Use of trime11 protein in preparation of medicine for treating spinocerebellar ataxia 51

Abstract

The application discloses application of TRIM11 protein in preparation of a medicine for treating spinocerebellar ataxia 51. The application adopts an SCA51 specific cell model, and identifies, through a large number of screening experiments, that the TRIM11 protein has the function of significantly removing mutant THAP11 protein and aggregates thereof. Experimental data show that the mutant protein removal efficiency of the TRIM11 treatment group is significantly better than that of the control group. The application verifies the removal effect of TRIM11 on the SCA51 pathological protein from the two dimensions of protein expression level and subcellular localization through Western blotting quantitative analysis and immunofluorescence co-localization technology. The three-dimensional reconstruction image clearly shows that the mutant protein aggregates are significantly reduced. The application first establishes a complete research system from target discovery to mechanism analysis, and provides a new intervention strategy for precise treatment of SCA51.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological drugs, and particularly relates to application of TRIM11 protein in preparation of a drug for treating spinocerebellar ataxia type 51. BACKGROUND

[0002] Spinocerebellar ataxia type 51 (SCA51) is an autosomal dominant neurodegenerative disease, and its pathogenesis is abnormal expansion (repeat number > 38) of CAG trinucleotide repeats in the THAP11 gene, which leads to the production of mutant THAP11 protein containing abnormally expanded polyglutamine (polyQ) chains.

[0003] Studies have shown that mutant THAP11 protein is widely distributed in the central nervous system, especially showing specific high expression in cerebellar tissue, which may be an important reason why the cerebellar brain region is particularly sensitive to mutant THAP11 protein toxicity. There is sufficient evidence that mutant THAP11 protein is a key factor in triggering neuronal damage, so targeted clearance of the mutant protein to reduce neurotoxicity has become a promising treatment strategy, which has been widely verified in the treatment of various neurodegenerative diseases.

[0004] SCA diseases are caused by abnormal repeat expansion of CAG trinucleotides in specific genes, and the mutant proteins caused by CAG repeat expansion in different SCA diseases have their own unique pathological characteristics and significant differences in structure and function. It is completely unpredictable whether TRIM11 can interact with these different mutant SCA proteins and promote their clearance. It is worth noting that as a newly discovered rare disease, SCA51 currently has no effective clinical treatment drugs. SUMMARY

[0005] The application provides application of TRIM11 protein in preparation of a drug for treating spinocerebellar ataxia type 51.

[0006] The purpose of the application is achieved by the following technical solutions:

[0007] Application of overexpressed TRIM11 protein in preparation of a drug for treating spinocerebellar ataxia type 51.

[0008] Application of a gene expressing TRIM11 protein in preparation of a drug for treating spinocerebellar ataxia type 51.

[0009] The application of the overexpression vector carrying the TRIM11 protein gene in the preparation of a drug for treating spinocerebellar ataxia type 51

[0010] Preferably, the overexpression vector carrying the TRIM11 protein gene is ssAAV-TRIM11-HA, and the sequence is shown as SEQ ID NO. 3.

[0011] The present application adopts an SCA51 specific cell model, and through a large number of screening experiments, the TRIM11 protein is first identified to have the functional property of significantly removing mutant THAP11 protein and its aggregates. Experimental data show that the mutant protein removal efficiency of the TRIM11 treatment group is significantly better than that of the control group.

[0012] The present application verifies the TRIM11 removal effect on the SCA51 pathological protein from two dimensions of protein expression level and subcellular localization through Western blotting quantitative analysis and immunofluorescence co-localization technology. The three-dimensional reconstruction image clearly shows that the mutant protein aggregates are significantly reduced.

[0013] The present application comprehensively uses co-immunoprecipitation (Co-IP), ubiquitination detection and autophagy pathway inhibitor experiments to clarify that TRIM11 plays a role through the following molecular pathways: directly combining with mutant THAP11 protein; promoting ubiquitination modification thereof; and completing final degradation through the autophagy-lysosome pathway.

[0014] The present application first establishes a complete research system from target discovery to mechanism analysis, and provides a new intervention strategy for the precise treatment of SCA51.

