RNF130 gene responding to BMAA signal, coded protein and application

Abstract

The invention discloses an RNF130 gene responding to a BMAA signal, an encoded protein and application, and the RNF130 gene is found to be a gene strongly responding to the BMAA signal through high-throughput screening. After the RNF130 gene is knocked out, cell proliferation inhibition caused by BMAA is reduced for mammalian cells. In addition, the RNF 130 transduction the BMAA signal to the downstream GSK-3beta protein, thereby correlating with the Wnt signaling pathway. After the activity of GSK-3beta is inhibited by using a small-molecule GSK-3beta inhibitor, the reaction of RNF130 to a BMAA signal is changed, and the influence of cell proliferation inhibition caused by BMAA can be relieved by cells. According to the present invention, the cell proliferation inhibition caused by the BMAA can be relieved by the knockout of the RNF130 gene and the inhibition of the GSK-3beta activity, such that the key response protein and the molecular mechanism of the cell proliferation toxicity caused by the algal toxin BMAA are provided so as to provide the important reference target for the research and development of the antidote for preventing or treating the BMAA toxicity or the research and development of the drug aiming at the RNF130 related structural domain in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the RNF130 gene, the encoded protein, and applications thereof that respond to BMAA signals, and more specifically to the cell proliferation inhibition effect caused by the RNF130 gene, the encoded protein, and the downstream signaling pathway under the treatment of the algal toxin β-methylamino-L-alanine (BMAA). Background Art

[0002] The algal toxin β-methylamino-L-alanine (BMAA), isolated from cycads in 1967, is believed to be a potential cause of Guam-type amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC). Administration of BMAA to mice, rats, chickens, and monkeys via feeding and injection has resulted in varying degrees of neuronal cell damage and neurodegenerative disease symptoms. Therefore, ingestion of BMAA carries a potential risk of causing neurodegenerative diseases.

[0003] There are many speculations about the molecular mechanism by which BMAA causes nerve damage, including the hypothesis that BMAA forms glutamate analogs that cause neuronal excitotoxicity, and that it is incorrectly incorporated into serine sites during protein synthesis. However, no BMAA receptor has been found in animals, that is, no receptor or protein has been found that responds to BMAA signals in the first place.

[0004] BMAA is widely distributed in the environment. It is produced by most algae, including cyanobacteria, dinoflagellates, and diatoms, which make up a significant portion of the biosphere. Furthermore, BMAA can accumulate in large quantities in fish, shellfish, and even chickens through the food chain, contaminating human food. Ingestion of BMAA by humans can also cause some degree of neurological damage.

[0005] At the cellular level, BMAA treatment inhibits the proliferation of both neural and non-neuronal cells. This inhibition of cell proliferation is a sign of cellular damage. Leveraging this phenotype, we used high-throughput screening to identify a BMAA-responsive protein on the cell membrane. This protein responds to BMAA signaling during BMAA treatment, and in its absence, the BMAA-induced inhibition of cell proliferation is largely eliminated.

[0006] GSK-3β and the Wnt signaling pathway are crucial signaling pathways in cell proliferation. The discovery of the RNF130 gene linked BMAA signaling to the Wnt signaling pathway via GSK-3β, explaining the mechanism by which BMAA inhibits cell proliferation.

[0007] Based on existing research at home and abroad, the present invention studies the molecular mechanism of the toxicity of the algal toxin BMAA, discovers that the cell surface protein RNF130 is highly responsive to BMAA signals, and finds the downstream related signaling pathways that RNF130 responds to BMAA signals. This is of great significance for the development of related drugs and the prevention or treatment of acute or long-term nerve damage and related neurodegenerative diseases caused by BMAA. Summary of the Invention

[0008] The purpose of the present invention is to provide an RNF130 gene that responds to BMAA signals, the encoded protein and applications.

