Polypeptide targeting TRAF6 and use thereof

Abstract

Provided are a polypeptide targeting TRAF6 and use thereof. Provided is use of a polypeptide in preparing a TRAF6 inhibitor. The polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The provided polypeptide can effectively inhibit TRAF6, thereby achieving the inhibition of neurodegenerative disease-related aggregates. The present disclosure has good prospects in improving, alleviating, and / or treating neurodegenerative diseases.

Description

A polypeptide targeting TRAF6 and its use

[0001] This application claims priority to Chinese invention patent application 202410068760X, “A polypeptide targeting TRAF6 and its use,” filed on January 17, 2024, which is incorporated by reference in its entirety. Technical Field

[0002] The present invention relates to the field of neurodegenerative diseases, and in particular to a polypeptide targeting TRAF6 and uses thereof. Background Art

[0003] Transactivation response (TAR) DNA / RNA binding protein 43 (TDP-43) is a 414-amino acid protein encoded by the TARDBP gene. TDP-43 plays an important role in RNA transcription, splicing, transport, translation, and mRNA stability regulation in cells.

[0004] TDP-43 is a highly conserved, ubiquitously expressed protein with strictly self-regulated expression levels. It constantly shuttles between the nucleus and cytoplasm and, under physiological conditions, is primarily localized in the nucleus. In a variety of neurodegenerative diseases (such as ALS and FTLD), most TDP-43 proteins show abnormal aggregation and localization in the cytoplasm and neurons. It has been reported that TDP-43 aggregation is closely related to autonomous degenerative changes in neurons, and its aggregation in neurons and glial cells of the central nervous system has become a pathological hallmark of neurodegenerative diseases such as ALS and FTLD-TDP.

[0005] Currently, there is a lack of effective treatments for TDP-43 protein diseases (such as ALS and FTLD). Over the past few decades, approximately 40 clinical trials on ALS patients have not shown good treatment prospects, and these clinical trials have not significantly improved the progression of the disease or patient survival. Riluzole is currently the only drug recognized to be effective in alleviating the progression of ALS. It can improve the patient's survival status, but it cannot prevent the progression of ALS. It is reported that riluzole can only prolong the average survival of patients by 3-6 months, but it will bring more side effects, including nausea, vomiting, diarrhea and liver toxicity. In addition, the clinical treatment of FTLD is mainly symptomatic treatment of the behavior, movement, cognitive disorders, etc. of FTLD patients. These treatments can only relieve some symptoms of FTLD patients, but cannot curb the progression of the disease. Summary of the Invention

[0006] In a first aspect, the present invention provides a use of a polypeptide for the preparation of a TRAF6 inhibitor, characterized in that the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. TRAF6 has been reported to promote the aggregation of various neurodegenerative disease-associated proteins, such as mutant SOD1, α-synuclein, mutant DJ-1, and huntingtin, thereby affecting a variety of neurodegenerative diseases. The polypeptide provided by the present invention is capable of competitively interacting with TRAF6 to specifically and effectively target TRAF6, thereby achieving improvement, alleviation, and / or treatment of neurodegenerative diseases.

[0007] In some embodiments, the TRAF6 inhibitor is used to treat and / or prevent a neurodegenerative disease. In some embodiments, the neurodegenerative disease comprises one or more of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0008] In some embodiments, the neurodegenerative disease comprises a TDP43 proteinopathy.

[0009] It should be emphasized that the experiments of the present invention have shown that TRAF6 can interact with TDP43 in living cells and promote its aggregation in the cytoplasm. Neurodegenerative diseases also commonly have the pathological characteristics of abnormal aggregation of TDP43 in the cytoplasm (such as neurons and glial cells). The polypeptides provided by the present invention can not only interact with TRAF6 competitively, but also specifically and effectively reduce the binding of TRAF6 to TDP43 and / or TDP43 mutants, thereby achieving the inhibition of aggregates associated with neurodegenerative diseases, for example, including: (a) inhibiting the cytoplasmic aggregation of TDP43 and / or TDP43 mutants; (b) inhibiting the cytoplasmic aggregation of insoluble TDP35; and / or (c) inhibiting the abnormal aggregation of proteins associated with neurodegenerative diseases. In summary, the polypeptides provided by the present invention can inhibit the formation of cytoplasmic insoluble substances (especially neuronal insoluble substances) associated with various neurodegenerative diseases, protect neurons from the effects of cytoplasmic aggregates to a certain extent, and can better treat various neurodegenerative diseases, especially TDP43 protein diseases.

[0010] In some embodiments, the polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NO: 1 and the entire amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NO: 1 and the entire amino acid sequence set forth in SEQ ID NO: 3.

