Lactylated NBS1 protein and uses thereof

Abstract

A lactylated NBS1 protein and uses thereof. A lactylation site is lysine 388 of an NBS1 protein. Lactylation at the K388 site of the NBS1 protein activates a DNA repair pathway, and enables various uses including promoting drug resistance of tumor cells in radiotherapy and chemotherapy and enhancing DNA homologous recombination repair. By means of targeted inhibition of the lactylated NBS1 protein, drug resistance of tumors in radiotherapy or chemotherapy can be reversed, providing a potential target for reversing drug resistance of tumors in radiotherapy or chemotherapy.

Description

Lactylation modification of NBS1 protein and its application Technical Field

[0001] The present invention relates to the field of tumor radiotherapy, and in particular to lactic acid-modified NBS1 protein and applications thereof. Background Art

[0002] DNA double-strand breaks (DSBs) are considered the most severe form of DNA damage, as a single unrepaired DSB is sufficient to cause permanent growth arrest or cell death. When a DSB occurs, cells immediately initiate DNA repair to maintain DNA integrity in order to survive. The MRN (MRE11-RAD50-NBS1) complex plays a crucial role in DNA damage sensing and repair. Upon DSB formation, the MRN complex forms immediately and is rapidly recruited to the site of DNA damage to initiate DNA repair. The MRN complex is composed of the proteins MRE11, RAD50, and NBS1. MRE11 and RAD50 are responsible for cleaving DNA breaks, while NBS1 regulates the functions of MRE11 and RAD50. Therefore, NBS1 is a regulator of the MRN complex. The MRN complex maintains genomic stability and prevents malignant transformation of normal cells. Its role in cancer development and its potential as an anticancer target have been extensively explored in various cancer types. Analysis of gastric cancer specimens surgically resected after chemotherapy showed that low expression levels of the MRN complex were associated with a robust response to chemotherapy and surgical resection. In vitro experiments showed that disruption of the MRN complex or its components could confer sensitivity to cisplatin in cancer cells, with increased DNA damage and cytotoxicity.

[0003] NBS1 is a member of the MRN complex and a regulator of the MRN complex, responsible for regulating the function of the MRN complex. NBS1 mutations are the main cause of Nijmegen breakage syndrome (NBS). Patients with Nijmegen breakage syndrome have symptoms of slow development and a high susceptibility to cancer. In tumorigenesis studies, NBS1 mutations have been observed in breast cancer, prostate cancer, lung cancer, liver cancer, and intrahepatic bile duct cancer, and are associated with increased cancer susceptibility. In studies of chemotherapy resistance mechanisms, downregulated expression of NBS1 is associated with hypoxia-induced chemotherapy resistance, inhibition of the double-strand break repair pathway, and p53 activation in medulloblastoma cells. In studies of radioresistance mechanisms, NBS1 can stabilize hypoxia-inducible factors and promote cancer cell migration and invasion under ionizing radiation.

[0004] Protein lactylation plays a key role in human physiological and pathological processes. Given the important role of NBS1 in tumorigenesis and progression, further research on the lactylation of NBS1 is of great significance.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a lactic acid modified NBS1 protein and its application. The lactic acid modification of NBS1 protein plays an important role in DNA damage repair. Targeted inhibition of lactic acid modified NBS1 protein can reverse tumor radiotherapy or chemotherapy resistance.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides the use of a reagent for detecting the expression level of lactic acid modification of NBS1 protein in the preparation of a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, wherein the lactic acid modification site is lysine 388 of the NBS1 protein.

[0009] Through lactation-modified proteomics, the present invention discovered that lysine (K) at position 388 in the NBS1 protein is lactated. The resulting lactated NBS1 protein promotes the formation of the MRN complex, thereby promoting tumor resistance to radiotherapy or chemotherapy. Immunohistochemical data statistical results show that NBS1 protein lactation is significantly higher in chemotherapy-resistant gastric cancer tissues compared to chemotherapy-sensitive gastric cancer tissues. Therefore, higher expression of lactated NBS1 protein is associated with poorer chemotherapy efficacy in cancer patients.

[0010] Preferably, the lactylation modification level of the NBS1 protein is regulated by lysine lactoyltransferase TIP60.

[0011] This study used immunoprecipitation and immunoblotting techniques to confirm the relationship between the lysine lactoyltransferase TIP60 and the NBS1 protein. Overexpression of TIP60 increased the expression of NBS1 protein lactylation, while knockdown of TIP60 reduced the expression of NBS1 protein lactylation. This suggests that TIP60 interacts with NBS1 protein and regulates NBS1 protein lactylation.

