Use of ATP6V0A1 K303 succinylation site

Abstract

This invention relates to the field of biomedicine and discloses the application of the ATP6V0A1 K303 succinylation site, including its application in the preparation of a kit for screening candidate drugs for treating neurodegenerative diseases, an in vitro method for screening candidate drugs for treating neurodegenerative diseases, and a kit for screening candidate drugs for treating neurodegenerative diseases. The amino acid sequence of the ATP6V0A1 protein is shown in SEQ ID NO:1, with K303 being lysine at position 303. The detection reagent includes reagents for LC-MS / MS detection of the modified peptide NWFVK[Succinyl]VR. This invention uses the succinylation modification level at this site for candidate drug screening, making the screening results correspond to the succinylation modification level at this site, thus providing a technical basis for screening candidate drugs with increased modification levels at this site.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of the ATP6V0A1 K303 succinylation site. Background Technology

[0002] Neurodegenerative diseases are a group of diseases characterized by the progressive loss of neuronal structure and function in the central nervous system, clinically manifesting as cognitive impairment, motor dysfunction, and behavioral abnormalities. Based on the affected brain regions and clinical presentations, neurodegenerative diseases mainly include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). Current treatments have limited effectiveness in intervening in the progression of neurodegenerative diseases, and the screening of candidate drugs still requires detection indicators that can reflect disease-related cellular functional changes.

[0003] Lysosomes participate in intracellular degradation, autophagy, and protein homeostasis. Lysosomal dysfunction is associated with pathological protein deposition in neurodegenerative diseases. Under pathological conditions, decreased lysosomal acidity and inhibited autophagy affect intracellular protein clearance, causing changes in autophagy-related indicators such as ubiquitinated proteins, p62 proteins, and Ref(2)P proteins. In existing drug candidate screening methods, the detection indicators for changes related to lysosomal autophagy function still need further clarification to establish a correlation between drug candidate screening and lysosomal function improvement.

[0004] Protein succinylation modification can alter the charge state of lysine residues and affect protein function. The ATP6V0A1 protein is associated with lysosomal acidity, and its K303 succinylation modification level is correlated with lysosomal acidity, autophagy-related protein levels, and pathological protein deposition. Currently, there is a lack of kits and in vitro methods that use the ATP6V0A1 protein K303 succinylation modification level as a detection indicator for screening candidate drugs for the treatment of neurodegenerative diseases. Therefore, establishing a candidate drug screening scheme based on the ATP6V0A1 protein K303 succinylation modification level is a problem that needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of the ATP6V0A1 K303 succinylation site, solving the problem that existing technologies lack kits and in vitro methods for screening candidate drugs for the treatment of neurodegenerative diseases using the succinylation modification level of the ATP6V0A1 protein K303 site as a detection indicator.

[0006] To address the above problems, the present invention provides the following technical solution: The first aspect of this invention provides the application of the ATP6V0A1 K303 succinylation site in the preparation of a kit for screening candidate drugs for the treatment of neurodegenerative diseases, using the following technical solution: The application of the ATP6V0A1 K303 succinylation site in the preparation of a kit for screening candidate drugs for the treatment of neurodegenerative diseases, the kit comprising an assay for the level of succinylation modification at the K303 site of the ATP6V0A1 protein, the amino acid sequence of the ATP6V0A1 protein being shown in SEQ ID NO:1, and the K303 site being lysine at position 303 of the amino acid sequence shown in SEQ ID NO:1.

[0007] By adopting the above technical solution, the succinylation modification level of the K303 site of the ATP6V0A1 protein is used as the detection target in the screening of candidate drugs. This allows the kit to detect the change in the succinylation modification level of the K303 site of the ATP6V0A1 protein before and after treatment with candidate drugs. The increase in the succinylation modification level of this site is used as the basis for judging candidate drugs, thereby making the ATP6V0A1 K303 succinylation site correspond to the screening of candidate drugs for the treatment of neurodegenerative diseases.

[0008] Preferably, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS).

[0009] By adopting the above technical solution, the neurodegenerative diseases in the application are limited to Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis, making the candidate drug screening targets of the kit clearer and facilitating the screening of candidate drugs around the above-mentioned neurodegenerative diseases.

[0010] Preferably, the succinylation modification level detection reagent at the K303 site of the ATP6V0A1 protein includes a reagent for LC-MS / MS detection of the modified peptide NWFVK[Succinyl]VR, wherein K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:1.

[0011] By adopting the above technical solution, the modified peptide NWFVK[Succinyl]VR is used as the LC-MS / MS detection target for the succinylation modification level of the K303 site of the ATP6V0A1 protein. It is clarified that K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:1, so that the succinylation modification level of the K303 site of the ATP6V0A1 protein can be detected by the corresponding modified peptide, thereby providing a detection method for comparing the succinylation modification level of the K303 site of the ATP6V0A1 protein before and after treatment with candidate drugs.

[0012] Preferably, the kit further includes an immunoassay kit for the ATP6V0A1 protein succinylation modification level, the immunoassay kit comprising an ATP6V0A1 antibody and a succinylated antibody for detecting succinylation modification.

[0013] By adopting the above technical solution, the kit includes an immunoassay kit for detecting the succinylation modification level of ATP6V0A1 protein. This allows the kit to detect not only the succinylation modification level at the K303 site of the ATP6V0A1 protein, but also the succinylation modification level of the ATP6V0A1 protein using an ATP6V0A1 antibody and a succinylation antibody for detecting succinylation modification, thereby providing an auxiliary detection method for candidate drug screening.

[0014] A second aspect of this invention provides an in vitro method for screening candidate drugs for treating neurodegenerative diseases, employing the following technical solution: An in vitro method for screening candidate drugs for the treatment of neurodegenerative diseases includes the following steps: Take a system containing ATP6V0A1 protein, the amino acid sequence of which is shown in SEQ ID NO:1, and the K303 site is the 303rd lysine in the amino acid sequence shown in SEQ ID NO:1; Add the candidate drug to the system containing ATP6V0A1 protein; The candidate drug was incubated with the system containing ATP6V0A1 protein; After incubation, the succinylation level of the K303 site of the ATP6V0A1 protein in the system containing the ATP6V0A1 protein after treatment with the candidate drug was detected. The succinylation modification level of the ATP6V0A1 protein K303 site in the system containing ATP6V0A1 protein after treatment with the candidate drug is compared with the succinylation modification level of the ATP6V0A1 protein K303 site in the system containing ATP6V0A1 protein before treatment with the candidate drug. The candidate drugs were screened to increase the level of succinylation modification at the K303 site of the ATP6V0A1 protein.

[0015] By adopting the above technical solution, candidate drugs are added to a system containing ATP6V0A1 protein and incubated. By detecting and comparing the succinylation modification level of the K303 site of ATP6V0A1 protein before and after treatment with candidate drugs, the change in the succinylation modification level of the K303 site of ATP6V0A1 protein can be used as a basis for candidate drug screening, and candidate drugs that increase the succinylation modification level of the K303 site of ATP6V0A1 protein can be screened.

[0016] Preferably, the succinylation modification level of the K303 site of the ATP6V0A1 protein in the system containing the ATP6V0A1 protein before the candidate drug treatment is detected before the candidate drug is added to the system containing the ATP6V0A1 protein.

[0017] By adopting the above technical solution, the succinylation modification level of the ATP6V0A1 protein K303 site before the addition of the candidate drug is obtained, so that the detection results after the candidate drug treatment have a comparative basis, thereby enabling the in vitro method to complete the candidate drug screening by comparing the succinylation modification level of the ATP6V0A1 protein K303 site before and after treatment.

[0018] Preferably, the system containing ATP6V0A1 protein is a cell system containing the ATP6V0A1 protein.

[0019] By adopting the above technical solution, the system containing ATP6V0A1 protein is limited to a cell system, so that the candidate drug can be incubated in a cell environment containing ATP6V0A1 protein, and the succinylation modification level of the K303 site of ATP6V0A1 protein can be detected in the cell system, thereby making the in vitro method applicable to candidate drug screening in cell systems.