[0011] It has been verified that not only the first polypeptide fragment (SEQ ID NO: 1, a fusion protein formed by the second and third polypeptide fragments) provided by the present invention can inhibit the cytoplasmic aggregation of TDP43 and / or TDP43 mutants, but also the second polypeptide fragment (SEQ ID NO: 2) or the third polypeptide fragment (SEQ ID NO: 3) alone can inhibit the cytoplasmic aggregation of TDP43 and / or TDP43 mutants to a certain extent. In other words, it is expected that a fusion protein comprising a portion of the first polypeptide fragment and the entire second polypeptide fragment, as well as a fusion protein comprising a portion of the first polypeptide fragment and the entire third polypeptide fragment, should have inhibitory activity against the cytoplasmic aggregation of TDP43 and / or TDP43 mutants.

[0012] In some embodiments, the TRAF6 inhibitor is used to target TRAF6 to inhibit the interaction of TRAF6 with TDP43 and / or TDP43 mutants, thereby inhibiting the formation of TDP43 and / or TDP43 mutant aggregates.

[0013] In some embodiments, the interacting region comprises the zinc finger domain and / or coiled-coil domain of TRAF6. Experimental validation indicates that the polypeptides provided herein do not target the RING domain of TRAF6, which is associated with its ubiquitin ligase activity. In other words, the polypeptides provided herein not only effectively inhibit TRAF6 but also do not interfere with its ubiquitin ligase function.

[0014] In summary, the polypeptides provided herein can act as TRAF6 inhibitors, effectively inhibiting TRAF6 (particularly inhibiting the binding of TRAF6 to TDP43 and / or TDP43 mutants), thereby inhibiting neurodegenerative disease-associated aggregates from different dimensions (for example, inhibiting TRAF6 and / or TDP43). These polypeptides demonstrate significant potential for improving, alleviating, and / or treating neurodegenerative diseases, and are expected to improve the survival rate of patients with these diseases. Furthermore, the polypeptides provided herein are human in origin, exhibiting low immunogenicity and minimal side effects.

[0015] In a second aspect, the present invention provides a use of a polypeptide in preparing a medicament for treating and / or preventing a neurodegenerative disease, characterized in that the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.

[0016] In some embodiments, the polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NO: 1 and the entire amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NO: 1 and the entire amino acid sequence set forth in SEQ ID NO: 3.

[0017] As used herein, "portion of an amino acid sequence" refers to at least one amino acid in the referenced amino acid sequence.

[0018] In some embodiments, the neurodegenerative disease comprises one or more of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and Parkinson's disease. In some embodiments, the neurodegenerative disease comprises a TDP43 proteinopathy.

[0019] In some embodiments, the use comprises one or more of the following: (a) inhibiting the cytoplasmic aggregation of TDP43 and / or TDP43 mutants; (b) inhibiting the cytoplasmic aggregation of insoluble TDP35; (c) inhibiting the abnormal aggregation of proteins associated with the neurodegenerative disease.

[0020] In some embodiments, the TDP43 mutants include, but are not limited to, one or more of A90V, P112H, G295S, G298S, S379A, and I383. It has been demonstrated that the polypeptides provided herein exhibit excellent inhibitory effects on the cytoplasmic aggregation of various TDP43 mutants.

[0021] In some embodiments, the neurodegenerative disease-associated protein comprises one or more of Tau protein, mutant SOD1 protein, α-synuclein, mutant DJ-1 protein, and Huntington protein. It has been demonstrated that the polypeptides provided herein can also effectively inhibit the formation of insoluble intracellular aggregates of proteins associated with neurodegenerative diseases.

[0022] In a third aspect, the present invention provides a polypeptide targeting TRAF6, characterized in that the polypeptide comprises:

[0023] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0024] (2) comprising a portion of the amino acid sequence shown in SEQ ID NO: 1 and the entire amino acid sequence shown in SEQ ID NO: 2; or

[0025] (3) comprising a portion of the amino acid sequence shown in SEQ ID NO: 1 and completely comprising the amino acid sequence shown in SEQ ID NO: 3.

[0026] In some embodiments, the amino acid sequence of the polypeptide is at least 95% identical to SEQ ID NO: 1.

[0027] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the above polypeptide.

[0028] In some embodiments, the nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO: 4. In some embodiments, the nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0029] In a fifth aspect, the present invention provides a gene expression vector, characterized in that the gene expression vector comprises the above-mentioned nucleic acid molecule.

[0030] In a sixth aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a first polypeptide fragment, wherein the amino acid sequence of the first polypeptide fragment comprises SEQ ID NO: 1. As described above, it has been verified that the first polypeptide fragment provided by the present invention (SEQ ID NO: 1) can inhibit the cytoplasmic aggregation of TDP43 and / or TDP43 mutants, thereby achieving the treatment of neurodegenerative diseases.