[0009] In order to achieve the above object, the technical solution of the present invention is as follows:

[0010] The RNF130 gene, whose nucleotide sequence is shown in SEQ ID NO.1, consists of 1260 bases. The encoded protein includes an extracellular domain, a transmembrane sequence and an intracellular signal transduction domain, with a total length of 419 amino acids. The amino acid sequence is shown in SEQ ID NO.2.

[0011] The extracellular domain is encoded by the sequence of SEQ ID NO.3, and the sequence of the intracellular signal transduction domain is encoded by the sequence of SEQ ID NO.4.

[0012] The sequence of the extracellular domain of RNF130 protein is shown in SEQ ID NO.5, and the sequence of the intracellular signal transduction domain is shown in SEQ ID NO.6

[0013] Due to post-transcriptional modifications, the RNF130 protein exists in multiple isoforms, including one or more isoforms that do not contain the complete extracellular sequence of SEQ ID NO. 3 in addition to the full-length protein. The protein encoded by RNF130 may also include an RNF130-derived protein having BMAA-responsive function formed by substitution, deletion, or addition of one or more (e.g., 1-30; preferably 1-20; more preferably 1-10, such as 5 or 3) amino acid residues of the amino acid sequence of SEQ ID NO. 2; or an RNF130-derived protein having 80% (preferably greater than 90%, such as 95%, 98%, 99% or higher) homology with the protein sequence of SEQ ID NO. 2 and having BMAA signaling response function.

[0014] One of the most important points of the present invention is the disclosure of the response function of RNF130 to the algal toxin BMAA and the downstream proteins and their signaling pathways that cooperate in the response. The present invention obtains RNF130 gene knockout mutants through gene editing, and uses the phenotype of cell proliferation inhibition caused by BMAA. Through cell proliferation counting, it is found that the RNF130 gene knockout mutant reduces the cell proliferation inhibition caused by BMAA. Through truncation experiments, it is confirmed that the extracellular structure of RNF130 includes the sequence portion of SEQ ID NO.3, and this domain directly or indirectly contacts BMAA and responds to BMAA; the intracellular structure of RNF130 includes the sequence portion of SEQ ID NO: 4, and this domain directly or indirectly interacts with GSK-3β, and this interaction affects the degradation of RNF130 itself and the traditional substrates of GSK-3β.

[0015] Therefore, the present invention mainly protects the use of the RNF130 gene or protein and the expression cell line containing the gene or protein in the inhibition of cell proliferation caused by responding to the algal toxin BMAA signal, wherein the cells include 293T cells and nerve cells. The specific manifestation of responding to the algal toxin BMAA signal is: under the stimulation of the BMAA signal (that is, when BMAA poisoning is present in the cell), the proliferation rate of cells containing the RNF130 gene slows down; after inhibiting the expression of the RNF130 gene or inhibiting the activity of the RNF130 protein or destroying the RNF130 protein structure, the cell proliferation rate is no longer inhibited, and the toxicity of BMAA to the cells is reduced.

[0016] During high-throughput screening and validation, mutations in multiple genes related to the Wnt signaling pathway all led to a weakening of the proliferation inhibition caused by BMAA. This established a link between RNF130 and the Wnt signaling pathway. The present invention also discovered during the experimental process that GSK-3β acts as a transit protein that transduces signals to the Wnt signaling pathway after BMAA receives a response from the RNF130 gene, effectively reducing the proliferation inhibition caused by BMAA in 293T cells and neural cells. Specifically,

[0017] (1) GSK-3β responds to BMAA signaling in the presence of RNF130;

[0018] (2) To undertake the signal transduction of RNF130 after contact with BMAA;

[0019] (3) After receiving the BMAA signal from RNF130, it takes on the degradation function within the cell and affects the Wnt signaling pathway.

