldolase A, B and C, is expressed at specific levels in different tissues of the human body, and collectively regulates 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-diphosphate into glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), which is essential for maintaining cellular energy metabolism balance. Previous high-sensitivity post-translational modification proteome experiments have shown that the post-translational level of aldolase A at position 330 is higher in lung cancer patient tumor tissues than in adjacent tissues. Although aldolase A is most abundantly expressed in adult muscle, it is widely distributed in various tissues and its expression is up-regulated in various tumor types, suggesting that aldolase A may be involved in the process of metabolic reprogramming of tumor cells and plays a key role in the growth and survival of tumor cells.

[0005] Notably, although the important role of aldolase A in tumors has been gradually recognized, research on the post-translational modification mechanism of aldolase A in regulating tumor cell growth is still relatively scarce. SUMMARY

[0006] In view of the fact that no important post-translational modification amino acid of aldolase A that regulates tumor cell growth has been found, the main purpose of the present application is to provide an application of post-translational protein modification of amino acid at position 330 of aldolase A protein for inhibiting tumor growth and a protein mutant of post-translational modification of lysine at position 330 of aldolase A protein, which not only achieves the purpose of successfully preparing the protein mutant of post-translational modification of lysine at position 330 of aldolase A protein for the first time, but also achieves the purpose of providing a new method for the diagnosis and treatment of diseases in the future.

[0007] Our research team found that lysine 330 of aldehyde dehydrogenase A (ALDOA) is a potential new tumor treatment target, which is of great significance for treating tumors. By using high-sensitivity post-translational modification group quantitative proteomics technology (SCASP-PTM technology), we observed that the post-translational modification level of lysine 330 of aldehyde dehydrogenase A in the tumor tissue of lung cancer patients was significantly higher than that in the surrounding non-cancerous tissue, which provided a new clue for the mechanism of tumor occurrence and development.

[0008] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0009] In a first aspect, the present application provides an aldehyde dehydrogenase A protein mutant, which is obtained by substitution of the amino acid sequence shown in SEQ ID No. 1.

[0010] The lysine at position 330 is substituted with other amino acids.

[0011] Further, the other amino acid is arginine.

[0012] Further, the aldehyde dehydrogenase A protein GenBank accession number: Ensembl: ENSG00000149925.

[0013] The aldehyde dehydrogenase A protein is UniProtKB-P04075, which is the amino acid sequence shown in SEQ ID No. 1, specifically: MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFVSNHAY.

[0014] Further, the amino acid sequence of the aldehyde dehydrogenase A protein mutant is the amino acid sequence shown in SEQ ID No. 2. Specifically:

[0015] MPYQYPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFHETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSALAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACTQKFSHEEIAMATVTALRRTVPPAVTGITFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENLKAAQEEYVRRALANSLACQGKYTPSGQAGAAASESLFVSNHAY.

[0016] Further, the aldolase A protein mutant has 80% or more homology to the amino acid sequence shown in SEQ ID No. 2 except for the mutation position.

[0017] Further, the aldolase A protein mutant has at least one amino acid substituted, deleted, inserted and / or added to the amino acid sequence shown in SEQ ID No. 2 except for the mutation position.

[0018] Further, the aldolase A protein mutant is further linked to a transcriptional regulator protein or domain.

[0019] Further, the transcriptional regulator is selected from a transcriptional activator, a transcriptional silencer or a transcriptional repressor.

[0020] In a second aspect, the present application provides a cell comprising a polynucleotide encoding the amino acid sequence of the aldolase A protein mutant as described in the first aspect.

[0021] The cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples of useful host cells are E. coli, Streptomyces; fungal cells such as yeast; CHO cells, and the like. Transformation of a host cell with a recombinant expression vector can be performed using conventional techniques known to those skilled in the art. When the host is a prokaryote, such as E. coli, a suitable host cell can be made competent and harvested during the exponential phase of growth, treated with a reagent such as CaCl2, and the like, and the procedures used are well known in the art. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like, can be used. In the present embodiment, wild-type 293T cell lines and K330R mutant 293T cell lines are constructed, and K330R knock-in A375, A549, 4T1 and B16F10 cells are constructed.