[0031] In some embodiments, the pharmaceutical composition further comprises a second polypeptide fragment and / or a third polypeptide fragment, wherein the second polypeptide fragment comprises SEQ ID NO: 2 and the third polypeptide fragment comprises SEQ ID NO: 3. It has been verified that the second polypeptide fragment (SEQ ID NO: 2) or the third polypeptide fragment (SEQ ID NO: 3) alone can also inhibit the cytoplasmic aggregation of TDP43 and / or TDP43 mutants to a certain extent, thereby achieving the treatment of neurodegenerative diseases. Therefore, the pharmaceutical composition provided by the present invention can inhibit the cytoplasmic aggregation of TDP43 and / or TDP43 mutants, thereby achieving the treatment of neurodegenerative diseases.

[0032] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0033] In a seventh aspect, the present invention provides a method for treating and / or preventing a neurodegenerative disease in a subject, characterized in that the method comprises administering an effective amount of the above-mentioned polypeptide, the above-mentioned gene expression vector, or the above-mentioned pharmaceutical composition to a subject in need.

[0034] In some embodiments, the neurodegenerative disease comprises a TDP43 proteinopathy. In some embodiments, the neurodegenerative disease comprises one or more of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and Parkinson's disease.

[0035] As used herein, "subject," "individual," or "patient" refers to any organism upon which the present invention can be used or applied. Typical subjects include mammals, such as mice, rats, rabbits, non-human primates, and humans.

[0036] As used herein, "therapeutically effective amount" refers to the amount of a therapeutic agent that is associated with or reasonably expected to achieve a specific therapeutic effect observed in a relevant population. In some embodiments, the therapeutically effective amount can be an amount that (a) inhibits the cytoplasmic aggregation of TDP43 and / or TDP43 mutants; (b) inhibits the cytoplasmic aggregation of insoluble TDP35; and / or (c) inhibits the abnormal aggregation of proteins associated with the neurodegenerative disease.

[0037] In an eighth aspect, the present invention provides a method for reducing the cytoplasmic aggregation of TDP43 and / or TDP43 mutants in cells, characterized in that the method comprises administering an effective amount of the above-mentioned polypeptide, the above-mentioned gene expression vector, or the above-mentioned pharmaceutical composition to the cells.

[0038] In some embodiments, the cell is a neuronal cell or a non-neuronal cell. In some embodiments, the cell expresses TDP43 and / or a TDP43 mutant. In some embodiments, the cell further expresses one or more of Tau protein, mutant SOD1 protein, α-synuclein, mutant DJ-1 protein, and Huntington protein.

[0039] In a ninth aspect, the present invention provides a method for reducing the level of a protein associated with a neurodegenerative disease in a cell, characterized in that the method comprises administering an effective amount of the above-mentioned polypeptide, the above-mentioned gene expression vector, or the above-mentioned pharmaceutical composition to the cell.

[0040] In some embodiments, the cell is a neuronal cell or a non-neuronal cell. In some embodiments, the cell expresses one or more of Tau protein, mutant SOD1 protein, α-synuclein, mutant DJ-1 protein, and Huntington protein. In some embodiments, the cell further expresses TDP43 and / or a TDP43 mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying any creative work.

[0042] FIG1a is a graph showing experimental results showing that TRAF6 specifically immunoprecipitates endogenous TDP43;

[0043] Figure 1b is an experimental result showing that TRAF6 promotes abnormal aggregation of TDP43 in cells;

[0044] Figure 1c shows the quantitative results of the experiment shown in Figure 1b (left: dynamic droplets; right: aggregates);

[0045] FIG1d is a graph showing experimental results showing that FRAP analysis further confirms that TRAF6 promotes the aggregation and accumulation of TDP43 in cells;

[0046] Figure 1e is a graph showing experimental results showing that TRAF6 significantly increases the accumulation of TDP43 in the insoluble fraction;

[0047] FIG1f is an experimental result showing that TRAF6 promotes the phase transition of cytoplasmic TDP43 in living cells;

[0048] FIG2 a is a graph showing experimental results showing that the ubiquitination level of TDP43 is not affected by TRAF6;

[0049] FIG2 b is an experimental result showing that TRAF6 does not rely on its E3 ligase activity to regulate TDP43;

[0050] Figure 2c is a schematic diagram of the structure of TRAF6-related constructs;

[0051] Figure 2d shows that TRAF6 ZC and TRAF6 WT Figure 1 shows the experimental results of interaction with TDP43;

[0052] Figure 2e shows that TRAF6 ZC Figure 1 shows the experimental results of promoting the aggregation of TDP43;

[0053] Figure 3a is a schematic diagram of the structure of TDP43-related constructs;

[0054] FIG3 b is a diagram showing the experimental results verifying the effect of the RRM1-RRM2 domain on the interaction;

[0055] Figure 3c is a diagram showing the experimental results verifying the effect of the K102-G277 region on the interaction;

[0056] Figure 3d is a graph showing the experimental results for verifying the effect of deleting the L region and FR2 region on the interaction;

[0057] FIG3e is a diagram showing the experimental results for verifying the effect of the first polypeptide fragment provided by the present invention on the interaction;

