Application of targeted amyloid precipitation degradation technology in treatment of neurodegenerative diseases

Abstract

The invention relates to the field of lysosome targeted degradation, in particular to application of a technology for targeted degradation of amyloid precipitation in cells in preparation of drugs for treating neurodegenerative diseases. The invention provides an application of a nano chimera in preparation of a medicine for treating neurodegenerative diseases, the nano chimera comprises a first protein and an ABIcs protein tag, and the first protein is selected from a group consisting of alpha-synuclein, tau protein, A beta protein precursor, huntingtin and TAR DNA binding protein 43. The nano chimera provided by the invention not only can target the intracellular amyloid aggregate, but also the amyloid aggregate inserted into the nano chimera can be efficiently degraded through a lysosome way, so that the safety is high.

Description

Technical Field

[0001] The present invention relates to the field of lysosome targeted degradation, and in particular to application of a targeted degradation technology for intracellular amyloid precipitation in the preparation of drugs for neurodegenerative diseases. Background Art

[0002] Targeted protein degradation (TPD) technology includes proteasome-based degradation technology and lysosome-based degradation technology. Proteasome-based degradation technology relies on the ubiquitin-proteasome pathway, which requires ubiquitination of the target protein to be degraded so that the target protein can be degraded by the proteasome. Lysosome-based degradation, especially autophagy-based degradation methods, have been developed in recent years. Mechanistically, autophagy relies on the formation of autophagosomes to encapsulate the target protein to be degraded, and after fusion with lysosomes, autophagolysosomes are formed to achieve the degradation of these target proteins. Autophagy-based degradation technologies include AUTAC, AUTOTAC and ATTEC, all of which use chemical small molecules to induce the degradation of target proteins. However, these small molecules have problems such as complex chemical structure, difficulty in synthesis and preparation, off-target effects, and are unable to target the degradation of intracellular amyloid deposits.

[0003] Chinese patent application CN116836295A discloses an autophagy-targeted nanoantibody chimera, which is composed of a nanoantibody connected to an autophagy marker. It uses nanoantibodies instead of chemical small molecules to achieve targeted recognition of intracellular targets, but it is also unable to target and degrade intracellular amyloid deposits. Summary of the invention

[0004] In a first aspect, the present invention provides a use of a nanochimera in the preparation of a drug for treating a neurodegenerative disease, characterized in that the nanochimera comprises a first protein and an ABIcs protein tag, and the first protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43.

[0005] In some embodiments, the nanochimera is used in combination with an autophagy promoter and abscisic acid, wherein the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag.

[0006] In some embodiments, the use specifically comprises targeted degradation of amyloid aggregates via the lysosomal pathway.

[0007] In some embodiments, the abscisic acid is added after the nanochimera and the autophagy promoter are added for a first time.

[0008] In some embodiments, the first time comprises 1-7 days.

[0009] In some embodiments, the first time is 24-48 hours.

[0010] In some embodiments, the first time is 3-7 days.

[0011] In some embodiments, a fluorescent protein is further included between the first protein and the ABIcs protein tag.

[0012] In some embodiments, a fluorescent protein is further included between the LC3B autophagy marker and the PYLcs protein tag.

[0013] In some embodiments, the nanochimera is used to target amyloid aggregates formed by aggregation of a second protein, the second protein being identical to the first protein.

[0014] In some embodiments, the second protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43.

[0015] In some embodiments, the nanochimera is used to target amyloid aggregates formed by aggregation of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein, or TAR DNA binding protein 43.

[0016] As used herein, "amyloid aggregates" or "amyloid deposits" refer to insoluble aggregates formed by pathological misfolding and aggregation of specific amyloid proteins. In some embodiments, the amyloid protein includes α-synuclein, tau protein, Aβ protein precursor, huntingtin protein, or TAR DNA binding protein 43.

[0017] In some embodiments, the amyloid aggregate is intracellular.

[0018] In some embodiments, the amyloid aggregates are insoluble.

[0019] In some embodiments, the neurodegenerative disease comprises one or more of Parkinson's disease, Lewy body disease, multiple system atrophy, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Prussner-Wilk's disease, and Pick's disease.

[0020] In some embodiments, when the first protein is α-synuclein, the neurodegenerative disease is Parkinson's disease; when the first protein is tau protein, the neurodegenerative disease is frontotemporal dementia with Parkinson's disease; when the first protein is Aβ protein precursor, the neurodegenerative disease is Alzheimer's disease; when the first protein is Huntington protein, the neurodegenerative disease is Huntington's disease; when the first protein is TAR DNA binding protein 43, the neurodegenerative disease is amyotrophic lateral sclerosis.

[0021] In some embodiments, nanochimeras comprising different first proteins can be used in combination to target the degradation of different types of amyloid aggregates (for example, amyloid aggregates formed by both α-synuclein and amyloid aggregates formed by tau protein), thereby achieving the treatment of different neurodegenerative diseases.