[0020] Preferably, the method for detecting the succinylation modification level at the K303 site of the ATP6V0A1 protein includes LC-MS / MS detection, wherein the target of the LC-MS / MS detection includes the modified peptide NWFVK[Succinyl]VR, and K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:1.

[0021] By adopting the above technical solution, the succinylation modification level of the K303 site of the ATP6V0A1 protein is detected by LC-MS / MS in the in vitro method, and the modified peptide NWFVK[Succinyl]VR is used as the detection target, so that the succinylation modification level of the K303 site of the ATP6V0A1 protein before and after the candidate drug treatment can be detected and compared by the corresponding modified peptide.

[0022] Preferably, the in vitro method further includes an immunoassay step for the level of ATP6V0A1 protein succinylation modification.

[0023] By adopting the above technical solution, an immunoassay step for the succinylation modification level of ATP6V0A1 protein is further set in the in vitro method, so that in addition to detecting the succinylation modification level of ATP6V0A1 protein at the K303 site, the in vitro method can also detect the succinylation modification level of ATP6V0A1 protein, thereby providing an auxiliary detection method for candidate drug screening.

[0024] Preferably, the immunoassay step for the succinylation modification level of the ATP6V0A1 protein includes immunoprecipitation of the ATP6V0A1 protein with an ATP6V0A1 antibody and detection of the succinylation modification level of the ATP6V0A1 protein with a succinylation antibody for detecting succinylation modification.

[0025] By adopting the above technical solution, the ATP6V0A1 protein is immunoprecipitated with an ATP6V0A1 antibody, and the succinylation modification level of the ATP6V0A1 protein is detected with a succinylation antibody for detecting succinylation modification. This allows the immunoassay step to detect the ATP6V0A1 protein itself and its succinylation modification level, thereby helping to determine the changes in the succinylation modification level of the ATP6V0A1 protein after treatment with candidate drugs.

[0026] This invention provides the application of the ATP6V0A1 K303 succinylation site. It has the following beneficial effects: 1. This invention uses the succinylation modification level of the K303 site of the ATP6V0A1 protein to prepare a kit for screening candidate drugs for the treatment of neurodegenerative diseases, so that the screening of candidate drugs corresponds to the succinylation modification level of the K303 site of the ATP6V0A1 protein, providing a technical basis for screening candidate drugs that increase the succinylation modification level of the K303 site of the ATP6V0A1 protein.

[0027] 2. This invention uses LC-MS / MS to detect the modified peptide NWFVK[Succinyl]VR, and K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:1. This allows the succinylation modification level at the K303 site of the ATP6V0A1 protein to be detected using the modified peptide as the detection target, providing a detection method for comparing the succinylation modification level at the K303 site of the ATP6V0A1 protein before and after treatment with candidate drugs.

[0028] 3. This invention provides an auxiliary detection method for detecting the succinylation modification level of ATP6V0A1 protein by setting up an immunoassay reagent kit for the ATP6V0A1 protein succinylation modification level and using ATP6V0A1 antibody and succinylation antibody for detecting succinylation modification. Attached Figure Description

[0029] Figure 1 Image showing the results of succinylation immunofluorescence staining of the brain in Drosophila, an AD model in which neurons overexpress Aβ42; Figure 2 The results of reducing the level of succinylation in the brain of Drosophila through genetic means on lysosomal function-related indicators are shown in the figure. Among them, (A) is the result of immunofluorescence staining of succinylation in the brain of Drosophila; (B) is the result of immunoblotting detection of succinylation in the brain of Drosophila; (C) is the result of immunofluorescence staining of ubiquitinated protein in the brain of Drosophila; (D) is the result of immunofluorescence staining of Ref(2)P in the brain of Drosophila. Figure 3 The results of the effect of exogenous succinic acid supplementation on the succinylation level of Drosophila brain on lysosomal function-related indicators are shown in the figure; (A) is the immunofluorescence staining result of succinylation in Drosophila brain; (B) is the immunofluorescence staining result of ubiquitinated protein and Ref(2)P in Drosophila brain. Figure 4 The results of reducing the succinylation level of HeLa cells using RNA interference technology on lysosomal function-related indicators are shown in the figure. Among them, (A) is the immunofluorescence staining result of succinylation in HeLa cells; (B) is the immunofluorescence staining result of p62 protein in HeLa cells; (C) is the detection result of the half-life of p62 and LC3 proteins in HeLa cells; and (D) is the detection result of lysosomal acidity in HeLa cells. Figure 5 The diagram shows the identification results of targets affected by succinylation on lysosomal function; (A) shows the results of differential protein analysis in proteomics / succinylation modification proteomics; (B) shows the results of differential protein GO analysis; (C) shows the results of immunoprecipitation verification of target protein succinylation levels; (D) shows the results of mass spectrometry identification of target protein succinylation modification sites; and (E) shows the results of interspecies conservation analysis of succinylation modification sites. Figure 6 Figure 1 shows the results of lysosomal function-related indicators after transfecting ATP6V0A1 KO cells with wild-type and point-mutant plasmids; (A) shows the results of immunofluorescence staining of p62 protein; (B) shows the results of lysosomal acidity detection. Figure 7The results of AD-related phenotype detection after supplementing succinic acid in Drosophila model of AD are shown in the figure. Among them, (A) is the life count result; (B) is the climbing ability test result; (C) is the cognitive function test result; and (D) is the brain Aβ immunofluorescence staining result. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Examples 1-2: Example 1: This embodiment provides a method for screening candidate drugs for treating neurodegenerative diseases, including the following steps: Take a system containing ATP6V0A1 protein. The amino acid sequence of ATP6V0A1 protein is shown in SEQ ID NO:1, and the encoding nucleotide sequence of ATP6V0A1 protein is shown in SEQ ID NO:2. Add the candidate drug to the system containing ATP6V0A1 protein; The candidate drug was incubated with a system containing ATP6V0A1 protein; After incubation, the succinylation modification level at the K303 site of the ATP6V0A1 protein was detected; The succinylation level of the K303 site of the ATP6V0A1 protein after treatment with the candidate drug was compared with the succinylation level of the K303 site of the ATP6V0A1 protein before treatment with the candidate drug. Screening for candidate drugs that increase the level of succinylation modification at the K303 site of the ATP6V0A1 protein; The selected candidate drugs were identified as those with the potential to treat neurodegenerative diseases. Neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS).

[0032] Example 2: Example 2: This embodiment provides a kit for screening candidate drugs for the treatment of neurodegenerative diseases. The kit includes a succinylation modification level assay at the K303 site of the ATP6V0A1 protein.

[0033] The amino acid sequence of the ATP6V0A1 protein is shown in SEQ ID NO:1, and the K303 position is the 303rd lysine in the amino acid sequence shown in SEQ ID NO:1.

[0034] The ATP6V0A1 protein K303 site succinylation modification level detection reagent is used for LC-MS / MS detection of the modified peptide NWFVK[Succinyl]VR, where K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:1.

[0035] The kit also includes an immunoassay kit for the succinylation modification level of the ATP6V0A1 protein, which includes an ATP6V0A1 antibody and a succinylated antibody.

[0036] In the immunoassay step for the succinylation modification level of ATP6V0A1 protein, an antigen-antibody immune complex was formed using ATP6V0A1 antibody, and the ATP6V0A1 protein and the succinylation modification level of ATP6V0A1 protein were detected by Western blotting.

[0037] Comparative Examples 1-7: Comparative Example 1: This comparative example uses Nsyb-QF×W1118 Drosophila as a negative control in AD model-related tests. This comparative example does not involve Aβ42 overexpression or 2% succinate supplementation.

[0038] Comparative Example 2: This comparative example uses Nsyb-QF×QUAS-Aβ42 Drosophila as an AD model. This comparative example specifically overexpresses Aβ42 in Drosophila nerve cells without 2% succinate supplementation.

[0039] Comparative Example 3: This comparative example provides Act88F-Gal4×W1118 Drosophila as a negative control in the CG5214 knockdown-related test. This comparative example does not knock down CG5214, nor does it undergo 2% succinic acid supplementation.

[0040] Comparative Example 4: This comparative example provides Act88F-Gal4×UAS-CG5214RNAi Drosophila as a CG5214 knockdown model. This comparative example specifically knocks down CG5214 in Drosophila skeletal muscle, reducing succinate production and its transport to the brain, without 2% succinate supplementation.