[0058] Figure 3f is a diagram showing the predicted structure of the first polypeptide fragment provided by the present invention;

[0059] FIG4 a is a graph showing the experimental results of the interaction strength between the first polypeptide fragment and TRAF6;

[0060] FIG4 b is a graph showing the experimental results of the first polypeptide fragment alleviating the aggregation of TDP35 in cells;

[0061] FIG5 is a graph showing the experimental results of the inhibitory effects of the first polypeptide fragment, the second polypeptide fragment, and the third polypeptide fragment;

[0062] FIG6 a is a graph showing the results of an immunoblotting experiment showing the inhibitory effect of the first polypeptide fragment on the aggregation of TDP43 mutants;

[0063] FIG6 b is an immunofluorescence experiment result showing the inhibitory effect of the first polypeptide fragment on the aggregation of TDP43 mutants;

[0064] FIG6 c is a graph showing the results of an immunoblotting experiment showing the inhibitory effect of the first polypeptide fragment on the aggregation of TAU protein;

[0065] FIG6 d is a graph showing the immunofluorescence experiment results showing the inhibitory effect of the first polypeptide fragment on the aggregation of TAU protein. DETAILED DESCRIPTION

[0066] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0067] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0068] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0069] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0070] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0071] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0072] Example 1: Materials and Methods

[0073] Plasmids and Antibodies: The TDP-43 (Gene ID: 23435), TRAF6 (Gene ID: 7189), and TAU (Gene ID: 4137) genes were amplified from a cDNA library derived from HEK 293T cells. For protein expression in mammalian cells, these genes were inserted into the pcDNA3.1 or pCMV plasmids. For expression in Escherichia coli, these genes were placed into the pET28a plasmid. TDP43 and its N-terminally fused EGFP mutants were cloned into the pLKO.1-CMV plasmid. The pMD2.G and psPAX2 plasmids were used for lentiviral production. The pX330 and PUC19 plasmids were used to construct knockout cell lines. The DNA sequences of all plasmids were verified. Strep II antibody (ABT2230) was purchased from Abbkine. Strep II antibody (HA500061) was purchased from Huabio. Ubiquitin antibodies (sc-8017) and GFP antibodies (sc-9996) were purchased from Santa Cruz. Anti-HA (M20003), anti-Flag (M20008), and HRP-conjugated anti-His (M20020S) antibodies were purchased from Abmart. Immunofluorescence secondary antibodies (A32732, A32733, and A32742) were purchased from Thermo Fisher Scientific.

[0074] Cell culture: HEK 293T cells (catalog #GNHu17) and SH-SY5Y cells (catalog #SCSP-5014) were obtained from the National Model and Specialty Laboratory Cell Resource Bank. HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. SH-SY5Y cells were cultured in Mini-Medium (MEM) supplemented with 44.5% Ham's F-12 medium, 10% FBS, and 1% NEAA. Transient transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions.

[0075] Immunoblotting: Samples were denatured in loading buffer and separated on SDS-PAGE gels at the appropriate concentration. After transfer to a PVDF membrane, the membrane was blocked with 5% nonfat milk for one hour at room temperature and then incubated with the primary antibody overnight at 4°C. The membrane was then exposed after incubation with an HRP-conjugated secondary antibody for 1-2 hours at room temperature.

[0076] Immunofluorescence: Wash cells three times with PBS and fix with methanol (pre-chilled at -20°C) for five minutes. Then, block with 10% goat serum (dissolved in PBS with 0.3% Triton X-100) at room temperature for one hour. Incubate with primary antibody overnight at 4°C. After incubation with fluorescent secondary antibody for one hour at room temperature, wash the membrane three times with PBS. Mount the slides with mounting medium containing DAPI. Image the samples using a fluorescence microscope. Quantify the immunofluorescence data using ImageJ.

[0077] Immunoprecipitation and pulldown experiments: For immunoprecipitation, cells were harvested and lysed on ice for 30 minutes using lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 5% glycerol, and 1% protease inhibitor cocktail). After centrifugation at 15,000 rpm / min for 30 minutes at 4°C, Flag or Strep II affinity gel was added to the sample, and Flag microbeads were bound overnight at 4°C, while Strep II microbeads were bound for 3 hours at room temperature. The beads were washed with lysis buffer and denatured with loading buffer for immunoblot analysis. For pulldown experiments, the purified protein was incubated with nickel beads in binding buffer (25 mM HEPES pH 8.0, 500 mM NaCl, 10% glycerol, and 1 mM DTT) for 2 hours at 4°C. The nickel beads were washed three times with binding buffer and denatured with loading buffer for immunoblot analysis.