[0020] In view of the conclusion of the above study, the present invention also protects a method for reducing the cytotoxicity of BMAA, which comprises the following (1) or (2) or (3):

[0021] (1) Reduce the activity and content of RNF130 protein in normal cells, thereby reducing the proliferation inhibition caused by BMAA;

[0022] (2) Inhibit the expression of RNF130 gene in normal cells, thereby reducing the proliferation inhibition caused by BMAA;

[0023] (3) Inhibit GSK-3β activity and inhibit the downstream of intracellular BMAA signaling, thereby reducing the proliferation inhibition caused by BMAA on cells;

[0024] Among them, the ways to inhibit the expression of RNF130 gene in normal cells include:

[0025] (1) Knockout the RNF130 gene to obtain an RNF130 knockout mutant;

[0026] (2) using small RNA interference and other methods to reduce RNF130 gene expression;

[0027] (3) Mutate the sequence of the RNF130 gene to change the structure of RNF130 and affect its response to BMAA signals.

[0028] The present invention proves through experiments that the RNF130 protein containing an extracellular domain and an intracellular domain is required to respond to the BMAA signal. If the complete structure of RNF130 is destroyed, RNF130 will not respond to the BMAA signal.

[0029] The complete structure of RNF130 can be destroyed by truncating the portion of the RNF130 sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0030] Therefore, through the response of RNF130 on the cell membrane to the extracellular BMAA signal, the signal is transduced to GSK-3β and the Wnt signaling pathway is affected: drugs developed for RNF130 and its downstream signaling pathways including GSK-3β to alleviate or prevent cell damage caused by BMAA can relieve the acute or long-term cell damage caused by BMAA; or drugs or compounds developed for RNF130 to regulate the activity of GSK-3β are all inspired by the present invention and therefore fall within the scope of protection of the present invention.

[0031] Advantages of the present invention:

[0032] This study first revealed a novel function of the RNF130 gene, which can respond to BMAA signals and lead to a phenotype of cell proliferation inhibition. The study also discovered that RNF130, as a BMAA-responsive protein, transduces signals to the GSK-3β and Wnt signaling pathways, playing a crucial role in BMAA-induced cell proliferation toxicity.

[0033] The present invention discovered through high-throughput screening that RNF130 is a gene that strongly responds to BMAA signals. After knocking out the RNF130 gene, mammalian cells reduced the cell proliferation inhibition caused by BMAA. In addition, RNF130 transduces BMAA signals to the downstream GSK-3β protein, thereby associating with the Wnt signaling pathway. After using a small molecule GSK-3β inhibitor to inhibit GSK-3β activity, the response of RNF130 to BMAA signals changes, and the cells can also partially relieve the effects of cell proliferation inhibition brought by BMAA. In addition, the present invention also learned through experiments that the response to BMAA signals requires an RNF130 protein containing an extracellular domain and an intracellular domain, and when the signal appears, it interacts with the intracellular GSK-3β to affect the cells. Therefore, knockout of the RNF130 gene and inhibition of GSK-3β activity can both relieve the cell proliferation inhibition caused by BMAA. The present invention provides the key response proteins and molecular mechanisms of cell proliferation toxicity caused by the algal toxin BMAA, providing important reference targets for the future development of antidotes for preventing or treating BMAA toxicity or the development of drugs targeting RNF130-related domains. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Evidence of RNF130 gene knockout;

[0035] Figure 2 Evidence that BMAA inhibits the proliferation of non-neuronal 293T cells;

[0036] Figure 3 Evidence that BMAA causes inhibition of neuronal proliferation;

[0037] Figure 4 Evidence that RNF130 knockout cells relieved BMAA-induced cell proliferation inhibition;

[0038] Figure 5 Evidence that GSK-3β inhibitors relieve BMAA-induced inhibition of 293T cell proliferation;