[0022] The polynucleotide encodes the amino acid sequence of the aldolase A protein mutant as described in the first aspect.

[0023] 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 transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to a suitable promoter in the expression vector to direct mRNA synthesis. The recombinant expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. In the present embodiment, 3 Flag-aldolase A and K330R 3 Plasmid of Flag-aldolase A.

[0024] In a third aspect, the present application provides a method for preparing the aldolase A protein mutant, which comprises:

[0025] 1) transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide as described in the second aspect;

[0026] 2) culturing the host cell in a suitable medium;

[0027] 3) isolating and purifying the protein from the medium or the cells.

[0028] In a fourth aspect, the present application provides a use of a drug containing the mutant of the targeting aldolase A protein in the preparation of an anti-tumor drug. Further, the use can include: a use for an immune sensitization target drug, a use for an anti-tumor active ingredient; a use for an anti-tumor inhibitor.

[0029] Further, the drug of the mutant of the targeting aldolase A protein is an ALDOA K330 site inhibitor.

[0030] Further, the use of the anti-tumor drug for an 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 down-regulating the expression level of the 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 the ubiquitination and acetylation of the ALDOA K330 site to treat tumors; specifically, the drug treatment reduces the ubiquitination and acetylation level of the ALDOA K330 site to inhibit tumor cell growth. In some embodiments, the ubiquitination and acetylation modification of the ALDOA K330 site is highly expressed in human lung cancer tissue samples.

[0031] Further, the use of the anti-tumor drug for an immune sensitization target drug includes using the mutant of the aldolase A protein (ALDOA K330 site inhibitor) as an anti-tumor active ingredient, or using the ALDOA K330 site inhibitor in the preparation of an anti-tumor drug.

[0032] Further, the use 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 down-regulating the expression level of the 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 the ubiquitination and acetylation of the ALDOA K330 site to treat tumors; specifically, the drug treatment reduces the ubiquitination and acetylation level of the ALDOA K330 site to inhibit tumor cell growth.

[0033] Further, the use for an anti-tumor inhibitor includes using an ALDOA K330 site ubiquitination and acetylation modification detection reagent for tumor screening / prognosis determination reagent, or for the preparation of a therapeutic effect / prognosis determination kit.

[0034] Further, the tumor is selected from lung cancer, liver cancer, intestinal cancer, gastric cancer, breast cancer, malignant melanoma, nasopharyngeal carcinoma, ovarian cancer, prostate cancer, basal cell carcinoma, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, kidney cancer, etc.

[0035] Further, the anti-tumor drugs further include alkylating agents (such as nimustine, carmustine, lomustine, cyclophosphamide, ifosfamide, and chlorambucil, etc.), anti-metabolites (such as deoxyfluorouridine, doxifluridine, fluorouracil, mercaptopurine, methotrexate, and nucleotide analogs), anti-tumor antibiotics (such as actinomycin D, doxorubicin, and daunorubicin, etc.), anti-tumor plant and animal components (such as vinorelbine, paclitaxel, harringtonine, irinotecan, docetaxel, and vinblastine, etc.), anti-tumor hormones (such as atamestane, anastrozole, aminoglutethimide, letrozole, formestane, and tamoxifen, etc.), and common chemotherapy drugs (such as cisplatin, dacarbazine, oxaliplatin, leucovorin, carboplatin, oxaliplatin, mitoxantrone, and procarbazine, etc.).

[0036] Further, the anti-tumor drugs further include targeted drugs. The targeted drugs include EGFR blockers such as gefitinib (Iressa and Iressa), and erlotinib (Tarceva), monoclonal antibodies of specific cell markers such as cetuximab (Erbitux), and anti-HER-2 mAb (Herceptin), tyrosine kinase receptor inhibitors such as crizotinib (Xalkori), anti-tumor angiogenesis drugs such as bevacizumab, endostatin, and bevacizumab, etc., Bcr-Abl tyrosine kinase inhibitors such as imatinib and dasatinib, anti-CD20 mAb 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.