[0015] The present application has the following advantages and effects relative to the prior art:

[0016] 1. Pioneering treatment target: as the first specific treatment strategy for SCA51 in the world, the present application fills the blank in the treatment field of the disease. Compared with the condition that there is no effective intervention means in the prior art, the present application first confirms the removal effect of TRIM11 on the pathogenic THAP11 mutant protein, and provides a key breakthrough for clinical treatment.

[0017] 2. Mechanism innovation: the present application first clarifies the mechanism that TRIM11 removes mutant protein through the ubiquitination-autophagy pathway, and the discovery is not only suitable for the treatment of SCA51, but also provides a new research idea for the treatment of other polyQ related neurodegenerative diseases.

[0018] 3. Treatment specificity: the present application experimentally confirms that TRIM11 has specific binding to the mutant THAP11 protein, and significantly improves the safety expectation of treatment.

[0019] 4. Transformation medical value: The complete evidence chain from basic research to preclinical research has been established for the present application, which lays a solid foundation for subsequent drug development and clinical trials, and has a more clear transformation application prospect compared with the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the constituent elements of the TRIM11 overexpression vector and expression verification results; wherein A: a schematic diagram of the constituent elements of the TRIM11 overexpression vector, B: expression results of the TRIM11 overexpression vector in HEK293 cells.

[0021] Figure 2 is the interaction analysis of TRIM11 and mutant THAP11 protein in the examples; wherein A: Western Blot detection of protein interaction results; B: immunofluorescence staining experiment results.

[0022] Figure 3 is the analysis results of the interaction of TRIM11 and other PolyQ proteins and its clearance effect in the examples; wherein A: Western Blot detection of protein interaction results; B: Western Blot protein expression level; C and D: quantitative analysis of B figure results; E: Western Blot detection of protein interaction results; F: Western Blot protein expression level; G and H: quantitative analysis of F figure results.

[0023] Figure 4 is an immunofluorescence staining experiment; wherein A: immunofluorescence staining experiment results; B: quantitative analysis of A figure.

[0024] Figure 5 is the influence of overexpression and knockdown of TRIM11 on the solubility and aggregate homeostasis of mutant THAP11 protein in the examples; wherein A: Western Blot protein expression level; B and C are quantitative analysis figures of A figure, D: Western Blot protein expression level; E and F are quantitative analysis figures of D figure.

[0025] Figure 6 is the influence of TRIM11 on the expression of mutant THAP11 protein in the examples; wherein A: Western Blot detection of small ubiquitin-like protein expression level; B: quantitative analysis results of A figure; C: Western Blot detection of ubiquitin-like protein expression level; D: quantitative analysis of C figure; E: Western Blot protein expression level. DETAILED DESCRIPTION

[0026] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0027] Example 1: Construction of TRIM11 overexpression vector (ssAAV-TRIM11-HA)

[0028] According to the human TRIM11 gene sequence (NM_145214.3) included in the NCBI database, the specific primers are designed as follows:

[0029] Forward primer: 5'-ccgaccggtgccaccatggccgcccccgacctgtcc-3' (SEQ ID NO. 1)

[0030] Reverse primer: 5'-ccggaattcttaagcataatctggaacatcatatggatactggggagccagggtgtccc-3' (SEQ ID NO. 2, wherein the HA tag sequence is introduced)

[0031] PCR amplification is performed using high-fidelity enzymes to obtain the target fragment. Subsequently, the purified PCR product and the ssAAV-CMV-EGFP vector (purchased from PeiGen Biotechnology) are subjected to double enzyme digestion (37°C, 4 hours) using AgeI and EcoRI endonucleases, respectively. The enzyme digestion products are separated by 1% agarose gel electrophoresis, and the target band is cut and the DNA fragment is purified using a gel recovery kit, and the concentration is measured. The purified PCR fragment and the linearized vector are mixed at an appropriate ratio (1:4), and an equal volume (5 μL) of Solution I ligase system (containing T4 DNA ligase) is added for ligation reaction. The ligation product is transformed into Trans1-T1 competent cells, and plated on LB plates containing ampicillin (AMP) resistance and incubated at 37°C overnight. The next day, single colonies are picked for small-scale shake culture, and plasmid DNA is extracted using a plasmid extraction kit. After sequence verification (the sequence of ssAAV-TRIM11-HA is shown as SEQ ID NO. 3), the TRIM11 overexpression vector is successfully constructed (the schematic diagram of its constituent elements is shown as Figure 1 A), and subsequent protein expression verification experiments are performed.