[0041] Comparative Example 5: This comparative example provides HeLa cells treated with negative control siRNA as a negative control in DLST siRNA-related assays. In this comparative example, HeLa cells were transfected with the negative control siRNA, but not with DLST siRNA.

[0042] Comparative Example 6: This comparative example provides ATP6V0A1 KO cells and ATP6V0A1 KO cells transfected with an empty vector plasmid as controls in ATP6V0A1 complementation-related assays. ATP6V0A1 KO cells were constructed using CRISPR / Cas9 gene editing technology, and the nucleotide sequences of gRNA1 and gRNA2 used are shown in SEQ ID NO:20 and SEQ ID NO:21, respectively. This comparative example does not transfect the ATP6V0A1 wild-type plasmid or the ATP6V0A1K303E point mutant plasmid.

[0043] Comparative Example 7: This comparative example provides ATP6V0A1 KO cells transfected with either the ATP6V0A1K303R point mutant plasmid, the ATP6V0A1K295E point mutant plasmid, or the ATP6V0A1K295R point mutant plasmid, serving as a control group for the ATP6V0A1K303E point mutant plasmid treatment group. The amino acid sequences of the ATP6V0A1K303E, ATP6V0A1K303R, ATP6V0A1K295E, and ATP6V0A1K295R point mutant proteins are shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. This comparative example is used to differentiate the effects of simulated K303 succinylation, simulated K303 desuccinylation, and K295 mutation treatment on p62 protein levels and lysosomal acidity.

[0044] Test Examples 1-7: Test Example 1: Detection of Succinylation Levels in the Brain of Drosophila (AD Model) Experimental description: This test case was used to detect changes in succinylation levels in the brain of Drosophila following neuronal overexpression of Aβ42. Using the QF / QUAS transgenic system, a Drosophila AD model was constructed by specifically overexpressing Aβ42 in Drosophila neurons using the neuron-specific driver strain Nsyb-QF and the Aβ42-overexpressing strain QUAS-Aβ42. The Nsyb-QF×W1118 Drosophila (Comparative Example 1) served as the negative control group, while the Nsyb-QF×QUAS-Aβ42 Drosophila (Comparative Example 2) served as the AD model group. Differences in succinylation modification levels in the brains of the two groups were observed by immunofluorescence staining of the brains.

[0045] Experimental steps: The Nsyb-QF×W1118 fruit flies of Comparative Example 1 were used as the negative control group, and the Nsyb-QF×QUAS-Aβ42 fruit flies of Comparative Example 2 were used as the AD model group. The brains of Drosophila from each group were dissected in PBS. The brains were fixed at room temperature for 20 min with 4% paraformaldehyde, and then washed three times with PBS buffer containing 0.3% Triton X-100 for 10 min each time. Blocked with 5% bovine serum albumin for 1 hour, followed by succinylation primary antibody and incubation at 4°C for 48 hours; Add AlexaFluor-labeled secondary antibody and Hoechst33342 dye, and incubate at 4°C for 48 h; The slides were mounted using anti-fluorescence quenching mounting medium, and succinylated immunofluorescence images of the Drosophila brain were acquired using a confocal microscope.

[0046] Experimental conclusion: See attached document Figure 1 Nsyb-QF represents the neuron-specific driver strain, Ctrl represents the W1118 negative control Drosophila, and QUAS-Aβ42 represents Aβ42 overexpressing Drosophila. In the figure, green fluorescent signals represent succinylation modification signals, and blue fluorescent signals represent nuclear staining signals.

[0047] See attached document Figure 1 Compared with Comparative Example 1, Comparative Example 2 showed reduced green succinylation signaling in the Drosophila brain. This result indicates that neuronal overexpression of Aβ42 reduces succinylation levels in the Drosophila brain.

[0048] These results indicate that the level of succinylation in the brain of the AD model fruit fly was lower than that in the negative control group, suggesting that the decrease in brain succinylation level is associated with AD-related pathological conditions, and providing experimental evidence for subsequent analysis of the relationship between succinylation modification at the K303 site of ATP6V0A1 protein and neurodegenerative diseases.

[0049] Test Example 2: The effect of reducing succinylation levels in the Drosophila brain on lysosomal function Experimental description: This test case was used to evaluate the effect of reduced succinylation levels in the Drosophila brain on lysosomal function and autophagy-related indicators. Using the Gal4 / UAS transgenic system, the skeletal muscle-specific driver strain Act88F-Gal4 and the transgenic Drosophila strain UAS-CG5214RNAi were used to specifically knock down CG5214 in Drosophila skeletal muscle, reducing succinic acid production and its transport to the brain, thereby reducing succinylation levels in the Drosophila brain. Act88F-Gal4×W1118 Drosophila (Comparative Example 3) served as the negative control group, and Act88F-Gal4×UAS-CG5214RNAi Drosophila (Comparative Example 4) served as the CG5214 knockdown group. Brain succinylation levels, ubiquitinated protein levels, and Ref(2)P protein levels were measured.

[0050] Experimental steps: Act88F-Gal4×W1118 Drosophila from Comparative Example 3 was used as the negative control group, and Act88F-Gal4×UAS-CG5214RNAi Drosophila from Comparative Example 4 was used as the CG5214 knockdown group. The brains of Drosophila from each group were dissected in PBS. The brains were fixed at room temperature for 20 min with 4% paraformaldehyde, and then washed three times with PBS buffer containing 0.3% Triton X-100 for 10 min each time. Blocked with 5% bovine serum albumin for 1 hour, then succinylated primary antibody was added and incubated at 4°C for 48 hours; Add AlexaFluor-labeled secondary antibody and Hoechst33342 dye, incubate at 4°C for 48 h, mount with anti-fluorescence quenching mounting medium, and collect succinylated immunofluorescence images of Drosophila brain using a confocal microscope; Take 100 fruit flies, collect their heads, add pre-cooled RIPA lysis buffer, grind them, and place them on ice for lysis for 30 min, shaking once every 10 min during the process. Then centrifuge at 12000 rpm and 4℃ for 15 min, collect the supernatant, and obtain the total protein. Protein concentration was determined using the BCA protein quantification kit. The protein concentration of each sample was adjusted to be consistent. 5×SDS protein loading buffer was added, and the samples were heated at 95℃ for 10 min to denature the protein. Equal amounts of protein samples were subjected to SDS-PAGE gel electrophoresis and transferred to a membrane. The PVDF membrane was placed in 5% skim milk and blocked at room temperature for 1 hour. Succinylation primary antibody was added and incubated at 4°C overnight. The membrane was washed three times with TBST for 10 min each time, HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 h. The membrane was then washed three times with TBST for 10 min each time, ECL developing solution was added, and images were acquired using a chemiluminescence imager. The primary antibody against ubiquitinated proteins and the secondary antibody labeled with AlexaFluor were used to perform immunofluorescence staining of ubiquitinated proteins in the brains of each group of Drosophila. The remaining procedures were the same as those for the immunofluorescence staining of brain succinylation. The brains of each group of Drosophila were stained with Ref(2)P protein primary antibody using Ref(2)P immunofluorescence staining. The remaining procedures were the same as those for brain succinylation immunofluorescence staining.

[0051] Experimental data: Table 1. Effects of reducing succinylation levels in the Drosophila brain on autophagy-related indicators ; Experimental conclusion: See attached document Figure 2 Act88F-Gal4 represents the skeletal muscle-specific driving strain, Ctrl represents the W1118 negative control Drosophila, and UAS-CG5214RNAi represents the CG5214 knockdown Drosophila. Succinyllysine represents the succinyl lysine signal, Nucleus represents the nuclear signal, Ubiquitin represents the ubiquitinated protein signal, Ref(2)P represents the Drosophila autophagy substrate protein signal, Brain represents the brain sample, and β-actin represents the internal control protein.

[0052] See attached document Figure 2 A. Appendix Figure 2 As shown in B and Table 1, compared with Comparative Example 3, Comparative Example 4 showed reduced succinylation immunofluorescence signal in the Drosophila brain and weakened succinylation immunoblot bands. These results indicate that specific knockdown of CG5214 in Drosophila skeletal muscle can reduce succinylation levels in the Drosophila brain.