[0037] Further, the anti-tumor drugs are further combined with other tumor treatment methods for treatment, and the other tumor treatment methods can be selected from one or more of surgical resection, radiofrequency ablation, argon-helium cryosurgery, laser ablation therapy, high-intensity focused ultrasound, and radiotherapy including X-knife, R-knife, 3D-CRT, and IMRT.

[0038] In a fifth aspect, the present application provides a use of a drug targeting the mutant of aldolase A protein in the preparation of an anti-tumor therapeutic agent, wherein the drug targeting the mutant of aldolase A protein is a drug targeting the ubiquitination and acetylation modification of the K330 site of ALDOA.

[0039] Further, the application of the tumor therapeutic agent includes the application of the ALDOA K330 ubiquitination and acetylation inhibitor as a component of tumor treatment function, or the application of the ALDOA K330 ubiquitination and acetylation inhibitor in combination with an immunotherapy drug for anti-tumor application.

[0040] The tumor therapeutic agent function refers to the function of targeting ALDOA K330 ubiquitination and acetylation to slow down the cell proliferation rate, thereby inhibiting tumor growth.

[0041] 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.

[0042] The present application has the following advantages:

[0043] The present application provides an innovative treatment strategy, which mutates the lysine at position 330 of aldolase A to arginine by Prime-Editing technology, and the gene mutation can significantly reduce the growth rate of mouse malignant breast cancer cells 4T1 and mouse malignant melanoma cells B16-F10 in mice, and exhibits excellent anti-tumor activity. Experiments show that the lysine at position 330 of aldolase A is a potential tumor therapeutic agent. A new tumor treatment method is created, and the technical gap in this field is filled.

[0044] The present application has significant anti-tumor effect, and experiments show 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, and exhibits excellent anti-tumor activity.

[0045] The protein mutant of the present application is a potential tumor immunosensitizer, and the lysine at position 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.

[0046] The present application uses Prime-Editing technology to achieve high-precision editing of target gene sites, reduces off-target risk, and improves the safety and effectiveness of treatment.

[0047] The present application has wide application prospect, and is expected to be applied to the treatment of various types of malignant tumors.

[0048] The present application can reduce the influence on normal cells through precise gene editing, reduce the side effects brought by traditional treatment methods, and improve the quality of life of patients.

[0049] The application first discloses that lysine at position 330 of aldolase A is a key target affecting tumor growth, which not only deepens our understanding of the regulation mechanism of tumor metabolism, but also provides a theoretical basis for the development of targeted therapy 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, providing safer and more efficient treatment options for patients and further improving the effectiveness and survival rate of tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only one embodiment of the present application, and those skilled in the art can obtain other embodiment drawings according to the provided drawings without creating any inventive labor.

[0051] Figure 1 Mass spectrum analysis chart of ALDOA K330 ubiquitination peptide and acetylation peptide intensity in lung cancer tissue and its corresponding paracancerous tissue of patients in the present application;

[0052] Figure 2 Ubiquitination modification of lysine at position 330 of wild-type aldolase A protein expressed and purified in 293T in the present application;

[0053] Figure 3 Acetylation modification of lysine at position 330 of wild-type aldolase A protein expressed and purified in 293T in the present application;

[0054] Figure 4 In vitro enzyme activity determination of wild-type and K330R aldolase A protein expressed and purified in 293T in the present application;

[0055] Figure 5 Genome sequencing results of aldolase A K330R knock-in human melanoma cell A375 and lung cancer cell A549 in the present application;

[0056] Figure 6 Mass spectrum analysis chart of ubiquitination and acetylation modification of aldolase A K330 in A375 cells in the present application;

[0057] Figure 7 Enzyme activity curve of comparison of aldolase A activity in wild-type A375 and aldolase A K330R knock-in A375 cells in the present application; Figure 8 Enzyme activity curve of comparison of aldolase A activity in wild-type A549 and aldolase A K330R knock-in A549 cells in the present application;

[0058] Figure 9Figure for the proliferation rate of wild type A375 and A375 cells with aldolase A K330R knock-in according to the present application;

[0059] Figure 10 Figure for the migration rate of wild type A549 and A549 cells with aldolase A K330R knock-in according to the present application;