[0039] Figure 6 Evidence that the full-length RNF130 protein interacts with GSK-3β under BMAA treatment. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0041] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0043] At the cellular level, BMAA treatment leads to the inhibition of cell proliferation in both neural and non-neuronal cells. Cell proliferation inhibition is a manifestation of cell damage. Using this phenotype, we found the protein RNF130 on the cell membrane of BMAA through high-throughput screening. This protein can respond to BMAA signals under BMAA treatment. Once the protein receptor is missing, the cell proliferation inhibition caused by BMAA signals can be relieved. Therefore, during the experiment, we first obtained RNF130 gene knockout mutants, and compared the cell number of RNF130 gene knockout mutants with that of the control group to verify the function of RNF130 gene in inhibiting cell proliferation caused by the response to the algal toxin BMAA signal. In addition, the present invention also used GSK-3β inhibitor treatment, and the results showed that it can increase cell proliferation and relieve the cell proliferation inhibition caused by BMAA. Through the construction of RNF130 gene and its truncated form overexpression vector and subcellular co-localization experiment with GSK-3β, it was concluded that the response to BMAA signal requires RNF130 protein containing extracellular domain and intracellular domain. The specific experimental process is as follows:

[0044] Example 1 Acquisition of RNF130 gene knockout mutants

[0045] The full-length RNF130 gene contains 1260 nucleotides, as shown in SEQ ID NO.1. The full-length protein obtained by translation contains 419 amino acid residues, and the amino acid sequence is shown in SEQ ID NO.2.

[0046] 1. Construction of RNF130 gene knockout vector

[0047] Primers were designed according to sgRNA (Table 1). After annealing, double-stranded DNA with sticky ends was obtained, which was ligated into the expression plasmid using T4 nucleic acid ligase and expression was initiated by the U6 promoter.

[0048] Table 1. RNF130 mutant knockout primers

[0049]

[0050] 2. Construction of Cas9 Stable Expression Cell Line

[0051] The Cas9 protein coding sequence was cloned into a lentiviral expression vector, along with a nuclear localization signal and antibiotic resistance gene. The vector, along with a lentiviral packaging helper vector, was then transformed into a 293T cell line. After 48 hours, the cells and culture medium containing the lentivirus were harvested and filtered through a 0.45 μm pore size filter, harvesting only the cell culture supernatant containing the virus.

[0052] The virus-containing supernatant is then added to the culture medium of the target cell line to allow the virus to fully infect the cells. After 48 hours, the appropriate antibiotic is added. Antibiotic-resistant cells—those with the lentiviral expression vector integrated into their genomes—survive the antibiotic treatment, while cells without the expression vector are killed by the antibiotic. This results in a cell population stably expressing Cas9.

[0053] To obtain a stable cell line, the cell population needs to be isolated by flow cytometry or infinite dilution to obtain single cells, and then the single cells are cultured into cell lines to obtain monoclonal cell lines.

[0054] 3. Obtaining RNF130 gene knockout cell population by liposome transfection and antibiotic selection

[0055] RNF130 gene knockout cell populations were obtained in two ways: one was to introduce the sgRNA expression vector into the target cell line stably expressing Cas9 by transfection, and use an empty expression plasmid as a control to obtain RNF130 knockout cell populations through high-efficiency plasmid transfection; the other was to introduce the lentivirus containing the sgRNA sequence into the target cell line stably expressing Cas9 by infection, and use cells infected with the lentivirus packaged with the empty plasmid as a control group to obtain RNF130 knockout cell populations through efficient viral infection. This type of RNF130 knockout cell population can then be killed by adding corresponding antibiotics to cells that do not express sgRNA, thereby obtaining RNF130 gene knockout mutants with higher knockout efficiency.

[0056] The RNF130 gene knockout mutant obtained by the above method was verified by western blotting to verify the expression of RNF130 protein in the cells. Compared with wild-type 293T cells, the expression of GAPDH protein was used as a reference, and the RNF130 protein in the 293T cell line after CRISPR knockout decreased by more than 75% ( Figure 1 ), indicating that the RNF130 gene knockout mutant was successfully constructed.

[0057] Example 2 Effects of RNF130 gene and GSK-3β inhibitor on cell proliferation ability

[0058] 1. Cell Counting and Splitting

[0059] 293T cells: Disintegrate 293T cells into single cells using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) digestion solution. Stain the cells with 0.4% trypan blue solution and count them using a hemocytometer. Count 10,000-20,000 cells and distribute them into 24-well plates to allow them to grow.