[0012] Preferably, the higher the expression level of the lactic acid-modified NBS1 protein, the worse the chemotherapy efficacy of the tumor patient.

[0013] In a second aspect, the present invention provides the use of lactylated NBS1 protein as a target in the preparation of drugs for reversing tumor radiotherapy or reversing radiotherapy resistance, wherein the lactylated modification site is lysine 388 of the NBS1 protein.

[0014] In a third aspect, the present invention provides a combination of preparations for reversing tumor radiotherapy or reversing radiotherapy resistance, including a preparation that specifically inhibits lactic acid modification of NBS1 protein.

[0015] The present invention proves through CCK8 drug sensitivity experiment and flow cytometry apoptosis experiment that abolishing NBS1 protein lactylation modification can reverse tumor cell radiotherapy and chemotherapy resistance.

[0016] Preferably, the agent comprises small molecule inhibitors, nucleic acids, or polypeptides.

[0017] Preferably, the preparation further comprises a pharmaceutically acceptable carrier.

[0018] In a fourth aspect, the present invention provides the use of the above-mentioned combination of preparations for reversing tumor radiotherapy or reversing radiotherapy resistance in the preparation of drugs for reversing tumor radiotherapy or reversing radiotherapy resistance.

[0019] The beneficial effects of the present invention are:

[0020] This study, using lactylation proteomics, discovered that lysine 388 in the NBS1 amino acid sequence is lactated. This modification of the NBS1 protein plays a crucial role in DNA damage repair. Lactylation of the NBS1 protein promotes the formation of the MRN complex, activates the DNA repair pathway, and significantly enhances tumor resistance to radiotherapy and chemotherapy.

[0021] Lactic acid-modified NBS1 protein can be used as a biomarker to predict the efficacy of radiotherapy or chemotherapy in cancer patients; by targeted inhibition of lactic acid-modified NBS1 protein, tumor radiotherapy or chemotherapy resistance can be reversed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a structural diagram of the NBS1 protein.

[0023] Figure 2 shows the mass spectrometry results of NBS1 protein.

[0024] Figure 3 shows the results of the interaction between lysine lactoyltransferase TIP60 and NBS1 protein; A, endogenous immunoprecipitation experiment results; B, Western blot results of overexpressing TIP60; C, Western blot results of knocking down TIP60; where Flag represents the tag protein, NBS1-K388la represents the lactylation of NBS1 protein at position K388, β-tubulin and H4 represent internal controls, and H4K8ac represents the positive control.

[0025] Figure 4 shows the results of NBS1 protein lactylation modification promoting the formation of the MRN complex; A, first-generation sequencing results; B, Western blot results; where NBS1-K388la represents the lactylation of NBS1 protein at position K388, β-tubulin represents the internal control, and ATM and p-ATM (S1981) represent positive controls.

[0026] Figure 5 shows the results of abolishing the lactylation modification of NBS1 protein to reverse the radiotherapy and chemotherapy resistance of tumor cells; A, CCK8 experimental results; Cell viability represents cell survival rate; B, flow cytometry apoptosis experimental results; C, CCK8 experimental results.

[0027] Figure 6 shows the relationship between the lactic acid modification of the highly expressed NBS1 protein and the chemotherapy efficacy of tumor patients; A, immunohistochemical staining results; B, statistical graph of immunohistochemical data results. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] Example 1:

[0030] One million AGS gastric cancer cells were collected and lysed using RIPA lysis buffer to extract intracellular proteins. The proteins were then hydrolyzed into peptides using trypsin. Lactate modification was enriched using the lactate antibody Anti-L-Lactyl Lysine Rabbit pAb (purchased from Jingjie Company, product number: PTM-1401), and then detected by mass spectrometry.

[0031] The structure of NBS1 protein is shown in Figure 1, and the mass spectrometry detection results are shown in Figure 2. The results showed that lysine 388 of NBS1 protein was lactylated.

[0032] Example 2:

[0033] 1. Use immunoprecipitation and immunoblotting techniques to confirm the relationship between lysine lactoyltransferase TIP60 and NBS1 protein.

[0034] The specific steps are as follows:

[0035] (1) One million 293T cells were collected and lysed by adding 1 mL of cell lysis buffer. The cells were placed on ice for 30 min, vortexed every 15 min, and then centrifuged at 12,000 g for 20 min at 4°C. The pellet was discarded and the supernatant was collected.