[0053] See attached document Figure 2 C. Appendix Figure 2 As shown in Table 1 (D), compared to Comparative Example 3, Comparative Example 4 showed enhanced ubiquitination protein signals and Ref(2)P signals in the Drosophila brain. The accumulation of ubiquitinated proteins and Ref(2)P reflects impaired intracellular protein clearance; therefore, decreased succinylation levels in the Drosophila brain can lead to lysosomal dysfunction and autophagy inhibition.

[0054] Test Example 3: Effects of increased succinylation levels in the Drosophila brain on lysosomal function Experimental description: This test case was used to evaluate the effect of exogenous succinate supplementation on lysosomal function and autophagy-related indicators after increasing succinylation levels in the brain of Drosophila. Act88F-Gal4×W1118 Drosophila (Comparative Example 3) was used as a negative control, and Act88F-Gal4×UAS-CG5214RNAi Drosophila (Comparative Example 4) was used as a CG5214 knockdown control. Based on this, Act88F-Gal4×W1118 and Act88F-Gal4×UAS-CG5214RNAi Drosophila were treated with 2% succinate supplementation, and the levels of succinylation, ubiquitinated protein, and Ref(2)P protein in the Drosophila brain were measured.

[0055] Experimental steps: Act88F-Gal4×W1118 fruit flies from Comparative Example 3 were used as the negative control group. Act88F-Gal4×W1118 fruit flies from Comparative Example 3 were also given 2% succinic acid in their food as the negative control group supplemented with succinic acid. The Act88F-Gal4×UAS-CG5214RNAi Drosophila from Comparative Example 4 were used as the CG5214 knockdown group. The Act88F-Gal4×UAS-CG5214RNAi Drosophila from Comparative Example 4 were also given 2% succinic acid in their food as the CG5214 knockdown and succinic acid supplementation group. A standard laboratory diet was prepared using 14g agar, 165.4g malt extract, 41.4g dry yeast, 78.2g corn flour, 4.7mL propionic acid, 3g methyl 4-hydroxybenzoate and 1.5L water. Succinic acid was added to the standard laboratory diet at a rate of 2g / 100mL to obtain a 2% succinic acid diet. The brains of Drosophila from each group were dissected in PBS. The brains were fixed at room temperature for 20 min with 4% paraformaldehyde, and then washed three times with PBS buffer containing 0.3% Triton X-100 for 10 min each time. Blocked with 5% bovine serum albumin for 1 hour, then succinylated primary antibody was added and incubated at 4°C for 48 hours; Add AlexaFluor-labeled secondary antibody and Hoechst 33342 dye, incubate at 4°C for 48 h, mount with anti-fluorescence quenching mounting medium, and acquire succinylated immunofluorescence images of Drosophila brain using a confocal microscope; The primary antibody against ubiquitinated proteins and the secondary antibody labeled with AlexaFluor were used to perform immunofluorescence staining of ubiquitinated proteins in the brains of each group of Drosophila. The remaining procedures were the same as those for the immunofluorescence staining of brain succinylation. The brains of each group of Drosophila were stained with Ref(2)P protein primary antibody using Ref(2)P immunofluorescence staining. The remaining procedures were the same as those for brain succinylation immunofluorescence staining.

[0056] Experimental data: Table 2. Effects of increased succinylation levels in the Drosophila brain on autophagy-related indicators ; Experimental conclusion: See attached document Figure 3 Act88F-Gal4 represents the skeletal muscle-specific driving strain, Ctrl represents the W1118 negative control Drosophila, UAS-CG5214RNAi represents the CG5214 knockdown Drosophila, Succinate represents succinic acid treatment, Succinyllysine represents succinyl lysine signaling, Nucleus represents nuclear signaling, Ubiquitin represents ubiquitinated protein signaling, and Ref(2)P represents Drosophila autophagy substrate protein signaling.

[0057] See attached document Figure 3 As shown in Table A and Table 2, compared with Comparative Examples 3 and 4, succinic acid supplementation in Comparative Examples 3 and 4 both increased succinylation signals in the Drosophila brain. This result indicates that exogenous succinic acid supplementation can increase succinylation levels in the Drosophila brain.

[0058] See attached document Figure 3 According to Table B and Table 2, the levels of ubiquitinated protein and Ref(2)P were increased in the brain of Drosophila in Comparative Example 4; after supplementation with succinic acid in addition to Comparative Example 4, the levels of ubiquitinated protein and Ref(2)P decreased. These results indicate that increasing the level of succinylation in the brain of Drosophila can reduce the accumulation of ubiquitinated protein and Ref(2)P, thereby enhancing lysosomal autophagy.

[0059] Test Example 4: Effects of reduced succinylation levels on lysosomal function in mammalian cells Experimental description: This test was used to evaluate the effect of reduced succinylation levels in mammalian cells on lysosomal function and autophagy-related indicators. HeLa cells were transfected with DLST siRNA to silence DLST expression, reducing succinate production and consequently decreasing succinylation levels. Cells treated with NC siRNA (complementary ratio 5) served as the negative control group, while cells treated with DLST siRNA served as the DLST silencing group. Cellular succinylation levels, p62 protein levels, p62 and LC3 protein half-lives, and lysosomal acidity were measured.

[0060] Experimental steps: HeLa cells were seeded at an appropriate density on cell crawling sheets. After the cells adhered, NCsiRNA or DLST siRNA was transfected into the HeLa cells using Lipo8000 transfection reagent. The nucleotide sequences of the Sense and Antisense chains of DLST siRNA are shown in SEQ ID NO:18 and SEQ ID NO:19, respectively. 48 h after transfection, the culture medium was discarded, and the membrane was fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% PBST for 10 min, and blocked with 5% bovine serum albumin for 1 h. Add succinylated primary antibody and incubate overnight at 4°C. Then add Alexa Fluor-labeled secondary antibody and Hoechst 33342 dye and incubate at room temperature for 2 hours. Immunofluorescence images of succinylated cells were acquired using a confocal microscope after mounting with anti-fluorescence quenching mount. p62 protein primary antibody and AlexaFluor-labeled secondary antibody were used to perform p62 immunofluorescence staining on NCsiRNA-treated cells and DLST siRNA-treated cells. The remaining procedures were the same as those for cell succinylation immunofluorescence staining. HeLa cells were seeded at an appropriate density in 6-well plates. After the cells adhered, NCsiRNA or DLST siRNA was transfected into the HeLa cells using Lipo8000 transfection reagent. 48 hours after transfection, the culture medium was discarded, and the protein synthesis inhibitor cycloheximide was added to the cells at a concentration of 200 μg / mL. Total protein was extracted from cells at 0h, 2h, 4h and 8h, and the levels of p62 and LC3 proteins were detected by Western blotting. HeLa cells were seeded at an appropriate density on cell crawling sheets. After the cells adhered, NCsiRNA or DLST siRNA was transfected into the HeLa cells using Lipo8000 transfection reagent. 48 h after transfection, the culture medium was discarded, and LysoSensorGreenDND-189 probe diluted with culture medium preheated to 37 °C was added at a concentration of 1 μM. The mixture was then incubated at 37 °C for 30 min. Add Hoechst 33342 dye, incubate at 37°C for 30 min, mount with anti-fluorescence quenching mounting medium, and acquire lysosomal acidity staining images using a confocal microscope.

[0061] Experimental data: Table 3. Effects of reduced succinylation levels in mammalian cells on lysosomal function-related indicators ; Experimental conclusion: See attached document Figure 4 NC indicates negative control siRNA treatment, DLST siRNA indicates DLST siRNA treatment. Succinyllysine indicates succinyl lysine signal, Nucleus indicates nuclear signal, p62 indicates autophagy substrate protein signal, LC3 indicates autophagy-related protein signal, and LysoSensor indicates lysosomal acidity probe signal.

[0062] See attached document Figure 4 Tables A and 3 show that, compared to Comparative Example 5, the DLST silencing group exhibited reduced succinylation immunofluorescence signal in cells, indicating that DLST siRNA treatment can decrease the succinylation level in HeLa cells. (See Appendix) Figure 4 B and Table 3 show that the p62 protein signal is enhanced in the DLST silencing group, indicating the accumulation of autophagy substrates in the cells.