[0060] Neural cells: The mouse pluripotent stem cell line E14 was digested into single cells using 0.25% trypsin-EDTA digestion solution, and the cells were diluted to a density of 500 cells / 20μl using differentiation medium (15% fetal bovine serum, high glucose DMEM medium, β-mercaptoethanol, L-glutamine, antibiotics and sodium pyruvate), and placed on the cover of the culture dish in a hanging drop manner for 2 days. The droplets were then collected to obtain embryoid bodies of uniform size, which were placed in differentiation medium for 2 days. The embryoid bodies were collected using a centrifugation speed of 200g and placed in a differentiation medium containing 5μM retinoic acid (RA) for 4 days. These initially differentiated neural embryoid bodies were then collected using a centrifugation speed of 200g. The collected neural embryoid bodies were digested with trypsin and passed through a 40μm nylon mesh to obtain the digested cells. At 1.5*10 5 / em 2 Cells were cultured at a density of 100 μg / mL in N2 medium (DMEM / F12 medium, N2 supplement, 10 ng / mL bFGF, L-glutamine, and antibiotics). After one day, the N2 medium was replaced with N2B27 medium (50% DMEM / F12 medium, 50% Neural Basal medium, 1 / 2 N2 supplement, 1 / 2 B27 supplement, L-glutamine, and antibiotics). After one day, in vitro differentiated neural cells were obtained as material for the next step of the experiment.

[0061] 2. BMAA and GSK-3β Inhibitor Treatment

[0062] Dilute BMAA powder with sterile deionized water to obtain a final concentration of approximately 100 mM BMAA solution. Add the BMAA solution to the cell culture medium, while a control group containing sterile deionized water was used.

[0063] A GSK-3β inhibitor (purchased from the market) was diluted with DMSO and added to the cell culture medium. A control group was supplemented with the same amount of DMSO.

[0064] 3. Cell Counting

[0065] After a sufficient treatment time, 293T cells and neural cells were digested into single cells using 0.25% trypsin-EDTA digestion solution, and then the cells were stained with 0.4% trypan blue staining solution. The unstained living cells were counted using a hemocytometer.

[0066] BMAA significantly inhibits cell growth. Under a bright field microscope, it can be clearly observed that the number of 293T cells and neural cells treated with BMAA is significantly reduced compared to the control group treated with deionized water, indicating that BMAA has an inhibitory effect on cell proliferation. For 293T cells, the number of cells decreased significantly when treated with 1mM BMAA, and the reduction in cell number was even more significant when using a higher concentration of 3mM BMAA, indicating that higher concentrations of BMAA have a stronger inhibitory effect on cell proliferation ( Figure 2 In nerve cells, only 0.1mM concentration of BMAA can cause very significant inhibition of nerve cells, indicating that nerve cells are more sensitive to BMAA concentration ( Figure 3 BMAA can inhibit cell proliferation in different cell lines, indicating that the mechanism by which BMAA inhibits cell growth is quite extensive.

[0067] The RNF130 gene in 293T cells was knocked out using the method of Example 1, and then the cells were treated by adding BMAA to the cell culture medium using Example 2. Compared with the control group transfected with the empty plasmid, the RNF130 gene knockout mutant was no longer affected by BMAA, that is, the proliferation inhibition phenotype no longer appeared ( Figure 4 ), indicating that the RNF130 gene knockout mutant no longer responds to BMAA signaling, and thus also shows that the RNF130 gene plays a key role in the BMAA signaling response.