[0032] Example 2: Expression verification of TRIM11 overexpression vector

[0033] To confirm whether the TRIM11 protein overexpression plasmid with HA tag constructed in Example 1 can be correctly expressed in cells, it was transfected into HEK293 cells, and an empty plasmid was set as a control. After 48 hours of transfection, the cells were collected, total protein was extracted, and Western Blot analysis was performed. The results showed that both the specific antibody targeting TRIM11 and the specific antibody targeting HA tag detected characteristic bands at the expected protein molecular weight (B), confirming that the plasmid can correctly express the TRIM11 protein. Figure 1

[0034] Example 3: Co-immunoprecipitation verifies the interaction of TRIM11 with various mutant PolyQ proteins

[0035] To explore the interaction of TRIM11 with different mutant PolyQ proteins, the expression plasmids of mutant HTT (N171) (which has been disclosed in the literature LI C, et al. Adv Sci. 2023, 10(31): e2301120.), mutant ATAXIN3 (which has been disclosed in the literature CHANG J C, et al. Scientific reports, 2016, 6: 30436.), and mutant THAP11 (which has been disclosed in the literature RUAN E, et al. The Journal of clinical investigation, 2025, 135(14)) were co-transfected with the TRIM11 overexpression plasmid (i.e., the TRIM11 overexpression vector of Example 1; hereinafter the same) into HEK293 cells. After 24 hours of transfection, the cell lysate was collected, and immunoprecipitation was performed using a TRIM11 specific antibody, followed by Western Blot (WB) detection of the presence of corresponding mutant PolyQ proteins in the precipitated complex.

[0036] The results showed that mutant THAP11 protein could be detected in the TRIM11 immunoprecipitation group, while the control group IgG (Dr. De BA1045) had no corresponding signal (A), indicating that TRIM11 can specifically bind to mutant THAP11. Figure 2

[0037] However, in the co-immunoprecipitation experiment of mutant HTT (N171) and TRIM11, neither the TRIM11 precipitated group nor the IgG control group detected mutant HTT (N171) protein (A), indicating that there is no interaction (no specific binding) between TRIM11 and mutant HTT (N171). Figure 3

[0038] ​​​Likewise, in the co-immunoprecipitation experiment of mutant ATAXIN3 and TRIM11, no mutant ATAXIN3 protein was detected (Fig. 6B), suggesting no binding between TRIM11 and mutant ATAXIN3. Figure 3 E), suggesting no binding between TRIM11 and mutant ATAXIN3.

[0039] In summary, these results suggest that the interaction between TRIM11 and different variant PolyQ proteins is obviously selective, suggesting that it may have different functional characteristics in clearing different variant PolyQ proteins.

[0040] Example 4: Immunofluorescence experiment to detect the interaction between TRIM11 and mutant THAP11 protein.

[0041] To verify the interaction between TRIM11 and mutant THAP11 protein, the experiment was carried out according to the following steps:

[0042] First, pre-coated polylysine glass slides were placed in a 12-well plate, and then well-conditioned HEK293 cells were inoculated. After the cells were attached and grew to the appropriate density, the TRIM11 overexpression plasmid and mutant THAP11 protein expression plasmid constructed in Example 1 were co-transfected into HEK293 cells using Lipo3000 liposome transfection reagent. After 24 hours of transfection, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with blocking solution (3% BSA + 2% NGS) for one hour. Then, the mutant THAP11 protein antibody (Proteintech, Cat No. 23030-1-AP) and the TRIM11 protein antibody (Proteintech, Cat No. 66006-2- Ig) were added, respectively, and incubated overnight at 4°C on a horizontal shaker. The next day, the primary antibody was recovered, and the cells were washed with PBS three times for 10 minutes each time; then, according to the species origin of the primary antibody, fluorescent secondary antibodies were added (Jackson ImmunoResearch Inc, West Grove, PA, USA, antirabbit Alexa Fluor® 594, Cat No. 711-585-152; anti-mouse Alexa Fluor® 488, Cat No. 715-545-151), and incubated at room temperature for 1 hour in the dark. After incubation, the secondary antibody was removed, and the cells were washed with PBS again three times for 10 minutes each time. Finally, DAPI was used for nuclear staining, and after staining for 1 minute, ddH2O was used for washing three times, anti-quenching agent was added on the glass slide, the glass slide was covered, and the mounting was completed. Whether there was co-localization between TRIM11 protein and mutant THAP11 protein was observed and compared by fluorescence microscopy.