[0063] See attached document Figure 4 C. Appendix Figure 4 As shown in Tables D and 3, the half-lives of p62 and LC3 proteins were prolonged in the DLST silencing group, and LysoSensor Green DND-189 signaling was reduced. These results indicate that decreased succinylation levels in mammalian cells lead to decreased lysosomal acidity, accompanied by slowed degradation of autophagy-related proteins, thereby causing lysosomal dysfunction and autophagy inhibition.

[0064] Test Example 5: Identification of Targets Affecting Lysosomal Function by Succinylation Experimental description: This test case was used to screen and identify potential target proteins and their succinylation modification sites that regulate lysosomal function through succinylation. Act88F-Gal4×W1118 Drosophila (Comparative Example 3) served as the negative control group, and Act88F-Gal4×UAS-CG5214RNAi Drosophila (Comparative Example 4) served as the CG5214 knockdown group. Differentially expressed proteins with unchanged expression levels but decreased succinylation modification levels were screened using proteomics and succinylation modification proteomics. Candidate target proteins and their modification sites were then identified using GO analysis, immunoprecipitation verification, and interspecies conservation analysis.

[0065] Experimental steps: Take Drosophila samples from Comparative Examples 3 and 4, collect the heads and thoraxes of 500 Drosophila from each group, add liquid nitrogen and grind thoroughly. Add 4 times the volume of phenol extraction buffer to each group of samples for ultrasonic lysis, add an equal volume of Tris-equilibrated phenol, centrifuge at 5500g for 10 min at 4℃, and take the supernatant. Add 5 times the volume of 0.1M ammonium acetate / methanol solution to the supernatant and precipitate overnight. Wash the precipitate with methanol and acetone respectively, then redissolve the precipitate with 8M urea, and determine the protein concentration using a BCA kit. Take an equal amount of protein, add 20% trichloroacetic acid to precipitate, wash with acetone, dry the precipitate, add trypsin for enzymatic hydrolysis overnight, then reduce with dithiothreitol and alkylate with iodoacetamide. The enzymatically digested peptides were dissolved in IP buffer and transferred to resin conjugated with succinylated antibody. The resin was gently shaken and incubated overnight at 4°C. After incubation, the resin was washed four times with IP buffer and twice with deionized water. The resin-bound peptides were then eluted with 0.1% trifluoroacetic acid elution buffer. The elution buffer was vacuum-frozen and desalted. Data were acquired and analyzed by LC-MS / MS. Differential proteins with unchanged protein expression levels but decreased succinylation modification levels in the head and thorax of Drosophila were screened. GO analysis was performed on the differential proteins, and potential target proteins were identified by combining lysosomal function-related activities. The succinylation modification sites of the potential target proteins were then identified. Take fruit fly samples from Comparative Example 3 and Comparative Example 4, collect the heads of 400 fruit flies in each group, add pre-cooled RIPA lysis buffer, grind and lyse on ice for 30 min, shaking once every 10 min during the process. After lysis, centrifuge at 12000 rpm and 4℃ for 15 min, collect the supernatant to obtain total protein, and use BCA protein quantification kit to determine protein concentration, and adjust the protein concentration of each sample to be consistent. Take a small amount of protein sample and add 5×SDS protein loading buffer. Heat at 95℃ for 10 min to obtain the input sample. Take 1 mg of protein sample and add IgG control antibody or ATP6V0A1 antibody. Add buffer to 500 μL and incubate at 4℃ overnight to form antigen-antibody immune complex. Then add magnetic beads and incubate at room temperature for 1 h. Collect the magnetic beads, wash 3 times with buffer and wash once with ultrapure water. Add elution buffer, incubate at room temperature for 10 min, separate the magnetic beads, collect the supernatant, add neutralization buffer to neutralize the pH, add 5×SDS protein loading buffer, heat at 95℃ for 10 min, detect the levels of β-actin and ATP6V0A1 in the input sample by protein immunoblotting, and detect the levels of ATP6V0A1 and succinylation in the immunoprecipitated sample. The amino acid sequences of Vha100-1 and ATP6V0A1 from different species were retrieved from the UniProt database. The amino acid sequence of the Drosophila Vha100-1 protein is shown in SEQ ID NO:7, and the amino acid sequence of the ATP6V0A1 protein is shown in SEQ ID NO:1. Multiple sequence alignment was performed using the align function to locate succinylation modification sites and compare the amino acid types at the corresponding sites in different species.

[0066] Experimental data: Table 4. Target identification results of succinylation affecting lysosomal function ; Experimental conclusion: See attached document Figure 5 A. Appendix Figure 5 B and Table 4 show that proteomics and succinylation modification screening identified 228 differentially expressed proteins with unchanged expression levels but decreased succinylation modification levels. GO analysis revealed that the enriched pathway related to lysosomal function among the differentially expressed proteins was proton transmembrane transport. Based on lysosomal function-related activities, Vha100-1 was identified as a potential target protein of succinylation affecting lysosomal function, with its mammalian homolog being ATP6V0A1.

[0067] See attached document Figure 5 C. Appendix Figure 5 Table D and Table 4 show the immunoprecipitation results, indicating that the ATP6V0A1 protein level in Comparative Example 4 was comparable to that in Comparative Example 3, but the succinylation modification level of ATP6V0A1 was reduced. Mass spectrometry identification results showed that Vha100-1 and ATP6V0A1 have two succinylation modification sites at lysine positions 295 and 303. The modified peptide sequence corresponding to the K303 site is NWFVK[Succinyl]VR, with a localization probability of 1, no FDR, and a quantification ratio of 0.61563, indicating that the K303 site can be used as a succinylation modification site for ATP6V0A1 for localization and detection.

[0068] See attached document Figure 5 As shown in E and Table 4, interspecies conservation analysis revealed that lysine residues at positions 295 and 303 remained lysine in all species. These results indicate that Vha100-1 and ATP6V0A1 are candidate target proteins for succinylation affecting lysosomal function, and that lysine residue 303 of ATP6V0A1 is an important succinylation site for further verification of its regulatory role in lysosomal function.

[0069] Test Example 6: Target Validation of Succinylation's Effect on Lysosomal Function Experimental description: This test case was used to verify the role of succinylation modification of the ATP6V0A1 protein in regulating lysosomal function. ATP6V0A1 KO cells were constructed using CRISPR / Cas9 gene editing technology and transfected with the ATP6V0A1 wild-type plasmid, the ATP6V0A1K295E point mutant plasmid, the ATP6V0A1K295R point mutant plasmid, the ATP6V0A1K303E point mutant plasmid, and the ATP6V0A1K303R point mutant plasmid, respectively. The effects of different site mutations on lysosomal function were evaluated by detecting p62 protein levels and lysosomal acidity.

[0070] The amino acid sequence of the ATP6V0A1 protein corresponding to the wild-type plasmid is shown in SEQ ID NO:1, and the encoding nucleotide sequence is shown in SEQ ID NO:2. The amino acid sequences of the ATP6V0A1K303E point mutant protein, ATP6V0A1K303R point mutant protein, ATP6V0A1K295E point mutant protein, and ATP6V0A1K295R point mutant protein are shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0071] K303E and K303R are used to simulate different succinylation states at the K303 site of the ATP6V0A1 protein. Succinylation modification changes a positively charged lysine residue to a negatively charged state. K303E mutates lysine to glutamate, introducing a negative charge, to simulate the charge change after succinylation modification; K303R mutates lysine to arginine, retaining the positive charge and blocking succinylation modification at this site, to simulate the desuccinylation state. K295E and K295R are used to functionally distinguish them from the K303 site.