[0060] Figure 11 Figure for the proliferation rate of wild type A549 and A549 cells with aldolase A K330R knock-in according to the present application;

[0061] Figure 12 Figure for the migration rate of wild type A549 and A549 cells with aldolase A K330R knock-in according to the present application;

[0062] Figure 13 Figure for the genome sequencing result of 4T1 cells with aldolase A K330R knock-in according to the present application;

[0063] Figure 14 Figure for the genome sequencing result of B16F10 cells with aldolase A K330R knock-in according to the present application;

[0064] Figure 15 Figure for the tumor size measurement of 4T1 subcutaneous tumor according to the present application;

[0065] Figure 16 Figure for the tumor size measurement of B16F10 subcutaneous tumor according to the present application. DETAILED DESCRIPTION

[0066] The present application will be further described by the following embodiments in conjunction with the accompanying drawings. In the following embodiments, many details are described in order to provide a more thorough understanding of the present application. However, one skilled in the art will readily recognize that some of the features to which specific embodiments are directed are applicable and can be omitted or substituted for alternative features in other situations without departing from the scope of the present application. In some instances, some of the operations described in the specification have not been shown or described in order to avoid obscuring the core of the present application. It is not necessary to describe the related operations in detail in the specification, and one skilled in the art can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0067] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various embodiments. Meanwhile, the steps or actions in the method description can also be sequentially adjusted or adjusted in a manner that is apparent to one 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 do not mean that the sequence is necessary, unless otherwise stated that a certain sequence must be followed.

[0068] The terms "first", "second", etc. used herein for the serial numbers of components are only used to distinguish the described objects and do not have any sequential or technical meaning.

[0069] The term "aldolase A protein mutant" also includes variants of sequences having the same function as the aldolase A protein mutant. These variants include, but are not limited to, deletion, insertion and / or substitution of several (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, still more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or similar functional amino acids usually does not change the function of the protein. For another example, 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 the aldolase A protein mutant.

[0070] The term "aldolase A protein mutant" also includes proteins having 80% or more homology with the polypeptide sequence defined by the above GenBank accession number; more preferably 85% or more homology, such as 90%, 95%, 98% or 99% homology, and having the same function as the aldolase A protein mutant involved in the examples of the present application. Methods and tools for comparing sequence homology are also 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 as a percentage of positions that are identical.

[0071] Pharmaceutical composition

[0072] The present application also provides a pharmaceutical composition comprising 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 the gene encoding the same, and a pharmaceutically acceptable carrier.

[0073] As a preferred mode of the present application, there is provided a composition for inhibiting tumors, the composition comprising an effective amount of the aldolase A protein mutant or an inhibitor of the gene encoding the same, and a pharmaceutically acceptable carrier. Preferably, the inhibitor is a gene editing reagent that specifically knocks out the gene encoding the aldolase A protein mutant, which recognizes the aldolase A protein mutant or the gene encoding the same and knocks out the gene, or a construct capable of expressing or forming the gene editing reagent.

[0074] As used herein, the term "effective amount" means an amount that is functional or active in and acceptable to a human and / or animal. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to a carrier that is compatible with the active ingredient, and that does not itself induce significant toxicity when administered. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in a composition can include liquids such as water, saline, buffers. In addition, these carriers can also contain auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like. The carriers can also contain cell transfection reagents.

[0075] Upon learning of the use of the aldolase A protein mutant or its encoding gene inhibitor, various methods well known in the art can be used to administer the down-regulator or its encoding gene, or its pharmaceutical composition to a mammal or human.

[0076] Preferably, a genetic therapy approach can be used. For example, the aldolase A protein mutant or its encoding gene inhibitor can be directly administered to a subject by methods such as injection; or an expression unit (such as an expression vector or virus, or siRNA) carrying the aldolase A protein mutant or its encoding gene inhibitor can be delivered to the target site through a certain route, and the active aldolase A protein mutant or its encoding gene inhibitor is expressed. The specific case depends on the type of the inhibitor, which is well known to those skilled in the art.

