Application of TRIM11 protein in preparation of medicine for treating spinal cerebellar ataxia 51

Abstract

The invention discloses an application of TRIM11 protein in preparation of a medicine for treating spinal cerebellar ataxia 51 type. According to the application, an SCA51 specific cell model is adopted, and the fact that the TRIM11 protein has the functional characteristic of remarkably eliminating the mutant THAP11 protein and the aggregate thereof is identified for the first time through a large number of screening experiments. Experimental data show that the mutant protein removal efficiency of a TRIM11 treatment group is obviously superior to that of a control group. According to the application, through Western blotting quantitative analysis and an immunofluorescence co-localization technology, the removal effect of the TRIM11 on the SCA51 pathological protein is systematically verified from two dimensions of protein expression level and subcellular localization. A three-dimensional reconstructed image clearly shows that mutant protein aggregates are obviously reduced. According to the invention, a complete research system from target discovery to mechanism analysis is established for the first time, and a brand new intervention strategy is provided 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 area is particularly sensitive to mutant THAP11 protein toxicity. There is sufficient evidence that mutant THAP11 protein is a key factor in causing neuronal damage, so targeted clearance of the mutant protein to reduce neurotoxicity has become a very promising treatment strategy, which has been widely verified in the treatment of various neurodegenerative diseases.

[0004] SCA diseases are caused by abnormal 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: Application of overexpressed TRIM11 protein in preparation of a drug for treating spinocerebellar ataxia type 51.

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

[0008] Application of an overexpression vector carrying a TRIM11 protein gene in preparation of a drug for treating spinocerebellar ataxia type 51. Preferably, the overexpression vector carrying the TRIM11 protein gene is ssAAV-TRIM11-HA, and the sequence is shown as SEQ ID NO. 3.

[0009] The present application adopts the SCA51 specific cell model, and through a large number of screening experiments, the TRIM11 protein is first identified to have the function of significantly removing the 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.

[0010] The present application verifies the TRIM11 removal effect on the SCA51 pathological protein from the protein expression level and the 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.

[0011] The present application comprehensively uses the co-immunoprecipitation (Co-IP), ubiquitination detection and autophagy pathway inhibitor experiment to clarify that the TRIM11 plays a role through the following molecular pathways: directly combining with the mutant THAP11 protein; promoting the ubiquitination modification; and completing the final degradation through the autophagy-lysosome pathway.

[0012] 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.

[0013] The present application has the following advantages and effects relative to the prior art: 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.

[0014] 2. Mechanism innovation: the present application first clarifies the mechanism that the TRIM11 removes the 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.

[0015] 3. Treatment specificity: the present application experimentally confirms that the TRIM11 has specific binding to the mutant THAP11 protein, and significantly improves the safety expectation of treatment.

[0016] 4. Value of translational medicine: the present application has established a complete evidence chain from basic research to preclinical research, and laid a solid foundation for subsequent drug development and clinical trials, and has a more definite application prospect in translation compared with the prior art. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the constituent elements of the TRIM11 overexpression vector and the expression verification results; where A: schematic diagram of the constituent elements of the TRIM11 overexpression vector, and B: expression results of the TRIM11 overexpression vector in HEK293 cells.

[0018] Figure 2 This is an example of the interaction analysis between TRIM11 and the mutant THAP11 protein; where A: Western Blot detection results of protein interaction; B: Immunofluorescence staining results.

[0019] Figure 3 Figure 1 shows the analysis results of the interaction between TRIM11 and other PolyQ proteins and their clearance effect in the examples; where A: Western Blot detection of protein interaction results; B: Western Blot protein expression level; C and D: Quantitative analysis of the results in Figure B; E: Western Blot detection of protein interaction results; F: Western Blot protein expression level; G and H: Quantitative analysis of the results in Figure F.

[0020] Figure 4 This is an immunofluorescence staining experiment; where A: results of the immunofluorescence staining experiment; B: quantitative analysis of figure A.