[0068] The cells were treated by adding BMAA or GSK-3β inhibitor to the cell culture medium in Example 2, with BMAA added to the same amount of deionized water as the control group (marked as not added), GSK-3β inhibitor added to the same amount of DMSO as the control group (marked as not added), and the group of BMAA and GSK-3B inhibitors added to the same amount of deionized water and DMSO as the control group (marked as not added). By comparison, it can be seen that BMAA has a significant inhibitory effect on 293T cells, and the inhibitory effect of the high concentration (3mM BMAA) group is more obvious than that of the low concentration (1mM BMAA) group. The GSK-3β inhibitor can increase the proliferation of 293T cells. Compared with the control group, the high concentration (50nM CHIR-99021) group has a more obvious promoting effect than the low concentration (20nM CHIR-99021) group. When both are added at the same time, compared with the control group with the same amount of deionized water and DMSO, 293T cells no longer show a significant inhibitory phenotype, that is, they are no longer sensitive to the signal of BMAA ( Figure 5 Experimental results show that GSK-3β inhibitors can reduce the cell proliferation inhibition caused by BMAA, indicating that the activity of GSK-3β is also critical for the response to BMAA signaling.

[0069] Example 3 Construction of RNF130 protein and its truncation overexpression vector and subcellular colocalization with GSK-3β

[0070] 1. Overexpression vector construction

[0071] Cellular RNA was extracted using Trizol, and eDNA was obtained by reverse transcription. The RNF130 gene was amplified by polymerase chain reaction (PCR) to obtain the full-length sequence SEQ ID NO. 1. A FLAG tag (DYKDDDDK) was added to the C-terminus using primers. Sequences encoding the extracellular domain of RNF130, SEQ ID NO. 3, and the intracellular domain, SEQ ID NO. 4, were also amplified using PCR. Primers were also used to add a FLAG tag to the C-terminus of the protein, and the protein was then recombinantly loaded into the pcDNA3.1 vector.

[0072] 2. Protein Expression

[0073] The recombinant vector was delivered into 293T cells by liposome transfection, and G418 was used for screening to enrich cells overexpressing the gene.

[0074] 3. Immunofluorescence Staining

[0075] The cells overexpressing the gene were fixed with 4% PFA for 10 minutes, then washed three times with PBS, and then the fixed cells were punched with a punching solution (PBS, 0.1% Triton X-100, 0.1% Tween-20) for 15 minutes, and then washed three times with PBS. The punched cells were treated with a blocking solution (PBS, 0.1% Tween-20, 3% BSA) to block non-specific sites. The primary antibody was diluted with the blocking solution and incubated overnight at 4°C. The next day, the cells were washed three times with PBS containing 0.1% Tween-20, and the secondary antibody was also diluted with the blocking solution, incubated at room temperature for more than 2 hours, and then washed three times with PBS containing 0.1% Tween-20. The cell nuclei were then stained with DAPI and washed three times with PBS. Finally, the sections were sealed with a mounting agent and observed using a fluorescence confocal microscope.

[0076] Taking advantage of the FLAG tag at the carboxyl terminus of the overexpressed protein, a FLAG tag antibody was used to recognize the overexpressed full-length and truncated RNF130 proteins, which were labeled with AlexaFluor 594 fluorescence (red), while GSK-3β was labeled with AlexaFluor 488 fluorescence (green), and the nuclear area marked by DAPI was labeled in blue.

[0077] Full-length RNF130 is localized in the cytoplasm. In the absence of BMAA, GSK-3β is also localized in the cytoplasm, but there is little co-localization between the two. However, after adding BMAA, it can be clearly seen that the red RNF130 and the green GSK-3β co-localize more in the cytoplasm (yellow), and a larger cytoplasmic complex is formed. At the same time, the RNF130 signal decreases significantly. This indicates that when the BMAA signal appears, the full-length RNF130 interacts with the GSK-3β in the cytoplasm, thereby affecting downstream cellular functions. The extracellular domain of RNF130 is localized on the cytoplasmic membrane. After adding BMAA, there is no significant change in the co-localization signal with GSK-3β in the cytoplasm, and the signal on the cell membrane does not show obvious degradation, indicating that expressing only the extracellular domain of RNF130 cannot fully respond to the BMAA signal. Similarly, the intracellular domain of RNF130 is located in the cytoplasm rather than on the cytoplasmic membrane. When BMAA is added, the intracellular domain protein of RNF130 in the cytoplasm does not show obvious changes, indicating that the intracellular domain of RNF130 alone cannot respond to the BMAA signal. Figure 6 ).