[0078] Protein expression, purification, and fluorescent labeling: TRAF6 and TDP43 proteins were purified from the BL21 (DE3) strain. IPTG was added at a final concentration of 0.5 mM to induce protein expression overnight at 16°C. The collected bacteria were resuspended in 25 mM HEPES buffer (pH 8.0) containing 10 mM imidazole, 10% glycerol, and 1 mM DTT (1 M NaCl was used for purification of TDP43 and 150 mM NaCl was used for purification of TRAF6), and then sonicated. The supernatant was collected after centrifugation at 13,000 rpm / min for 30 minutes and incubated with nickel beads or GST microbeads. TDP43 protein and TRAF6 were eluted with buffer containing 300 mM imidazole or 10 mM reduced glutathione (GSH), respectively. The eluted TDP43 sample was further purified using starch resin (NEB) and then eluted with 25 mM HEPES pH 8.0, 1 M NaCl, 10% glycerol, 10 mM maltose, and 1 mM DTT. The purified protein was aliquoted and stored at -80°C. TDP43-MBP-His was fluorescently labeled using AF647 succinimidyl ester (D10157, Duofluor Inc.). Labeling experiments were performed according to the provided instructions.

[0079] FRAP analysis: For FRAP analysis, cells expressing GFP-tagged proteins were cultured in glass-bottomed culture dishes. A Zeiss LSM880 confocal microscope was used. The intensity at each time point was normalized to the initial intensity before photobleaching. FRAP data were analyzed using Zen software and GraphPad.

[0080] Isolation of the insoluble fraction: After three PBS washes, cells were lysed on ice for 20 minutes in pre-chilled lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 5% glycerol, and 1% protease inhibitors) followed by sonication. The lysate was centrifuged at 15,000 rpm / min for 30 minutes at 4°C, and the supernatant was collected as the soluble fraction. The pellet was washed with lysis buffer and sonicated three times. The pellet was dissolved in lysis buffer containing 8 M urea at 60°C for 2 hours. The lysate was then centrifuged at 15,000 rpm / min for 30 minutes, and the supernatant was collected as the insoluble fraction.

[0081] Differential interference contrast (DIC) microscopy: DIC images were acquired on an Olympus BX53 microscope.

[0082] In addition, the experiments related to the first polypeptide fragment, the second polypeptide fragment and the third polypeptide fragment in the present invention can be performed with reference to the general methods above (for example, with reference to the experimental methods related to TDP43 protein).

[0083] The specific information of the materials used in the present invention is shown in Table 1.

[0084] Table 1:

[0085] The amino acid sequence of the first polypeptide fragment is: LPNSKQSQDEPLRSAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFG (SEQ ID NO: 1). The amino acid sequence of the second polypeptide fragment is: LPNSKQSQDEPLRS (SEQ ID NO: 2). The amino acid sequence of the third polypeptide fragment is: AQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFG (SEQ ID NO: 3).

[0086] The nucleotide sequence of the first polypeptide fragment is: cttcctaattctaagcaaagccaagatgagcctttgagaagcgcgcagtctctttgtggagaggacttgatcattaaaggaatcagcgttcatatatccaatgccgaacctaagcacaatagcaatagacagttagaaagaagtggaagatttggt (SEQ ID NO: 4). The nucleotide sequence of the second polypeptide fragment is: cttcctaattctaagcaaagccaagatgagcctttgagaagc (SEQ ID NO: 5). The nucleotide sequence of the third polypeptide fragment is: gcgcagtctctttgtggagaggacttgatcattaaaggaatcagcgttcatatatccaatgccgaacctaagcacaatagcaatagacagttagaaagaagtggaagatttggt (SEQ ID NO: 6).

[0087] Example 2: TRAF6 interacts with TDP43 in living cells and promotes its aggregation

[0088] To evaluate whether TRAF6 (tumor necrosis factor receptor-associated factor 6) binds to TDP43, this example transfected HEK293T cells with a plasmid encoding Flag-TRAF6 and immunoprecipitated endogenous TDP43 in the cells. Figure 1a shows that TRAF6 can specifically immunoprecipitate TDP43, indicating that TRAF6 can interact with TDP43 in living cells. TRAF6 expression levels have been reported to be elevated in patients with ALS and PD and is associated with a variety of age-related neurodegenerative diseases. Furthermore, this example further verified through pulldown experiments that purified TDP43 can directly interact with TRAF6 in vitro. Based on this, this example demonstrates that TRAF6 can interact with TDP43 in living cells.

[0089] To investigate the potential role of TRAF6 in regulating TDP43, this example co-expressed EGFP-TDP43 and TRAF6 in HEK293T cells. As shown in Figure 1b, EGFP-TDP43 formed tiny dots in living cells but did not aggregate, indicating that TDP43 itself does not form aggregates in cells (Figure 1b, first row). However, 24 hours after co-transfection with TRAF6, TRAF6 expression caused EGFP-TDP43 to form dynamic droplets (which readily transitioned to a solid form) in 21% of cells and aggregates in 25% of cells (Figure 1b, third row, Figure 1c (Ctr: control)). Figure 1b, second row, shows the results of the experiment 12 hours after co-transfection. This example further verified using neural cells that TRAF6 also similarly promotes the aggregation of TDP43 in SH-SY5Y human neuroblastoma cells.