[0021] Figure 5 The examples illustrate the effects of overexpression and knockdown of TRIM11 on the solubility and aggregate homeostasis of mutant THAP11 protein; where A represents Western Blot protein expression levels; B and C are quantitative analysis plots of plot A; D represents Western Blot protein expression levels; and E and F are quantitative analysis plots of plot D.

[0022] Figure 6 The following figures illustrate the effect of TRIM11 on the expression of mutant THAP11 protein in the examples; where A: Western Blot detection of small ubiquitin-like protein expression level; B: Quantitative analysis results of Figure A; C: Western Blot detection of ubiquitin-like protein expression level; D: Quantitative analysis of Figure C; E: Western Blot protein expression level. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] Example 1: Construction of TRIM11 overexpression vector (ssAAV-TRIM11-HA) Based on the human TRIM11 gene sequence (NM_145214.3) in the NCBI database, the following specific primers were designed: Forward primer: 5'-ccgaccggtgccaccatggccgcccccgacctgtcc-3' (SEQ ID NO. 1) Reverse primer: 5'-ccggaattcttaagcataatctggaacatcatatggatactggggagccagggtgtccc-3' (SEQ ID NO. 2, in which an HA tag sequence is introduced) PCR amplification was performed using high-fidelity enzymes to obtain the target fragment. Subsequently, the purified PCR product and the ssAAV-CMV-EGFP vector (purchased from Paizhen Biotechnology) were double-digested with AgeI and EcoRI restriction enzymes (37℃, 4 hours). The digested products were separated by 1% agarose gel electrophoresis, and the target band was excised and purified using a gel extraction kit to determine its concentration. The purified PCR fragment and the linearized vector were mixed at an appropriate ratio (1:4), and an equal volume (5 μL) of Solution I ligase system (containing T4 DNA ligase) was added for ligation. The ligation product was transformed into Trans1-T1 competent cells, plated on LB agar plates containing ampicillin (AMP) resistance, and incubated overnight at 37℃ inverted. The next day, single colonies were picked for small-scale shake culture, and plasmid DNA was extracted using a plasmid miniprep kit. After sequencing verification confirmed the sequence was correct (the sequence of ssAAV-TRIM11-HA is shown in SEQ ID NO. 3), the TRIM11 overexpression vector was successfully constructed (its constituent elements are shown in the schematic diagram below). Figure 1 As shown in A), subsequent protein expression verification experiments were conducted.

[0025] Example 2: Expression validation of TRIM11 overexpression vector To confirm whether the HA-tagged TRIM11 protein overexpression plasmid constructed in Example 1 could be correctly expressed in cells, it was transfected into HEK293 cells, with an empty plasmid used as a control. Cells were collected 48 hours after transfection, and total protein was extracted and analyzed by Western blotting. The results showed that both the specific antibody targeting TRIM11 and the specific antibody targeting the HA tag exhibited characteristic bands at the expected protein molecular weight. Figure 1 (B) This confirms that the plasmid can correctly express the TRIM11 protein.

[0026] Example 3: Immunoprecipitation verification of the interaction between TRIM11 and various variant PolyQ proteins To investigate the interaction between TRIM11 and different variant PolyQ proteins, we co-transfected HEK293 cells with expression plasmids of mutant HTT (N171) (disclosed in LI C, et al. Adv Sci. 2023, 10(31): e2301120.), mutant ATAXIN3 (disclosed in CHANG JC, et al. Scientific reports, 2016, 6: 30436.), and mutant THAP11 (disclosed in RUAN E, et al. The Journal of clinical investigation, 2025, 135(14).) and TRIM11 overexpression plasmids (i.e., the TRIM11 overexpression vector in Example 1; hereinafter the same). Cell lysates were collected 24 hours after transfection, and immunoprecipitation was performed using a TRIM11-specific antibody. The presence of the corresponding variant PolyQ protein in the precipitate complex was then detected by Western Blot (WB).