[0022] In some embodiments, the first protein may also be a mutant.

[0023] In some embodiments, the nanochimera is used to insert into the amyloid aggregates, and under the action of an autophagy promoter and abscisic acid, the amyloid aggregates inserted into the nanochimera are degraded through a lysosomal pathway.

[0024] In a second aspect, the present invention provides a system for targeted degradation of amyloid aggregates, comprising: a) a nanochimera comprising a first protein and an ABIcs protein tag, wherein the first protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43; b) autophagy promoters, which include LC3B autophagy marker and PYLcs protein tag; c) Abscisic acid.

[0025] In some embodiments, the amyloid aggregates are formed by aggregation of a second protein, which is the same as the first protein.

[0026] In some embodiments, the nanochimera is used to target amyloid aggregates formed by aggregation of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein, or TAR DNA binding protein 43.

[0027] In some embodiments, the amyloid aggregate is intracellular.

[0028] In some embodiments, the amyloid aggregates are insoluble.

[0029] In a third aspect, the present invention provides a method for targeted degradation of intracellular amyloid aggregates for non-therapeutic purposes, characterized in that the specific steps are as follows: S101 co-transfecting the first expression vector and the second expression vector into cells; S102: adding abscisic acid to the cells after the first co-transfection time, thereby achieving targeted degradation of amyloid aggregates in the cells; Among them, the first expression vector includes a gene encoding a nanochimera, and the second expression vector includes a gene encoding an autophagy promoter; the nanochimera includes a first protein and an ABIcs protein tag, and the first protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43; the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag.

[0030] In some embodiments, the first time comprises 24-48 hours.

[0031] In some embodiments, the first expression vector and the second expression vector are plasmids.

[0032] In some embodiments, the mass ratio of the first expression vector to the second expression vector comprises 1:1-3.

[0033] In some embodiments, the mass ratio of the first expression vector to the second expression vector is 1:2.

[0034] In some embodiments, the cell comprises a Hela cell or a neuroblastoma cell.

[0035] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects: Existing methods for degrading target proteins such as α-synuclein based on autophagy mainly use specific antibodies or small chemical molecules to induce the degradation of soluble α-synuclein. Chinese patent application CN116836295A discloses an autophagy-targeted nanoantibody chimera, which uses nanoantibodies to target and recognize target proteins in cells. The inventors found that the above-mentioned prior art can only degrade some soluble proteins or prevent the accumulation of toxic proteins, and cannot degrade the already formed amyloid aggregates. For nanoantibodies, since they can only recognize specific target sites (i.e., fixed antigenic determinants), and the actual structure of amyloid aggregates in cells is ever-changing, it is difficult to effectively target and degrade intracellular amyloid aggregates, and thus it is difficult to apply them to the treatment of neurodegenerative diseases. The prior art has not yet reported how to use autophagy to degrade intracellular amyloid aggregates.

[0036] The present invention breaks through the conventional idea of ​​using autophagy-targeted nanoantibody chimeras to target and recognize soluble proteins, and creatively replaces nanoantibodies with exogenous amyloid proteins. Unexpectedly, the present invention found that nanochimeras including exogenous amyloid proteins and ABIcs protein tags can not only target intracellular amyloid aggregates, but also do not affect the normal physiological activity of soluble amyloid proteins in cells. In addition, the present invention can control the addition time of abscisic acid so that the exogenous amyloid protein of the nanochimera is fully inserted into the intracellular amyloid aggregates, and then add abscisic acid, so that the autophagy promoter will recognize the chimera and then perform efficient degradation, such as Fig. 9 As shown. Finally, the amyloid aggregates inserted into the nano-chimera can be efficiently degraded through the lysosomal pathway. The degradation products of the amyloid aggregates degraded by the lysosomes are non-toxic and will not trigger the aggregation of amyloid proteins again, and are highly safe. Therefore, the present invention has a wide range of application scenarios in the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is 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 creative labor.

[0038] Figure 1 The results of plasmid transfection alone (BF: bright field, DF: dark field); Figure 2 This is the result of plasmid co-transfection (BF: bright field); Figure 3 This is the result of intracellular fluorescence localization without ABA; Figure 4 This is the result of intracellular fluorescence localization without ABA; Figure 5 This is the result of intracellular fluorescence localization after adding ABA; Figure 6 This is the result of chase experiment after transfection; Figure 7 It is a schematic diagram of the structure of plasmid α-syn-GFP-ABI; Figure 8 It is a schematic diagram of the structure of plasmid LC3B-mCherry-PYL; Fig. 9 Schematic diagram of the mechanism by which nanochimeras degrade amyloid aggregates. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0040] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0041] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0042] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0043] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0044] 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.

[0045] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges 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.