[0090] Fluorescence recovery after photobleaching (FRAP) analysis showed (Figure 1d) that TDP43 droplets recovered to 40% within 8 seconds, while TDP43 aggregates in HEK293T cells hardly recovered, further confirming that TRAF6 promotes the aggregation and accumulation of TDP43 in cells.

[0091] Next, in this example, cell lysates from HEK293T cells co-expressing EGFP-TDP43 and TRAF6 were separated into Triton X-100-soluble and insoluble fractions. The immunoblot results in Figure 1e show that TRAF6 significantly increases TDP43 accumulation in the insoluble fraction. In summary, this example demonstrates that TRAF6 can enhance TDP43 aggregation in living cells.

[0092] Example 3: TRAF6 induces phase transition of cytoplasmic TDP43 in living cells

[0093] Mislocalization and aggregation of TDP43 in the cytoplasm are pathological features of frontotemporal dementia-TDP (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). It has been reported that TDP43 inclusion bodies in the cytoplasm are observed in up to 97% of ALS patients.

[0094] In this example, TRAF6 was combined with EGFP-cTDP43 ΔNLS The mutant (TDP43 (cTDP43) mutant lacking nuclear localization signal and localized in the cytoplasm) was co-expressed. The experimental results were similar to those in Example 2. As shown in Figure 1f, EGFP-TDP43 ΔNLS It forms dots but does not aggregate in the cytoplasm (Figure 1f, left, first row); 24 hours after TRAF6 expression, 14% of cells produce TDP43 ΔNLS 26% of the cells formed aggregates (of which the fluidity of the hollow rings was slightly higher than that of the aggregates). The second and third rows of the left figure in Figure 1f show the experimental results 12 hours and 24 hours after co-expression, respectively. The above results show that TRAF6 can promote the phase transition of cytoplasmic TDP43 in living cells. In addition, as verified by immunoblotting, overexpression of TRAF6 can significantly increase the amount of cytoplasmic TDP43 that is insoluble in the detergent Triton X-100. In summary, this example proves that TRAF6 can regulate cytoplasmic TDP43.

[0095] Example 4: TRAF6 regulates the phase transition of TDP43 independently of its E3 ligase activity

[0096] TRAF6 is an E3 ubiquitin ligase. It has been reported that the ubiquitination of TDP43 is closely related to its aggregation. When His-TDP43 and TRAF6 (TRAF6) are overexpressed in cells, WT , wild-type TRAF6) or TRAF6 C70A (TRAF6 E3 ubiquitin ligase inactive mutant), and then denatured and purified His-TDP43. This example found that after the TRAF6 E3 ubiquitin ligase was inactivated, the ubiquitination level of TDP43 did not change significantly (Figure 2a). In addition, this example also found that TRAF6 lacking E3 ligase activity C70A The mutant still promoted TDP43 insolubility. Immunofluorescence results also confirmed that TRAF6 ubiquitin ligase activity is not required for TRAF6-mediated TDP43 aggregation (Figure 2b). In summary, this example found that TRAF6 does not rely on its E3 ligase activity to regulate TDP43.

[0097] TRAF6 contains a RING domain, four zinc finger domains (ZnF1-ZnF4), a coiled-coil (CC) domain and a MATH domain (Figure 2c). The E3 ubiquitin ligase active site of TRAF6 is located in the RING domain. ZC (The RING and MATH domains were deleted, leaving only the zinc finger and coiled-coil domains) showed similar binding ability to TDP43 (Figure 2d) and was able to significantly induce TDP43 aggregation in cells and in vitro (Figure 2e). The above experimental results reveal that the RING domain is not essential for TRAF6-induced TDP43 aggregation, and TRAF6 may promote TDP43 aggregation in cells through its zinc finger and / or coiled-coil domains (i.e., the interaction domains between TRAF6 and TDP43 may be the zinc finger and / or coiled-coil domains).

[0098] Example 5: The interaction between TDP43 and TRAF6 depends on its TDP6 fragment