[0027] The results showed that mutant THAP11 protein could be detected in the TRIM11 co-precipitation group, while no corresponding signal was found in the control group IgG (Boster BA1045). Figure 2 A) indicates that TRIM11 can specifically bind to the mutant THAP11.

[0028] However, in the co-precipitation experiment of mutant HTT(N171) and TRIM11, mutant HTT(N171) protein was not detected in either the TRIM11 precipitation group or the IgG control group. Figure 3 A) indicates that there is no interaction between TRIM11 and the mutant HTT (N171) (there is no specific binding).

[0029] Similarly, in the co-precipitation experiment of mutant ATAXIN3 and TRIM11, mutant ATAXIN3 protein was not detected. Figure 3 E), indicating that there is no binding between TRIM11 and the mutated ATAXIN3.

[0030] In summary, these results indicate that the interaction between TRIM11 and different variant PolyQ proteins is highly selective, suggesting that it may have different functional properties in clearing different variant PolyQ proteins.

[0031] Example 4: Immunofluorescence assay to detect the interaction between TRIM11 and mutant THAP11 protein.

[0032] To verify the interaction between TRIM11 and the mutant THAP11 protein, the experiment was conducted according to the following steps: First, pre-coated poly-L-lysine slides were placed in 12-well plates, followed by seeding with healthy HEK293 cells. After the cells adhered and grew to a suitable density, HEK293 cells were co-transfected with the TRIM11 overexpression plasmid constructed in Example 1 and the mutant THAP11 protein expression plasmid using Lipo3000 liposome transfection reagent. Twenty-four hours after transfection, the culture medium was discarded, and 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. Subsequently, antibodies targeting the mutant THAP11 protein (Proteintech, Cat No. 23030-1-AP) and the TRIM11 protein antibody (Proteintech, Cat No. 66006-2-Ig) were added, and the cells were incubated overnight at 4°C in a horizontal shaker. The next day, the primary antibody was recovered, and cells were washed three times with PBS for 10 minutes each time. Then, according to the species of the primary antibody, fluorescent secondary antibodies (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) were added, and the cells were incubated at room temperature in the dark for 1 hour. After incubation, the secondary antibody was removed, and the cells were washed three more times with PBS for 10 minutes each time. Finally, the cell nuclei were stained with DAPI, and after staining for 1 minute, the cells were washed three times with ddH2O. Antiquenching agent was added to the slides, which were then covered and mounted. The presence of co-localization between TRIM11 protein and mutant THAP11 protein was observed and compared using a fluorescence microscope.

[0033] Experimental results showed that the green fluorescence signal of TRIM11 protein and the red fluorescence signal of mutant THAP11 protein significantly overlapped. Figure 2 (B) The results showed that TRIM11 protein and mutant THAP11 protein co-localized, indicating that TRIM11 protein and mutant THAP11 protein interact.

[0034] Example 5: Immunofluorescence assay to detect the scavenging effect of TRIM11 on mutant THAP11 protein aggregates To verify the clearance effect of TRIM11 on the SCA51 pathogenic protein (mutant THAP11 protein), the experiment was conducted according to the following steps: First, pre-coated poly-L-lysine slides were placed in 12-well plates, and then healthy HEK293 cells were seeded. After the cells adhered and grew to a suitable 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. Twelve hours after transfection, the culture medium was discarded, and 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 mutant THAP11 protein and TRIM11 were added, and the cells were incubated overnight at 4°C in a horizontal shaker. The next day, the primary antibody was recovered, and the cells were washed three times with PBS for 10 minutes each time. Then, fluorescent secondary antibody was added according to the species of the primary antibody, and the cells were incubated at room temperature in the dark for one hour. After incubation, the secondary antibody was discarded, and the cells were washed three more times with PBS for 10 minutes each time. Finally, DAPI was used for nuclear staining, followed by washing three times with ddH2O after one minute of staining, and then mounting the slides. The aggregation of mutant THAP11 protein in the TRIM11 group and the control group was observed and compared using fluorescence microscopy to evaluate the clearance effect of TRIM11.