[0072] Experimental steps: ATP6V0A1 KO cells were constructed using CRISPR / Cas9 gene editing technology. The nucleotide sequences of gRNA1 and gRNA2 used are shown in SEQ ID NO:20 and SEQ ID NO:21, respectively. HeLa cells were used as the negative control group, ATP6V0A1 KO cells were used as the ATP6V0A1 deletion model group, and ATP6V0A1 KO cells were transfected with an empty vector plasmid as the empty vector group. The wild-type plasmid ATP6V0A1 was prepared based on the nucleotide sequence encoding ATP6V0A1 shown in SEQ ID NO:2. It was amplified using WT Forward primer and WT Reverse primer. The nucleotide sequences of WT Forward primer and WT Reverse primer are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively. The ATP6V0A1K295E point mutant plasmid was prepared using the K295E forward primer and the K295E reverse primer. The nucleotide sequences of the K295E forward primer and the K295E reverse primer are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively. The ATP6V0A1K295R point mutant plasmid was prepared using the K295R forward primer and the K295R reverse primer. The nucleotide sequences of the K295R forward primer and the K295R reverse primer are shown in SEQ ID NO:12 and SEQ ID NO:13, respectively. The ATP6V0A1K303E point mutant plasmid was prepared using the K303E forward primer and the K303E reverse primer. The nucleotide sequences of the K303E forward primer and the K303E reverse primer are shown in SEQ ID NO:14 and SEQ ID NO:15, respectively. The ATP6V0A1K303R point mutant plasmid was prepared using the K303R forward primer and the K303R reverse primer. The nucleotide sequences of the K303R forward primer and the K303R reverse primer are shown in SEQ ID NO:16 and SEQ ID NO:17, respectively. ATP6V0A1 KO cells were transfected with ATP6V0A1 wild-type plasmid, ATP6V0A1K295E point mutant plasmid, ATP6V0A1K295R point mutant plasmid, ATP6V0A1K303E point mutant plasmid or ATP6V0A1K303R point mutant plasmid respectively. The cells in each group were immunofluorescence stained with p62 protein primary antibody and Alexa Fluor-labeled secondary antibody, and p62 immunofluorescence images of the cells in each group were acquired by confocal microscopy. Lysosomal acidity of cells in each group was detected using the LysoSensorGreenDND-189 probe, and lysosomal acidity staining images of cells in each group were acquired using confocal microscopy.

[0073] Experimental data: Table 5. Basis for simulating different succinylation states using K303E and K303R mutations ; Table 6. Effects of different ATP6V0A1 treatments on p62 levels and lysosomal acidity ; Experimental conclusion: See attached document Figure 6 In ATP6V0A1 KO cells, p62 protein levels were elevated and lysosomal acidity was decreased; however, after ATP6V0A1 KO cells were transfected with an empty vector plasmid, p62 protein levels and lysosomal acidity did not recover significantly. Combined with Table 6, it can be concluded that ATP6V0A1 deficiency leads to the accumulation of the autophagy substrate p62 and affects lysosomal acidification.

[0074] See attached document Figure 6 As shown in Table 6, after transfection of ATP6V0A1 wild-type plasmid into ATP6V0A1 KO cells, p62 protein levels and lysosomal acidity were restored to levels similar to those of HeLa cells; transfection of ATP6V0A1 K303E point mutant plasmid also restored p62 protein levels and lysosomal acidity. These results indicate that the restoration of ATP6V0A1 protein expression and the succinylation-mimicking state at the K303 site are both beneficial for maintaining lysosomal function.

[0075] See attached document Figure 6 Tables 5 and 6 show that the ATP6V0A1 K303R point mutant plasmid could not effectively restore p62 protein levels and lysosomal acidity, while both the ATP6V0A1 K295E and ATP6V0A1 K295R point mutant plasmids could restore the relevant phenotypes. These results indicate that the succinylation state of lysine at position 303 of the ATP6V0A1 protein is more directly related to lysosomal acidity and function, and that the K303 site is an important succinylation site for ATP6V0A1 in regulating lysosomal function.

[0076] Test Example 7: Effects of succinic acid supplementation on AD-related phenotypes in Drosophila with Alzheimer's disease (AD). Experimental description: This test case was used to evaluate the effects of exogenous succinate supplementation on related phenotypes in an AD model of Drosophila. Using the QF / QUAS transgenic system, the neuron-specific driver strain Nsyb-QF and the Aβ42 overexpression strain QUAS-Aβ42 were employed to specifically overexpress Aβ42 in Drosophila neurons, thus constructing an AD model. Based on Comparative Examples 1 and 2, Drosophila were treated with 2% succinate supplementation, and their lifespan, climbing ability, cognitive function, and brain Aβ deposition were assessed.

[0077] Experimental steps: Nsyb-QF×W1118 Drosophila from Comparative Example 1 were fed standard food and served as the negative control group; Nsyb-QF×W1118 Drosophila from Comparative Example 1 were fed food containing 2% succinic acid and served as the negative control supplemented with succinic acid group; Nsyb-QF×QUAS-Aβ42 Drosophila from Comparative Example 2 were fed standard food and served as the AD model group; Nsyb-QF×QUAS-Aβ42 Drosophila from Comparative Example 2 were fed food containing 2% succinic acid and served as the AD model supplemented with succinic acid group. Each group of fruit flies was separated by sex and transferred to plastic rearing bottles, 100 flies per bottle. They were reared at 25℃ and 60% humidity. The fruit fly food was changed every 2 days, and the number of dead fruit flies was recorded until all the fruit flies in the bottle died. Prism statistical software was used to analyze the lifespan data, Kaplan-Meier method was used to draw survival curves, and Log-rank test was used for intergroup comparisons. Take 4 tubes of fruit flies for each group, totaling 80 flies. Transfer them to climbing tubes marked 6cm above the bottom. After training 3 times, gently tap the bottom of the tube to make the fruit flies fall to the bottom. Start timing immediately and count the number of fruit flies that climb over the 6cm mark in each tube within 10 seconds. Calculate the percentage of fruit flies that climb over the mark. 400 fruit flies were collected for each group, and 80 fruit flies were placed in a cognitive function testing device. Two different odors were applied to both ends of the device. During the training phase, no treatment was performed when odor A was applied, and an electric shock was applied simultaneously when odor B was applied. The association between odor B and electric shock was established after 3 training sessions. During the testing phase, only odor A and odor B were applied, and the number of fruit flies at the odor A end, the odor B end, and the middle area was counted. Fruit flies in the middle area were not included in the calculation. The learning and memory index was calculated by subtracting the number of fruit flies at the odor A end from the number of fruit flies at the odor B end, and then dividing the difference by the sum of the number of fruit flies at the odor A end and the number of fruit flies at the odor B end. The brains of Drosophila in each group were stained with Aβ immunofluorescence using a β-Amyloid protein primary antibody. The remaining procedures were the same as those for brain succinylation immunofluorescence staining. Confocal microscopy was used to acquire Aβ immunofluorescence images of the brains of Drosophila in each group.

[0078] Experimental data: Table 7. Effects of succinic acid supplementation on AD-related phenotypes in Drosophila aeruginosa. ; Experimental conclusion: See attached document Figure 7 And Table 7, Nsyb-QF represents the neuron-specific driving strain, Ctrl represents the W1118 negative control fruit fly, QUAS-Aβ42 represents the Aβ42 overexpressing fruit fly, and Succinate represents succinic acid treatment. (Appendix) Figure 7 A corresponds to the lifetime count result, attached. Figure 7B corresponds to the climbing ability test results, attached. Figure 7 C corresponds to the cognitive function test results, attached. Figure 7 D corresponds to the brain Aβ immunofluorescence staining result.

[0079] See attached document Figure 7 A. Appendix Figure 7 B. Appendix Figure 7 As shown in Table C and Table 7, compared with Comparative Example 1, Comparative Example 2 showed a shorter lifespan, reduced climbing ability, and a lower learning and memory index. Supplementing Comparative Example 2 with succinic acid extended the lifespan of the AD model fruit flies, and improved their climbing ability and learning and memory index. These results indicate that succinic acid supplementation can improve the survival, movement, and cognitive phenotypes of the AD model fruit flies.

[0080] See attached document Figure 7 As shown in Tables D and 7, no Aβ signaling was observed in the brains of Drosophila in Comparative Example 1 and after succinic acid supplementation. Extensive Aβ deposition was observed in the brains of Drosophila in Comparative Example 2. After succinic acid supplementation in addition to Comparative Example 2, Aβ deposition in the brains of Drosophila decreased. Combined with the results on lifespan, climbing ability, and cognitive function, it can be concluded that increasing the succinylation level at the ATP6V0A1K303 site can improve the relevant phenotypes in the AD model Drosophila and reduce Aβ deposition.