[0077] The effective amount of the aldolase A protein mutant or its encoding gene inhibitor of the present application can vary depending on the mode of administration and the severity of the disease to be treated. Preferably, the selection of the effective amount can be determined by those of ordinary skill in the art according to various factors (e.g. through clinical trials). The factors include but are not limited to: pharmacokinetic parameters of the aldolase A protein mutant or its encoding gene inhibitor such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.

[0078] Materials and methods are as follows:

[0079] Commonly used drugs and reagents:

[0080] Most of the drugs and reagents used in this specification were purchased from Sigma, Shanghai Sangon Biotech (Sangon) Co., Ltd. and MedChemExprss (MCE) Co., Ltd. respectively. The antibodies used for Western Blot analysis were purchased from Sigma, Cell Signaling (CST) and Proteintech.

[0081] Example 1 High-sensitivity quantitative mass spectrometry analysis of ubiquitination and acetylation levels in lung cancer clinical samples and lung cancer adjacent normal tissues

[0082] (1) Experimental procedure

[0083] First, clinical samples and their adjacent normal tissues from lung cancer patients were collected from the body as the basis of the control experiment. To ensure the accuracy of the experimental data, uniform homogenate processing procedures were performed on these two types of tissue samples, aiming to destroy the cell structure and release all the proteins inside the cells.

[0084] Next, trypsin was chosen as the main enzymatic reagent to cut the proteins in normal and cancer tissues into peptide segments. To ensure the efficiency and specificity of the enzymatic process, strict control conditions were set, including the accurate control of protein concentration, enzymatic temperature, pH value, and enzyme-to-substrate ratio. The implementation of this series of control measures aims to minimize experimental errors and ensure the consistency of the peptides extracted from normal and cancer cells in terms of quantity and quality.

[0085] Subsequently, the ubiquitinated and acetylated peptide segments were enriched using immunoaffinity chromatography technology. For the enrichment of ubiquitinated peptides, an antibody that specifically recognizes double-alanine on lysine was used to form an antibody-peptide complex through immunoaffinity, and the unbound peptides were washed away using a chromatography column. Finally, the ubiquitinated peptides were eluted from the antibody using appropriate elution conditions and collected. Similarly, acetylated peptide enrichment uses acetylated-specific antibodies or chemical groups with acetylated group affinity to effectively enrich and elute through affinity chromatography columns for subsequent mass spectrometry analysis.

[0086] (2) Data analysis

[0087] Based on the quantitative evaluation of modified peptide peak intensity, the relative contents of all ubiquitination and acetylation modifications in lung cancer tissues and adjacent normal tissues were compared. First, advanced mass spectrometry techniques were used to successfully obtain the mass information and sequence characteristics of a large number of ubiquitinated and acetylated peptides in these tissues. Subsequently, DIA-NN software was used for in-depth quantitative comparison to compare the peak intensities of the corresponding modified peptides in lung cancer tissues and adjacent normal tissues. The differences in peak intensity directly reflect the relative abundance of modified peptides in the two types of tissues, revealing the differences in ubiquitination and acetylation modification levels at the K330 position of aldolase A protein between lung cancer tissues and adjacent normal tissues.

[0088] (3) Experimental results

[0089] The present application detects a large number of acetylation modification and ubiquitination modification levels in lung cancer tissues and paracancer tissues by high sensitivity quantitative mass spectrometry, and finds that the acetylation and ubiquitination levels of ALDOA K330 are higher in lung cancer tissues than in lung cancer adjacent tissues. We have detected 6 acetylation sites and 5 ubiquitination sites on ALDOA, among which 5 acetylation sites and 4 ubiquitination sites do not change in tumor and paracancer tissues, and only the acetylation and ubiquitination levels at K330 position are higher than those in lung cancer adjacent tissues, as shown in Figure 1 .

[0090] Example 2

[0091] (1) Construction of plasmid

[0092] First, the DNA sequences of aldolase A gene and K330R mutant thereof are extracted from organisms by gene cloning technology, and amplified and purified by PCR technology. Then, a suitable plasmid vector is selected, which should have the ability to replicate autonomously in host cells, a selection marker gene and a cloning site for inserting foreign DNA. Then, the DNA sequence of aldolase A gene or K330R mutant thereof is fused with 3 Flag tag sequence to form a fusion gene, and inserted into the cloning site of the plasmid vector, thereby constructing 3 Flag-aldolase A and K330R 3 Flag-aldolase A plasmid.