[0036] (2) Add 2 μg of the corresponding NBS1 antibody to the supernatant and incubate at 4°C on a shaker for 2 h;

[0037] (3) Add 20 μL of protein A / G-beads (magnetic beads) to a 1.5 mL centrifuge tube, wash the beads with 800 μL of RIPA lysis buffer, discard the supernatant, repeat three times, add the magnetic beads to the incubation solution treated in step (2), and incubate overnight at 4°C in a shaker;

[0038] (4) The next day, the centrifuge tube was placed on a magnetic rack. After the magnetic beads were aggregated on the tube wall, the supernatant was carefully aspirated and washed with 800 μL RIPA lysis buffer. The washing was repeated 5 times. 80 μL RIPA lysis buffer and 20 μL 5× SDS loading buffer were added, and the tube was mixed by vortexing. The tube was heated at 99°C for 10 min for denaturation. The relationship between TIP60 protein and NBS1 protein was detected by Western blot. The results are shown in Figure 3A.

[0039] The results of endogenous immunoprecipitation experiments showed that lysine lactoyltransferase TIP60 interacted with NBS1 protein.

[0040] 2. In order to further confirm the relationship between lysine lactoyltransferase TIP60 and NBS1 protein lactylation modification, lysine lactoyltransferase TIP60 was overexpressed and knocked down respectively.

[0041] The specific experimental methods are:

[0042] (1) Overexpression of lysine lactyltransferase TIP60: The overexpression plasmid PLVX-Flag-TIP60 (constructed by Guangzhou Aiji Biotechnology Co., Ltd.) was transfected into AGS cells. 24 hours after transfection, the protein was collected and subjected to Western blot analysis. The results are shown in Figure 3B.

[0043] (2) Knockdown of lysine lactoyltransferase TIP60: Si-TIP60 primers (primer 1: ACGGAAGGUGGAGGUGGUU; primer 2: AAGAAGAUCCAGUUCCCCAAGTT) were transfected into AGS cells to achieve TIP60 knockdown, and then Western blot experiments were performed. The results are shown in Figure 3C.

[0044] Experimental results showed that overexpression of lysine lactoyltransferase TIP60 increased the expression level of NBS1 protein lactylation modification; knockdown of lysine lactoyltransferase TIP60 reduced the expression level of NBS1 protein lactylation modification.

[0045] In summary, lysine lactoyltransferase TIP60 interacts with NBS1 protein, and TIP60 regulates the lactylation modification of NBS1 protein.

[0046] Example 3:

[0047] 1. Use gene editing technology and immunoprecipitation technology to confirm whether lactylation modification of NBS1 protein can promote the formation of MRN complex.

[0048] The experimental process is as follows:

[0049] The AGS-NBS1-K388R gene-edited cell line was constructed, and the lysine 388 on the NBS1 gene was mutated to arginine, thereby abolishing the lactylation modification of the NBS1 protein.

[0050] The construction method of the AGS-NBS1-K388R gene-edited cell line is as follows:

[0051] (1) First, synthesize a spacer DNA sequence and an extension DNA sequence. The spacer DNA sequence is: 5'-GAAATCAAAGTCTCCAAAA-3'; the extension DNA sequence is: 5'-TTTTTGTTCCATTCTGGAGACTTTGAT-3';

[0052] (2) The pU6-pegRNA-GG plasmid (purchased from Addgene, #132777), the spacer DNA sequence, and the extension DNA sequence were ligated into a pU6-pegRNA-GG-NBS1-K388R plasmid using the Golden Gate assembly method;

[0053] (3) The pCMV-PE2 plasmid (purchased from Addgene, #132775) and pU6-pegRNA-GG-NBS1-K388R plasmid were transfected into the gastric cancer cell line AGS (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, #TCHu232);

[0054] (4) Monoclonal bacteria were picked from the transfected AGS cells and sequenced. The sequencing results are shown in Figure 4A.

[0055] The first-generation sequencing results showed that the present invention successfully constructed the AGS-NBS1-K388R gene-edited cell line.

[0056] 2. Exploring the relationship between lactic acid modification of NBS1 protein and the formation of MRN complex

[0057] Co-immunoprecipitation (experimental steps as shown in Example 2) and Western blot experiments confirmed that lactylation of NBS1 protein promoted the formation of the MRN complex, while abolishing lactylation of NBS1 protein significantly reduced the formation of the MRN complex ( FIG4B ).

[0058] Example 4:

[0059] The CCK-8 kit (brand: Fude Bio, catalog number: FD378) and Annexin V-FITC flow apoptosis kit (brand: abcam, catalog number: ab14085) were used to perform drug sensitivity experiments and flow apoptosis experiments on the AGS-NBS1-K388R gene-edited cell line and the AGS parental line. The results are shown in Figure 5.