[0078] In summary, the response to BMAA signaling requires the RNF130 protein, which contains both extracellular and intracellular domains. When the signal is present (i.e., when BMAA toxicity is present in cells), it interacts with intracellular GSK-3β to exert its effects on cells. Both RNF130 gene knockout and GSK-3β inhibition can relieve BMAA-induced cell proliferation inhibition, providing important information for the future development of drugs to prevent or treat BMAA toxicity.

[0079] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. The RNF130 gene responding to BMAA signaling is characterized by: The nucleotide sequence of the RNF130 gene is shown in SEQ ID NO.

1. The protein encoded by the RNF130 gene includes an extracellular domain, a transmembrane sequence and an intracellular signal transduction domain, wherein the extracellular domain is encoded by the SEQ ID NO.3 sequence and the sequence of the intracellular signal transduction domain is encoded by the SEQ ID NO.4 sequence.

2. The protein encoded by the RNF130 gene according to claim 1, characterized in that The amino acid sequence of the RNF130 protein is shown in SEQ ID NO. 2, wherein the sequence of the extracellular domain is shown in SEQ ID NO. 5, and the sequence of the intracellular signal transduction domain is shown in SEQ ID NO.

6.

3. Use of the RNF130 gene according to claim 1 or the protein according to claim 2 and a cell line expressing said gene or protein in inhibiting cell proliferation in response to a microalgal toxin BMAA signal, characterized in that: The cells are 293T cells and neural cells.

4. The use according to claim 3, characterized in that The specific manifestations of responding to the algal toxin BMAA signal are: under the stimulation of the BMAA signal, the proliferation rate of cells containing the RNF130 gene slows down; after inhibiting the expression of the RNF130 gene or inhibiting the activity of the RNF130 protein or destroying the RNF130 protein structure, the cell proliferation rate is no longer inhibited and the toxicity of BMAA to the cells is reduced.

5. The use according to claim 4, characterized in that GSK-3β acts as a transfer protein to the Wnt signaling pathway after BMAA receives RNF130 gene response, specifically manifested in: (1) GSK-3β responds to BMAA signaling in the presence of the RNF130 gene; (2) Initiate signal transduction of the RNF130 gene after exposure to BMAA; (3) After receiving the BMAA signal from the RNF130 gene, it takes on the degradation function within the cell and affects the Wnt signaling pathway.

6. A method for reducing the cytotoxicity of BMAA, characterized in that: The method includes the following (1) or (2) or (3): (1) Reduce the activity and content of RNF130 protein in normal cells, thereby reducing the proliferation inhibition caused by BMAA; (2) Inhibit the expression of RNF130 gene in normal cells, thereby reducing the proliferation inhibition caused by BMAA; (3) Inhibit GSK-3β activity and inhibit the downstream of intracellular BMAA signaling, thereby reducing the proliferation inhibition caused by BMAA on cells; The nucleotide sequence of the RNF130 gene is shown in SEQ ID NO.1, and the amino acid sequence of the RNF130 protein is shown in SEQ ID NO.

2.

7. The method for reducing cytotoxicity of BMAA according to claim 6, characterized in that: Ways to inhibit the expression of the RNF130 gene in normal cells include: (1) Knockout the RNF130 gene to obtain an RNF130 knockout mutant; (2) using small RNA interference to reduce RNF130 gene expression; (3) Mutate the sequence of the RNF130 gene to change the structure of RNF130.

8. The method for reducing cytotoxicity of BMAA according to claim 7, characterized in that: Changing the RNF130 structure means destroying the complete structure of RNF130, affecting its response to BMAA signals or affecting its signal transduction to GSK-3β.

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