[0093] (2) Construction of K330R mutant cell line

[0094] When 293T cells reach the logarithmic growth phase, the expression vectors encoding wild-type and K330R mutant aldolase A genes are introduced into cells by liposome-mediated transfection technology, and wild-type 293T cell line and K330R mutant 293T cell line are constructed. Then, 3 Flag-aldolase A and K330R 3 Flag-aldolase A plasmid is transfected into recipient cells to induce cells to express wild-type aldolase A protein and K330R mutant aldolase A protein with 3 Flag tag. Then, according to the specific binding principle between Flag tag and anti-Flag antibody, anti-Flag conjugated agarose beads are added to the cell lysate, and the target protein is tightly bound to the beads by affinity. Then, high-purity wild-type aldolase A protein and K330R mutant aldolase A protein are separated and obtained by affinity chromatography and other protein purification techniques.

[0095] (3) Analysis of the protease activity of wild-type and K330R mutant aldolase A proteins

[0096] At room temperature, the wild-type and K330R mutant aldolase A proteins were incubated with fructose diphosphate as a specific substrate. To accurately determine the activity of aldolase A, a reaction system and conditions matching the in vitro enzyme activity evaluation were used to determine the activity of the purified wild-type ALDOA and the purified K330R mutant ALDOA. During the determination, the absorption value was measured every 5 minutes using a UV-visible spectrophotometer at OD 240nm . Subsequently, the collected data was statistically analyzed, and an enzyme activity curve was plotted, in which time was taken as the abscissa and the absorption value at OD 240nm was taken as the ordinate. Through this graph, the activity differences of aldolase A in the six different cells can be directly evaluated and analyzed.

[0097] (4) Experimental results

[0098] In 293T, aldolase A has ubiquitination and acetylation modifications at the K330 position, as shown in Figure 2 and Figure 3 . The ubiquitination and acetylation of K330 inhibit in vitro enzyme activity, as shown in Figure 4 .

[0099] Example 3 Construction of K330R knock-in A375, A549, 4T1 and B16F10 cells

[0100] (1) Experimental procedure

[0101] First, PE-GFP was mixed with the K330R-pegRNA plasmid, and then the mixed plasmid was introduced into wild-type A375, A549, 4T1 and B16F10 cell lines using electroporation technology to prepare mutant cell lines. After the electroporation operation was completed, the cells were cultured in an incubator for 48 hours. After the end, flow cytometry was used to sort the cells for green fluorescence and red fluorescence to screen out cell populations that successfully transfected and expressed PE-GFP. Next, from the sorted cells, single clone cells were separated and cultured to obtain purified monoclonal cell lines, and cell samples were collected and genomic DNA was extracted. Then, specific primers for the K330 position of aldolase A gene were used for PCR amplification to obtain a DNA fragment containing the K330 position. The PCR amplification product was subjected to sequencing analysis, and the sequencing results were compared with the expected K330R sequence to obtain the complete sequence information of the DNA molecule.

[0102] (2) Experimental results

[0103] We successfully obtained ALDOA K330R knock-in A375, A549, 4T1 and B16F10 cells, and the results of genome sequencing are shown in Figure 5 、 Figure 13 and Figure 14 .

[0104] Example 4 Proliferation of WT and K330R knock-in A375 and A549 cells

[0105] 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, ensuring that the number of cells in each well was consistent. Then, an appropriate amount of CCK8 reagent was added to each well, which can react with the dehydrogenase in living cells to generate a colored formazan product, the amount of which is directly proportional to the number of living cells. Next, the 96-well plate was incubated at 37°C for one hour to allow sufficient reaction. After that, the OD absorbance value of each well was measured at 450 nm wavelength using a microplate reader, which reflects the number of living cells in each well. By repeating the above steps for several consecutive days and recording the OD 450nm absorbance value at each time point, a graph of the proliferation rates of wild-type and K330R mutant A375 and A549 cells can be drawn, where the horizontal axis represents the time points from the beginning to the end of the experiment, and the vertical axis represents the proliferation of the cells. Thus, the proliferation abilities of different cell lines can be compared intuitively.