[0081] Appendix: Amino acid sequence of ATP6V0A1 protein: SEQ ID NO:1: MGELFRSEEMTLAQLFLQSEAAYCCVSELGELGKVQFRDLNPDVNVFQRKFVNEVRRCEEMDRKLRFVEKEIRKANIPIMDTGENPEVPFPRDMIDLEANFEKIENELKEINTNQEALKRNFLELTELKFILRKTQQFFDEAELHHQQMADPDLLEESSSLLEPSEMGRGTPLRLGFVAGVINRERIPTFERMLWRVCRGNVFLRQAEIENPLEDPVTGDYVHKSVFIIFFQGDQLKNRVKKICEGFRASLYPCPETPQERKEMASGVSTRIDDLQMVLNQMEDHRQRVLQAAAKNIRVWFIKVRKMKAIYHTLNLCNIDVTQKCLIAEVWCPVTDLDSIQFALRRGTEHSGSTVPSILNRMQTNQTPPTYNKTNKFTYGFQNIVDAYGIGTYREINPAPYTIITFPFLFAVMFGDFGHGILMTLFAVWMVLRESRILSQKNENEMFSTVFSGRYIILLMGVFSMYTGLIYNDCFSKSLNIFGSSWSVRPMFTYNWTEETLRGNPVLQLNPALPGVFGGPYPFGIDPIWNIATNKLTFLNSFKMKMSVILGIIHMLFGVSLSLFNHIYFKKPLNIYFGFIPEIIFMTSLFGYLVILIFYKWTAYDAHTSENAPSLLIHFINMFLFSYPESGYSMLYSGQKGIQCFLVVVALLCVPWMLLFKPLVLRRQYLRRKHLGTLNFGGIRVGNGPTEEDAEIIQHDQLSTHSEDADEFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLSVKSLAGGLVLFFFFTAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFEE; Coding nucleotide sequence of ATP6V0A1: SEQ ID NO: 2: Amino acid sequence of ATP6V0A1 K303E mutant protein: SEQ ID NO: 3: MGELFRSEEMTLAQLFLQSEAAYCCVSELGELGKVQFRDLNPDVNVFQRKFVNEVRRCEEMDRKLRFVEKEIRKANIPIMDTGENPEVPFPRDMIDLEANFEKIENELKEINTNQEALKRNFLELTELKFILRKTQQFFDEAELHHQQMADPDLLEESSSLLEPSEMGRGTPLRLGFVAGVINRERIPTFERMLWRVCRGNVFLRQAEIENPLEDPVTGDYVHKSVFIIFFQGDQLKNRVKKICEGFRASLYPCPETPQERKEMASGVSTRIDDLQMVLNQMEDHRQRVLQAAAKNIRVWFIEVRKMKAIYHTLNLCNIDVTQKCLIAEVWCPVTDLDSIQFALRRGTEHSGSTVPSILNRMQTNQTPPTYNKTNKFTYGFQNIVDAYGIGTYREINPAPYTIITFPFLFAVMFGDFGHGILMTLFAVWMVLRESRILSQKNENEMFSTVFSGRYIILLMGVFSMYTGLIYNDCFSKSLNIFGSSWSVRPMFTYNWTEETLRGNPVLQLNPALPGVFGGPYPFGIDPIWNIATNKLTFLNSFKMKMSVILGIIHMLFGVSLSLFNHIYFKKPLNIYFGFIPEIIFMTSLFGYLVILIFYKWTAYDAHTSENAPSLLIHFINMFLFSYPESGYSMLYSGQKGIQCFLVVVALLCVPWMLLFKPLVLRRQYLRRKHLGTLNFGGIRVGNGPTEEDAEIIQHDQLSTHSEDADEFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLSVKSLAGGLVLFFFFTAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFEE; Amino acid sequence of ATP6V0A1 K303R mutant protein: SEQ ID NO: 4: MGELFRSEEMTLAQLFLQSEAAYCCVSELGELGKVQFRDLNPDVNVFQRKFVNEVRRCEEMDRKLRFVEKEIRKANIPIMDTGENPEVPFPRDMIDLEANFEKIENELKEINTNQEALKRNFLELTELKFILRKTQQFFDEAELHHQQMADPDLLEESSSLLEPSEMGRGTPLRLGFVAGVINRERIPTFERMLWRVCRGNVFLRQAEIENPLEDPVTGDYVHKSVFIIFFQGDQLKNRVKKICEGFRASLYPCPETPQERKEMASGVSTRIDDLQMVLNQMEDHRQRVLQAAAKNIRVWFIRVRKMKAIYHTLNLCNIDVTQKCLIAEVWCPVTDLDSIQFALRRGTEHSGSTVPSILNRMQTNQTPPTYNKTNKFTYGFQNIVDAYGIGTYREINPAPYTIITFPFLFAVMFGDFGHGILMTLFAVWMVLRESRILSQKNENEMFSTVFSGRYIILLMGVFSMYTGLIYNDCFSKSLNIFGSSWSVRPMFTYNWTEETLRGNPVLQLNPALPGVFGGPYPFGIDPIWNIATNKLTFLNSFKMKMSVILGIIHMLFGVSLSLFNHIYFKKPLNIYFGFIPEIIFMTSLFGYLVILIFYKWTAYDAHTSENAPSLLIHFINMFLFSYPESGYSMLYSGQKGIQCFLVVVALLCVPWMLLFKPLVLRRQYLRRKHLGTLNFGGIRVGNGPTEEDAEIIQHDQLSTHSEDADEFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLSVKSLAGGLVLFFFFTAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFEE; Amino acid sequence of ATP6V0A1 K295E mutant protein: SEQ ID NO: 5: MGELFRSEEMTLAQLFLQSEAAYCCVSELGELGKVQFRDLNPDVNVFQRKFVNEVRRCEEMDRKLRFVEKEIRKANIPIMDTGENPEVPFPRDMIDLEANFEKIENELKEINTNQEALKRNFLELTELKFILRKTQQFFDEAELHHQQMADPDLLEESSSLLEPSEMGRGTPLRLGFVAGVINRERIPTFERMLWRVCRGNVFLRQAEIENPLEDPVTGDYVHKSVFIIFFQGDQLKNRVKKICEGFRASLYPCPETPQERKEMASGVSTRIDDLQMVLNQMEDHRQRVLQAAAENIRVWFIKVRKMKAIYHTLNLCNIDVTQKCLIAEVWCPVTDLDSIQFALRRGTEHSGSTVPSILNRMQTNQTPPTYNKTNKFTYGFQNIVDAYGIGTYREINPAPYTIITFPFLFAVMFGDFGHGILMTLFAVWMVLRESRILSQKNENEMFSTVFSGRYIILLMGVFSMYTGLIYNDCFSKSLNIFGSSWSVRPMFTYNWTEETLRGNPVLQLNPALPGVFGGPYPFGIDPIWNIATNKLTFLNSFKMKMSVILGIIHMLFGVSLSLFNHIYFKKPLNIYFGFIPEIIFMTSLFGYLVILIFYKWTAYDAHTSENAPSLLIHFINMFLFSYPESGYSMLYSGQKGIQCFLVVVALLCVPWMLLFKPLVLRRQYLRRKHLGTLNFGGIRVGNGPTEEDAEIIQHDQLSTHSEDADEFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLSVKSLAGGLVLFFFFTAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFEE; Amino acid sequence of ATP6V0A1 K295R mutant protein: SEQ ID NO: 6: MGELFRSEEMTLAQLFLQSEAAYCCVSELGELGKVQFRDLNPDVNVFQRKFVNEVRRCEEMDRKLRFVEKEIRKANIPIMDTGENPEVPFPRDMIDLEANFEKIENELKEINTNQEALKRNFLELTELKFILRKTQQFFDEAELHHQQMADPDLLEESSSLLEPSEMGRGTPLRLGFVAGVINRERIPTFERMLWRVCRGNVFLRQAEIENPLEDPVTGDYVHKSVFIIFFQGDQLKNRVKKICEGFRASLYPCPETPQERKEMASGVSTRIDDLQMVLNQMEDHRQRVLQAAARNIRVWFIKVRKMKAIYHTLNLCNIDVTQKCLIAEVWCPVTDLDSIQFALRRGTEHSGSTVPSILNRMQTNQTPPTYNKTNKFTYGFQNIVDAYGIGTYREINPAPYTIITFPFLFAVMFGDFGHGILMTLFAVWMVLRESRILSQKNENEMFSTVFSGRYIILLMGVFSMYTGLIYNDCFSKSLNIFGSSWSVRPMFTYNWTEETLRGNPVLQLNPALPGVFGGPYPFGIDPIWNIATNKLTFLNSFKMKMSVILGIIHMLFGVSLSLFNHIYFKKPLNIYFGFIPEIIFMTSLFGYLVILIFYKWTAYDAHTSENAPSLLIHFINMFLFSYPESGYSMLYSGQKGIQCFLVVVALLCVPWMLLFKPLVLRRQYLRRKHLGTLNFGGIRVGNGPTEEDAEIIQHDQLSTHSEDADEFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLSVKSLAGGLVLFFFFTAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFEE; Amino acid sequence of Drosophila Vha100-1 protein: SEQ ID NO:7: MGSLFRSEEMALCQLFLQSEAAYACVSELGELGLVQFRDLNPDVNAFQRKFVNEVRRCDEMERKLRYLEKEIKKDGIPMLDTGESPEAPQPREMIDLEATFEKLENELREVNQNAEALKRNFLELTELKHILRKTQVFFDESVPTVYKSSGAYSSSKYRRYPQMADNQNEDEQAQLLGEEGVRASQPGQNLKLGFVAGVILRERLPAFERMLWRACRGNVFLRQAMIETPLEDPTNGDQVHKSVFIIFFQGDQLKTRVKKICEGFRATLYPCPEAPADRREMAMGVMTRIEDLNTVLGQTQDHRHRVLVAAAKNLKNWFVKVRKIKAIYHTLNLFNLDVTQKCLIAECWVPLLDIETIQLALRRGTERSGSSVPPILNRMQTFENPPTYNRTNKFTKAFQALIDAYGVASYREMNPAPYTIITFPFLFAVMFGDLGHGAIMALFGLWMIRKEKGLAAQKTDNEIWNIFFGGRYIIFLMGVFSMYTGLIYNDIFSKSLNIFGSHWHLSYNKSTVMENKFLQLSPKGDYEGAPYPFGMDPIWQVAGANKIIFHNAYKMKISIIFGVIHMIFGVVMSWHNHTYFRNRISLLYEFIPQLVFLLLLFFYMVLLMFIKWIKFAATNDKPYSEACAPSILITFIDMVLFNTPKPPPENCETYMFMGQHFIQVLFVLVAVGCIPVMLLAKPLLIMQARKQANVQPIAGATSDAEAGGVSNSGSHGGGGGHEEEEELSEIFIHQSIHTIEYVLGSVSHTASYLRLWALSLAHAQLAEVLWTMVLSIGLKQEGPVGGIVLTCVFAFWAILTVGILVLMEGLSAFLHTLRLHWVEFQSKFYKGQGYAFQPFSFDAIIENGAAAAEE; Mutant primer nucleotide sequence: SEQ ID NO: 8: TAGCGTTTAAACTTAAGCTTGCCACCATGGACTACAAGGACGACGA; SEQ ID NO: 9: CAGCGGGTTTAAACGGGCCCTCTAGActactcttcaaacttcccttccc; SEQ ID NO: 10: aggcagctgctgagaacatccgtgtctggttcatcaaagt; SEQ ID NO: 11: gaaccagacacggatgttctcagcagctgcctgcagaacc; SEQ ID NO: 12: ggttctgcaggcagctgctaGgaacatccgtgtctggttc; SEQ ID NO: 13: gaaccagacacggatgttcCtagcagctgcctgcagaacc; SEQ ID NO: 14: catccgtgtctggttcatcGaagtgcggaagatgaaggcc; SEQ ID NO: 15: ggccttcatcttccgcacttCgatgaaccagacacggatg; SEQ ID NO: 16: tccgtgtctggttcatcaGagtgcggaagatgaaggc; SEQ ID NO: 17: ttcatcttccgcactCtgatgaaccagacacggat; Nucleotide sequence of DLST siRNA: SEQ ID NO: 18: GCGUUUGCAGAAUCUGUCA(dT)(dT); SEQ ID NO: 19: UGACAGAUUCUGCAAACGC(dT)(dT); Nucleotide sequence of ATP6V0A1 KO gRNA1: SEQ ID NO: 20: TTTGGTGATGCAAGAACAACAGG; Nucleotide sequence of ATP6V0A1 KO gRNA2: SEQ ID NO:21: TGGCTGTGCACTCCTGGTAGAGG。