[0043] The experimental results show that the green fluorescence signal of the TRIM11 protein and the red fluorescence signal of the mutant THAP11 protein have obvious overlap (B), and the results show that the TRIM11 protein and the mutant THAP11 protein have co-localization, indicating that the TRIM11 protein and the mutant THAP11 protein have interaction. Figure 2 B), and the results show that the TRIM11 protein and the mutant THAP11 protein have co-localization, indicating that the TRIM11 protein and the mutant THAP11 protein have interaction.

[0044] Example 5: Immunofluorescence method for detecting the clearing effect of TRIM11 on mutant THAP11 protein aggregates

[0045] To verify the clearing effect of TRIM11 on SCA51 pathogenic protein (mutant THAP11 protein), the experiment was performed according to the following steps:

[0046] First, the pre-coated polylysine glass slides were placed in a 12-well plate, and then the well-grown HEK293 cells were inoculated. After the cells were attached and grew to the appropriate density, the TRIM11 overexpression plasmid constructed in Example 1 and the mutant THAP11 protein expression plasmid were co-transfected into HEK293 cells using Lipo3000 liposome transfection reagent. After 12 hours of transfection, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with blocking solution (3% BSA + 2% NGS) for one hour. Then, antibodies targeting the mutant THAP11 protein and TRIM11 were added, and incubated overnight at 4°C on a horizontal shaker. The next day, the primary antibody was recovered, and the cells were washed with PBS three times for 10 minutes each time; then, according to the species origin of the primary antibody, fluorescent secondary antibodies were added, and incubated at room temperature for 1 hour in the dark. After incubation, the secondary antibody was discarded, and the cells were washed with PBS again three times for 10 minutes each time. Finally, DAPI was used for nuclear staining, and after staining for 1 minute, ddH2O was used for washing three times, and the mounting was completed. The mutant THAP11 protein aggregate situation in the TRIM11 group and the control group was observed and compared by fluorescence microscope, to evaluate the clearing effect of TRIM11 on it.

[0047] The results show that no mutant THAP11 protein aggregates are observed in cells overexpressing TRIM11, while obvious mutant THAP11 protein aggregates are observed in control cells overexpressing GFP (A and B). This result indicates that overexpression of TRIM11 protein can significantly clear mutant THAP11 protein aggregates. Figure 4 A and B). This result indicates that overexpression of TRIM11 protein can significantly clear mutant THAP11 protein aggregates.

[0048] The above results show that overexpression of TRIM11 protein can significantly promote the clearing of mutant THAP11 protein aggregates.

[0049] Example 6: Evaluation of the scavenging effect of TRIM11 overexpression on HD, SCA3, and SCA51-related variant PolyQ proteins using Western Blot.

[0050] Western blotting was used to verify the clearance effect of TRIM11 on HD, SCA3 and SCA51-related variant PolyQ proteins.

[0051] In this experiment, expression plasmids of mutant HTT (N171), mutant ATAXIN3, and mutant THAP11 were co-transfected into HEK293 cells with the TRIM11 overexpression plasmid constructed in Example 1 (or GFP control). Cells were collected 48 hours after transfection, and the expression levels of mutant PolyQ protein in each group were detected by Western blotting.

[0052] The results showed that in the SCA51 cell model (i.e., the expression plasmid of mutant THAP11), the TRIM11 protein overexpression group significantly reduced the solubility and aggregate form of mutant THAP11 protein compared with the GFP control group (purchased from Paizhen Biotechnology). Figure 5 (A, B, and C); This result fully demonstrates that TRIM11 protein can promote the clearance of aggregates and soluble THAP11 protein.