[0099] In this example, different regions of the RNA binding region of TDP43 (K102-G277, KG) were removed (Figure 3a). This region mainly contains the RRM1-RRM2 domain and flanking sequences, among which the RNA recognition domains RRM1 and RRM2 are often considered to play a crucial role in the pathogenesis of TDP43. In Figure 3a, the red cross indicates the mutation of the nuclear localization signal (NLS); L indicates the linker between RRM1 and RRM2 (L177-R189); FR2 indicates the A240-G277 sequence after RRM2; and the dotted line indicates the deleted region. Figure 3b shows that the deletion of RRM1 (cTDP32 ΔRRM1 ), RRM2 domain (cTDP32 ΔRRM2 ) or both (cTDP32 ΔRRM1-2 ) had no significant effect on the interaction between cTDP43 and TRAF6. However, after deleting the K102-G277 region of TDP43, cytoplasmic TDP32ΔK102-G277 (cTDP43 ΔKG ) significantly reduced the interaction between the RRM1 and RRM2 domains (Figure 3c). ΔL+ΔFR2) will eliminate its interaction with TRAF6 (Figure 3d). In other words, this example found that TDP6 (which can also be understood as the first polypeptide fragment, a polypeptide formed by fusing the TDP43 linker (second polypeptide fragment) and FR2 (third polypeptide fragment) in the present invention) actually plays a key role in the interaction between TDP43 and TRAF6 (Figure 3e).

[0100] The first polypeptide fragment constructed in the present invention forms a basic pocket in the predicted structure, as shown in FIG3f (wherein the second polypeptide fragment is purple, the third polypeptide fragment is blue, and the amino acids marked in red are the more critical positively charged amino acids).

[0101] Example 6: The first polypeptide fragment constructed by the present invention can alleviate various pathological TDP43 mutants

[0102] This example found that the interaction strength between TRAF6 and TDP6 was much stronger than that between TRAF6 and TDP43 / TDP35 ( FIG. 4 a ). In other words, the first polypeptide fragment constructed in the present invention competitively inhibits the binding between TRAF6 and TDP43 / TDP35.

[0103] The confocal images in Figure 4b show that the incidence of EGFP-TDP35 aggregation was significantly reduced in HEK293T cells expressing the first polypeptide fragment. This indicates that the first polypeptide fragment provided by the present invention reduces TDP35 aggregation in cells. Similarly, immunoblotting experiments demonstrated that the first polypeptide fragment reduced insoluble TDP35. This indicates that the first polypeptide fragment provided by the present invention reduced the insolubility of TRAF6-dependent TDP35 in cells. Furthermore, it has been demonstrated that the first polypeptide fragment can similarly inhibit TDP35 aggregation in SHY5Y cells. These results demonstrate that the first polypeptide fragment provided by the present invention can inhibit TDP35 aggregation in living cells.

[0104] Next, this example further studies the contribution of the second and third polypeptide fragments in the first polypeptide fragment to the inhibitory effect of TDP43. As shown in Figure 5, by comparing the depth of the bands in the "+" and "-" parts of Figure 5, it can be seen that the second polypeptide fragment (Figure 5a) and the third polypeptide fragment (Figure 5b) alone can both produce a certain degree of inhibitory effect on the insoluble component of TDP43 (taking TDP35, one of the mutants of TDP43, as an example), among which the inhibitory effect of the third polypeptide fragment is worse than that of the second polypeptide fragment, and the inhibitory effect of the two fragments alone is not as good as the first polypeptide fragment (i.e., the polypeptide fragment formed by the fusion of the second and third polypeptide fragments) (Figure 5c). Next, the examples of the present invention conduct further inhibitory effect studies based on the first polypeptide fragment.

[0105] Pathological mutations in TDP43 have been widely found in patients with ALS and FTLD. This example further evaluated the effects of the first polypeptide fragment on different pathogenic TDP43 strains. Figures 6a and 6b show that TDP43 mutants with G295S, G298S, S379A, or I383V mutations aggregate in cells, and this is significantly alleviated by the first polypeptide fragment. In other words, the first polypeptide fragment provided by the present invention can effectively inhibit the cytoplasmic aggregation of pathological TDP43 and / or TDP43 mutants.

[0106] This example further evaluated the effects of the first polypeptide fragment on proteins associated with neurodegenerative diseases. Insoluble intracellular aggregates containing various proteins associated with neurodegenerative diseases, such as Tau, have been reported to be found in neurons of patients with various neurodegenerative diseases. Figures 6c and 6d demonstrate that the first polypeptide fragment can reduce Tau aggregation in cells. In other words, the first polypeptide fragment provided by the present invention can reduce the insolubility of various proteins associated with neurodegenerative diseases.

[0107] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. Use of a polypeptide in the preparation of a TRAF6 inhibitor, characterized in that, The polypeptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:

3.

2. The use according to claim 1, characterized in that, The TRAF6 inhibitor is used for treating and / or preventing neurodegenerative diseases.

3. The use according to claim 2, characterized in that, The neurodegenerative disease comprises TDP43 proteinopathy.

4. The use according to claim 1, characterized in that, The polypeptide: (1) comprises a part of the amino acid sequence shown in SEQ ID NO:1 and completely comprises the amino acid sequence shown in SEQ ID NO:2; or (2) comprises a part of the amino acid sequence shown in SEQ ID NO:1 and completely comprises the amino acid sequence shown in SEQ ID NO:

3.