[0106] 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 exhibited significantly slower growth, as shown in Figure 9 and Figure 11 .

[0107] Example 5 Comparison of migration rates of WT and K330R knock-in A375 and A549 cells

[0108] To compare the migration speed of wild type and K330R mutant A375 and A549 cells, first, cells of both types were evenly spread into 96-well plates, respectively, to ensure the same number of cells in each well. Then, a sterile gun tip was used to 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 plate was placed in the cell culture incubator for culture under standard culture conditions. Every 12 hours, the cells were photographed using a microscope, focusing on the cell gap area formed by the gun tip line. By comparing the photos taken at different time points, the migration of cells from the edge to the center of the gap can be observed, the width of the gap area in each time point photo is measured, and recorded. The time is taken as the horizontal coordinate, or the distance of cell migration as the vertical coordinate, to draw the migration speed graph of wild type and K330R mutant A375 and A549 cells. In this way, by comparing the migration curves of different cell lines, their migration ability can be clearly evaluated.

[0109] 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 exhibited significantly slower migration. K330R knock-in cells exhibited significantly slower migration, specifically, as shown in Figure 10 and Figure 12 WT cells can migrate faster to the intercellular gap area than K330R cells.

[0110] Example 6 Subcutaneous tumor implantation of WT and K330R knock-in 4T1 and B16F10 cells

[0111] To compare the growth rates of wild-type and K330R mutant 4T1 and B16F10 cells in mice, first, the two types of cells were suspended in an appropriate amount of normal saline and implanted into mice of the corresponding strain by subcutaneous injection. The concentration of the cell suspension should be consistent, and the injection amount for each mouse should be the same to ensure uniformity of the experiment. Starting on the 10th day after cell implantation, the size of the subcutaneous tumors in mice was measured every 2 days using a vernier caliper. During measurement, the long diameter (L) and short diameter (W) of the tumor were accurately recorded, and the tumor volume was calculated using the formula V = (L x 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. To visually show the growth rate of the tumor, the measured data were sorted and plotted as a graph with time as the horizontal coordinate and tumor volume as the vertical coordinate. By comparing the tumor growth curves formed by different cell lines, their growth rates can be clearly evaluated.

[0112] Experimental results: The growth rate of aldehyde dehydrogenase A K330R knock-in 4T1 and B16F10 in mice has significantly decreased compared to wild-type 4T1 and B16F10. As shown in Figure 15 and Figure 16 , the average volume of WT 4T1 and B16F10 cells reached 1 cubic centimeter at about 25 days and 16 days, while the average volume of K330R 4T1 and B16F10 was less than 200 cubic millimeters.

[0113] The present application verifies the potential of mutating the lysine at position K330 of the aldehyde dehydrogenase A protein to arginine in tumor treatment through high-sensitivity quantitative mass spectrometry analysis, in vitro enzyme activity determination, cell proliferation and migration experiments, and mouse tumor implantation experiments. The experimental results show that ubiquitination and acetylation modification at position K330 of the aldehyde dehydrogenase A protein significantly inhibit the enzyme activity of aldehyde dehydrogenase A and slow down the growth and migration speed of cells. Therefore, position K330 of the aldehyde dehydrogenase A protein is expected to become a new target for tumor treatment, providing new ideas and methods for tumor treatment.

[0114] The above is only a description of the preferred embodiments of the present application, which aims to demonstrate its core concept and application mode, but not to limit the broad applicability of the present application. Any form of modification, equivalent alternative, technical improvement or innovation should be considered as falling within the scope of the present application as claimed.

Claims

1. Use of an aldolase A protein mutant in the preparation of an anti-tumor drug, characterized in that: The amino acid sequence of the aldolase A protein mutant is the amino acid sequence shown in SEQ ID No. 2; The tumor is selected from malignant melanoma, malignant breast cancer and non-small cell lung cancer.

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