[0053] In the SCA3 cell model (i.e., the expression plasmid of mutant ATAXIN3), TRIM11 overexpression was able to simultaneously eliminate both soluble and aggregate forms of mutant ATAXIN3. Figure 3 F, G, and H), while in the HD cell model (i.e., the expression plasmid of mutant HTT (N171)), TRIM11 only cleared aggregates of mHTT (N171), and no significant clearing was observed for the soluble form of mHTT (N171). Figure 3 (B, C, and D).

[0054] Example 7: Western Blot was used to evaluate the effect of TRIM11 knockdown on the homeostasis of mutant THAP11 protein in SCA51.

[0055] In this experiment, the expression plasmid of the mutant THAP11 protein was co-transfected into HEK293 cells with the shTRIM11-1 plasmid (disclosed in Chinese patent application CN113908280A) and the control plasmid (Scramble shRNA, purchased from Guangzhou Aiji Biotechnology Co., Ltd.). Cells were collected 48 hours after transfection, and the expression levels of the mutant THAP11 protein in each group were detected by Western blotting.

[0056] The results show that: compared with the control group, the solubility and aggregate form of mutant THAP11 protein in the knockdown group are significantly increased Figure 5 D, E and F), which further proves that the TRIM11 protein can promote the clearance of mutant THAP11 protein.

[0057] Example 8: Detection of TRIM11 on post-translational modification of mutant THAP11

[0058] To explore the effect of TRIM11 overexpression on ubiquitination and small ubiquitin-like modification of mutant THAP11 protein, this study used Western blotting to analyze the regulatory effect of TRIM11 on post-translational modification of mutant THAP11 protein in SCA51 disease model.

[0059] The mutant THAP11 expression plasmid was co-transfected with the TRIM11 overexpression plasmid (or GFP control plasmid) constructed in Example 1 into HEK293 cells. After 48 hours of transfection, the cell samples were collected, and the changes in ubiquitination and SUMO modification levels of mutant THAP11 protein under TRIM11 overexpression were detected by Western Blot.

[0060] The results show that: overexpression of TRIM11 can significantly promote ubiquitination modification of mutant THAP11 protein, increase its degradation signal ( Figure 6 C and D), while no significant effect on SUMO modification was found ( Figure 6 A and B).

[0061] Example 9: Pathway inhibition experiment to analyze the molecular mechanism of TRIM11 clearing mutant THAP11 protein

[0062] To explore the molecular pathway on which TRIM11 depends to clear mutant THAP11 protein, this study conducted a pathway-specific inhibition experiment.

[0063] HEK293 cells in good growth condition were seeded in a six-well plate, and after the cell state was stable, mutant THAP11 and TRIM11 overexpression plasmid constructed in Example 1 were co-transfected. After 12 hours of transfection, different treatment groups were set: adding proteasome inhibitor MG132 (10 μM), autophagy inhibitor Bafilomycin A1 (100 nM), and no drug control group. After 12 hours of inhibitor treatment, the cells were collected, and the degradation of mutant THAP11 protein under different inhibition conditions was analyzed by Western Blot.

[0064] The results of the pathway inhibition experiment show that the removal of the aggregates and soluble THAP11 protein is not obviously weakened in the MG132 group, and the removal of the aggregates and soluble THAP11 protein is obviously hindered in the BafA1 group, which fully proves that TRIM11 mainly mediates the removal of the mutant THAP11 protein through the autophagy pathway Figure 6 E}.

[0065] Summary: The present study successfully constructs the overexpression vector (ssAAV-TRIM11-HA) of TRIM11. In vitro experiments prove that the expression of TRIM11 can effectively remove the aggregates and soluble forms of the SCA51 pathogenic protein mutant THAP11 protein, thereby significantly alleviating the neurotoxicity mediated thereby.

[0066] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. Application of an overexpression vector carrying the gene encoding TRIM11 protein in the preparation of a drug for treating spinocerebellar ataxia type 51.

2. The application according to claim 1, characterized in that: The overexpression vector carrying the gene encoding the TRIM11 protein is ssAAV-TRIM11-HA; The sequence of ssAAV-TRIM11-HA is shown in SEQ ID NO.3.

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