[0035] The results showed that no mutant THAP11 protein aggregates were observed in cells overexpressing TRIM11, while obvious mutant THAP11 protein aggregates were observed in control cells overexpressing GFP. Figure 4 (A and B). This result demonstrates that overexpression of TRIM11 protein can significantly clear mutant THAP11 protein aggregates.

[0036] The results above demonstrate that overexpression of TRIM11 protein can significantly promote the clearance of mutant THAP11 protein aggregates.

[0037] Example 6: Evaluation of the scavenging effect of TRIM11 overexpression on HD, SCA3, and SCA51-related variant PolyQ proteins using Western Blot. Western blotting was used to verify the clearance effect of TRIM11 on HD, SCA3 and SCA51-related variant PolyQ proteins.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] Example 7: Western Blot was used to evaluate the effect of TRIM11 knockdown on the homeostasis of mutant THAP11 protein in SCA51.

[0042] 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.

[0043] The results showed that the knockdown group had a significant increase in both the solubility and aggregate form of the mutant THAP11 protein compared to the control group. Figure 5 (D, E, and F) This result further demonstrates that the TRIM11 protein can promote the clearance of mutant THAP11 protein.

[0044] Example 8: Detection of post-translational modifications of mutant THAP11 by TRIM11 To investigate the effects of TRIM11 overexpression on ubiquitination and small ubiquitin-like modifications of mutant PolyQ protein, this study used Western blotting to focus on the regulatory role of TRIM11 in the post-translational modifications of mutant THAP11 protein in an SCA51 disease model.

[0045] In this experiment, the mutant THAP11 expression plasmid and the TRIM11 overexpression plasmid constructed in Example 1 (or the GFP control plasmid) were co-transfected into HEK293 cells. Cell samples were collected 48 hours after transfection, and the changes in ubiquitination and SUMOylation levels of the mutant THAP11 protein under TRIM11 overexpression conditions were detected by Western blotting.

[0046] The results showed that TRIM11 overexpression significantly promoted ubiquitination of mutant THAP11 protein and increased its degradation signaling. Figure 6 C and D), while the effect on SUMOylation modification was not significant ( Figure 6 (A and B).

[0047] Example 9: Pathway Inhibition Experiment to Analyze the Molecular Mechanism of TRIM11 in Clearing Mutant THAP11 Protein To investigate the molecular pathways through which TRIM11 clears mutant THAP11 protein, this study conducted pathway-specific inhibition experiments.

[0048] HEK293 cells in good growth condition were seeded in six-well plates. After the cells stabilized, they were co-transfected with the mutant THAP11 and the TRIM11 overexpression plasmid constructed in Example 1. Twelve hours after transfection, different treatment groups were set up: the proteasome inhibitor MG132 (10 μM), the autophagy inhibitor Bafilomycin A1 (100 nM), and the untreated control group. Cells were collected 12 hours after inhibitor treatment, and the degradation of mutant THAP11 protein under different inhibitory conditions was analyzed by Western blotting.

[0049] Pathway inhibition experiments showed that the clearance of aggregates and soluble THAP11 protein was not significantly reduced in the MG132 group, while the clearance of aggregates and soluble THAP11 protein was significantly inhibited in the BafA1 group. This result fully demonstrates that TRIM11 mainly mediates the clearance of mutant THAP11 protein through the autophagy pathway. Figure 6 E).

[0050] In summary, this study successfully constructed an overexpression vector for TRIM11 (ssAAV-TRIM11-HA). In vitro experiments confirmed that TRIM11 expression effectively cleared aggregates and soluble forms of the SCA51 pathogenic protein mutant THAP11, thereby significantly alleviating its mediated neurotoxicity.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

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

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

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

4. Use according to claim 3, characterized in that: The overexpression vector carrying the TRIM11 protein gene is ssAAV-TRIM11-HA.

5. Use according to claim 4, characterized in that: The sequence of the ssAAV-TRIM11-HA is shown in SEQ ID NO. 3.

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