Claims

1. The application of the ATP6V0A1 K303 succinylation site in the preparation of a kit for screening candidate drugs for the treatment of neurodegenerative diseases, characterized in that, The kit includes a succinylation modification level detection reagent at the K303 site of the ATP6V0A1 protein. The amino acid sequence of the ATP6V0A1 protein is shown in SEQ ID NO:1, and the K303 site is the 303rd lysine residue in the amino acid sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The neurodegenerative diseases mentioned are Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis (ALS).

3. The application according to claim 1, characterized in that, The assay reagent for detecting the succinylation modification level at the K303 site of the ATP6V0A1 protein includes a reagent for detecting the modified peptide NWFVK[Succinyl]VR by LC-MS / MS, wherein K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:

1.

4. The application according to claim 1, characterized in that, The kit also includes an immunoassay kit for the succinylation modification level of the ATP6V0A1 protein, which includes an ATP6V0A1 antibody and a succinylated antibody for detecting succinylation modification.

5. An in vitro method for screening candidate drugs for treating neurodegenerative diseases, characterized in that, Includes the following steps: Take a system containing ATP6V0A1 protein, the amino acid sequence of which is shown in SEQ ID NO:1, and the K303 position is the 303rd lysine in the amino acid sequence shown in SEQ ID NO:1; Add the candidate drug to the system containing ATP6V0A1 protein; The candidate drug was incubated with the system containing ATP6V0A1 protein; After incubation, the succinylation level of the K303 site of the ATP6V0A1 protein in the system containing the ATP6V0A1 protein after treatment with the candidate drug was detected. The succinylation modification level of the ATP6V0A1 protein K303 site in the system containing ATP6V0A1 protein after treatment with the candidate drug is compared with the succinylation modification level of the ATP6V0A1 protein K303 site in the system containing ATP6V0A1 protein before treatment with the candidate drug. The candidate drugs were screened to increase the level of succinylation modification at the K303 site of the ATP6V0A1 protein.

6. The in vitro method according to claim 5, characterized in that, In the system containing ATP6V0A1 protein before the candidate drug treatment, the succinylation modification level of the K303 site of the ATP6V0A1 protein was detected before the candidate drug was added to the system containing ATP6V0A1 protein.

7. The in vitro method according to claim 5, characterized in that, The system containing ATP6V0A1 protein is a cell system containing the ATP6V0A1 protein.

8. The in vitro method according to claim 5, characterized in that, The method for detecting the succinylation modification level at the K303 site of the ATP6V0A1 protein includes LC-MS / MS detection, wherein the target of the LC-MS / MS detection includes the modified peptide NWFVK[Succinyl]VR, and K[Succinyl] corresponds to lysine at position 303 of the amino acid sequence shown in SEQ ID NO:

1.

9. The in vitro method according to claim 5, characterized in that, The in vitro method also includes an immunoassay step to detect the level of ATP6V0A1 protein succinylation modification.

10. The in vitro method according to claim 9, characterized in that, The immunoassay step for the succinylation modification level of the ATP6V0A1 protein includes immunoprecipitation of the ATP6V0A1 protein with an ATP6V0A1 antibody, and detection of the succinylation modification level of the ATP6V0A1 protein with a succinylation antibody for detecting succinylation modification.

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