5. The use according to claim 1, characterized in that, The TRAF6 inhibitor is used to target TRAF6 to inhibit the interaction between TRAF6 and TDP43 and / or TDP43 mutants, thereby inhibiting the formation of aggregates of the TDP43 and / or TDP43 mutants.

6. The use according to claim 1, wherein The use comprises one or more of the following: (a) inhibiting the cytoplasmic aggregation of TDP43 and / or TDP43 mutants; (b) inhibiting the cytoplasmic aggregation of insoluble TDP35; (c) inhibiting the abnormal aggregation of proteins associated with the neurodegenerative disease.

7. Use of a polypeptide in the preparation of a medicament for treating and / or preventing neurodegenerative diseases, characterized in that, The polypeptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:

3.

8. The use according to claim 7, wherein The polypeptide: (1) comprises a part of the amino acid sequence shown in SEQ ID NO:1 and completely comprises the amino acid sequence shown in SEQ ID NO:2; or (2) comprises a part of the amino acid sequence shown in SEQ ID NO:1 and completely comprises the amino acid sequence shown in SEQ ID NO:

3.

9. The use according to claim 7, characterized in that, The neurodegenerative disease comprises one or more of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease and Parkinson's disease.

10. The use according to claim 7, characterized in that, The neurodegenerative disease comprises TDP43 proteinopathy.

11. The use according to claim 7, characterized in that, The use comprises one or more of the following: (a) inhibiting the cytoplasmic aggregation of TDP43 and / or TDP43 mutants; (b) inhibiting the cytoplasmic aggregation of insoluble TDP35; (c) inhibiting the abnormal aggregation of proteins associated with the neurodegenerative disease.

12. The use according to claim 11, characterized in that, The proteins associated with the neurodegenerative disease comprise one or more of Tau protein, mutant SOD1 protein, α-synuclein, mutant DJ-1 protein and huntingtin protein.

13. The use according to claim 11, characterized in that, The TDP43 mutants include one or more of A90V, P112H, G295S, G298S, S379A and I383.

14. A polypeptide targeting TRAF6, characterized in that, The polypeptide comprises: (1) the amino acid sequence shown in SEQ ID NO:1; or (2) comprises a part of the amino acid sequence shown in SEQ ID NO:1 and completely comprises the amino acid sequence shown in SEQ ID NO:2; or (3) comprises a part of the amino acid sequence shown in SEQ ID NO:1 and completely comprises the amino acid sequence shown in SEQ ID NO:

3.

15. The polypeptide according to claim 14, wherein, The amino acid sequence of the polypeptide has at least 95% identity with SEQ ID NO:

1.

16. A nucleic acid molecule encoding the polypeptide according to claim 14 or 15.

17. The nucleic acid molecule according to claim 16, wherein The nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO:

4.

18. The nucleic acid molecule according to claim 16, wherein The nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO:5 and / or SEQ ID NO:

6.

19. A gene expression vector, characterized in that, The gene expression vector comprises the nucleic acid molecule according to any one of claims 16 - 18.

20. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a first polypeptide fragment, wherein the amino acid sequence of the first polypeptide fragment comprises SEQ ID NO:

1.

21. The pharmaceutical composition according to claim 20, wherein, The pharmaceutical composition further comprises a second polypeptide fragment and / or a third polypeptide fragment, the second polypeptide fragment comprises SEQ ID NO:2, and the third polypeptide fragment comprises SEQ ID NO:

3.

22. A method for treating and / or preventing neurodegenerative diseases in a subject, characterized in that, The method comprises administering to a subject in need thereof an effective amount of the polypeptide according to claim 14 or 15, the gene expression vector according to claim 19, or the pharmaceutical composition according to claim 20 or 21.

23. The method according to claim 22, wherein The neurodegenerative disease comprises TDP43 proteinopathy.

24. The method according to claim 22, wherein, The neurodegenerative disease comprises one or more of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, and Parkinson's disease.

25. A method for reducing cytoplasmic aggregation of TDP43 and / or TDP43 mutants in cells, characterized in that, The method comprises administering to the cell an effective amount of the polypeptide according to claim 14 or 15, the gene expression vector according to claim 19, or the pharmaceutical composition according to claim 20 or 21.

26. The method according to claim 25, wherein, The cell is a neuronal cell or a non - neuronal cell.

27. The method according to claim 25, wherein, The cell expresses TDP43 and / or a TDP43 mutant.

28. The method according to claim 25, wherein The cell also expresses one or more of Tau protein, mutant SOD1 protein, α - synuclein, mutant DJ - 1 protein, and huntingtin protein.

29. A method for reducing a protein associated with neurodegenerative diseases in cells, characterized in that, The method comprises administering to the cell an effective amount of the polypeptide according to claim 16 or 17, the gene expression vector according to claim 21, or the pharmaceutical composition according to claim 22 or 23.

30. The method according to claim 29, wherein The cell is a neuronal cell or a non - neuronal cell.

Images