[0060] The results of CCK8 experiments showed that abolishing the lactylation modification of NBS1 protein could reverse chemotherapy resistance (Figure 5A); the results of flow cytometry apoptosis experiments showed that abolishing the lactylation modification of NBS1 protein could reverse chemotherapy resistance (Figure 5B); the results of CCK8 experiments showed that abolishing the lactylation modification of NBS1 protein could reverse radiotherapy resistance (Figure 5C).

[0061] Therefore, this example demonstrates through CCK8 drug sensitivity experiments and flow cytometry apoptosis experiments that abolishing the lactylation modification of NBS1 protein can reverse the radiotherapy and chemotherapy resistance of tumor cells.

[0062] Example 5:

[0063] In this example, 94 gastric cancer tissue samples were collected from the Digestive Medicine Center of the Seventh Affiliated Hospital of Sun Yat-sen University before neoadjuvant chemotherapy. Based on the principle of tumor regression grade (TRG), these 94 patient samples were divided into two groups: chemotherapy-sensitive (Sensitive) and chemotherapy-resistant (Resistant):

[0064] Chemosensitive group: TRG grade 0-2 was defined as chemotherapy sensitive;

[0065] Chemotherapy-resistant (Resistant) group: TRG grade 3 was defined as chemotherapy-resistant.

[0066] Subsequently, immunohistochemical staining was performed using NBS1-K388ka antibody (which can specifically recognize the lactylation modification of NBS1 protein), and the immunohistochemical image is shown in Figure 6A.

[0067] Immunohistochemical data showed that NBS1 protein lactylation was significantly higher in chemoresistant gastric cancer tissues compared to chemotherapy-sensitive gastric cancer tissues (Figure 6B). Therefore, higher levels of NBS1 lactylation are associated with poorer chemotherapy efficacy in cancer patients.

[0068] In summary, the present invention discovered through lactation-modified proteomics that lysine (K) at position 388 in the NBS1 protein is a lactated amino acid. Experiments in the present invention have confirmed that the lactated NBS1 protein not only promotes the formation of the MRN complex, but also has multiple uses such as promoting tumor cell radiotherapy and chemotherapy resistance and enhancing DNA homologous recombination repair. The lactated NBS1 protein has the following application prospects: (1) it can regulate the function of the NBS1 protein; (2) it can serve as a biomarker for predicting the efficacy of radiotherapy or chemotherapy in tumor patients; and (3) by targeted inhibition of the lactated NBS1 protein, it can reverse tumor radiotherapy or chemotherapy resistance.

[0069] Lactic acid modification of NBS1 protein can be used as a predictor of cancer patient prognosis and the efficacy of neoadjuvant chemotherapy. Lactic acid modification is of great significance in regulating NBS1 protein function and predicting the therapeutic efficacy of cancer patients.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a reagent for detecting the expression level of NBS1 protein lactylation modification in the preparation of a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, characterized in that: The lactylation modification site is lysine 388 of the NBS1 protein.

2. Use of the reagent for detecting the expression level of NBS1 protein lactylation modification according to claim 1 in the preparation of a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, characterized in that: The lactylation modification level of the NBS1 protein is regulated by the lysine lactoyltransferase TIP60.

3. Use of the reagent for detecting the expression level of NBS1 protein lactylation modification according to claim 1 in preparing a kit for predicting the efficacy of tumor radiotherapy or chemotherapy, characterized in that: The higher the expression level of the lactic acid-modified NBS1 protein, the worse the chemotherapy efficacy of tumor patients.

4. Use of lactylated NBS1 protein as a target in the preparation of a drug for reversing tumor radiotherapy or reversing radiotherapy resistance, characterized in that: The lactylation modification site is lysine 388 of the NBS1 protein.

5. A preparation combination for reversing tumor radiotherapy or reversing radiotherapy resistance, characterized in that: The invention comprises a preparation that specifically inhibits the lactylation modification of NBS1 protein.

6. The preparation combination for reversing tumor radiotherapy or reversing radiotherapy resistance according to claim 5, characterized in that: The preparations include small molecule inhibitors, nucleic acids, and polypeptides.

7. The preparation combination for reversing tumor radiotherapy or reversing radiotherapy resistance according to claim 6, characterized in that: Also included are pharmaceutically acceptable carriers.

8. Use of the combination of preparations for reversing tumor radiotherapy or reversing radiotherapy resistance as claimed in any one of claims 5 to 7 in the preparation of drugs for reversing tumor radiotherapy or reversing radiotherapy resistance.

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