[0046] Embodiment 1 Construction of a system for targeted degradation of amyloid aggregates a) a nanochimera, comprising a first protein and an ABIcs protein tag, wherein the first protein may be α-synuclein, tau protein, Aβ protein precursor, huntingtin protein or TAR DNA binding protein 43; b) Autophagy promoters, including LC3B autophagy marker and PYLcs protein tag; c) Abscisic acid.

[0047] like Fig. 9 As shown, in this example, α-synuclein was selected as the first protein, and plasmids expressing the nanochimera and the autophagy promoter were constructed, namely, the plasmid α-syn-GFP-ABI (as shown in Figure 7 as shown) and plasmid LC3B-mCherry-PYL (as Figure 8 as shown).

[0048] The amino acid sequence of alpha-synuclein is shown below: DVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQID NO: 1) The nucleotide sequence of alpha-synuclein is shown below: GATGTGTTCATGAAAGGTCTGAGCAAAGCGAAAGAAGGCGTAGTTGCTGCTGCTGAGAAAACCAAACAGGGCGTTGCTGAGGCAGCGGGTAAAACCAAAGAAGGCGTGCTGTACGTTGGCTCTAAGACTAAAGAAGGCGTTGTTCACGGTGTTGCCACTGTGGCGGAGAAAACTAAGGAGCAGGTAACCAATGTTGGTGGTGCGGTTGTAACCGGTGTAACCGCAGTTGCGCAGAAGACTGTTGAAGGTGCTGGTTCCATTGCAGCGGCGACTGGTTTCGTTAAGAAGGACCAGCTGGGTAAGAACGAGGAGGGTGCGCCGCAGGAAGGCATTCTGGAAGATATGCCGGTTGATCCAGACAACGAGGCTTATGAAATGCCGTCCGAGGAGGGTTACCAAGACTACGAGCCGGAAGCA (SEQ ID NO:2) The amino acid sequence of the eGFP protein is shown below: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:3) The nucleotide sequence of the eGFP protein is shown below: ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO:4) The amino acid sequence of the mCherry protein is as follows: VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQD GEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO:5) The nucleotide sequence of the mCherry protein is as follows: ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAG (SEQ ID NO:6) The amino acid sequence of LC3B protein (microtubule-associated protein 1 light chain 3 beta) is shown below: MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKDEDGFLYMVYASQETFGMKLSV (SEQ ID NO:7) The nucleotide sequence of LC3B protein is shown below: ATGCCGTCGGAGAAGACCTTCAAGCAGCGCCGCACCTTCGAACAAAGAGTAGAAGATGTCCGACTTATTCGAGAGCAGCATCCAACCAAAATCCCGGTGATAATAGAACGATACAAGGGTGAGAAGCAGCTTCCTGTTCTGGATAAAACAAAGTTCCTTGTACCTGACCATGTCAACATGAGTGAGCTCA TCAAGATAATTAGAAGGCGCTTACAGCTCAATGCTAATCAGGCCTTCTTCCTGTTGGTGAACGGACACAGCATGGTCAGCGTCTCCACACCAATCTCAGAGGTGTATGAGAGTGAGAAAGATGAAGATGGATTCCTGTACATGGTCTATGCCTCCCAGGAGACGTTCGGGATGAAATTGTCAGTGA (SEQ ID NO:8) The amino acid sequence of the ABIcs protein (protein phosphatase 2C 56) is shown below: VPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLS VDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO:9) The nucleotide sequence of the ABIcs protein (protein phosphatase 2C 56) is shown below: GTGCCTTTGTATGGTTTTACTTCGATTTGTGGAAGAAGACCTGAGATGGAAGATGCTGTTTCGACTATACCAAGATTCCTTCAATCTTCCTCTGGTTCGATGTTAGATGGTCGGTTTGATCCTCAATCCGCCGCTCATTTCTTCGGTGTTTACGACGGCCATGGCGGTTCTCAGGTAGCGAACTATTGTAGAGAGAGGATGCATTTGGCTTTGGCGGAGGAGATAGCTAAGGAGAAACCGATGCTCTGCGATGGTGATACGTGGCTGGAGAAGTGGAAGAAAGCTCTTTTCAACTCGTTCCTGAGAGTTGACTCGGAGATTGAGTCAGTTGCGCCGGAGACGGTTGGGTCAACGTCGGTGGTTGCCGTTGTTTTCCCGTCTCACATCTTCGTCGCTAACTGCGGTGACTCTAGAGCCGTTCTTTGCCGCGGCAAAACTGCACTTCCATTATCCGTTGACCATAAACCGGATAGAGAAGATGAAGCTGCGAGGATTGAAGCCGCAGGAGGGAAAGTGATTCAGTGGAATGGAGCTCGTGTTTTCGGTGTTCTCGCCATGTCGAGATCCATTGGCGATAGATACTTGAAACCATCCATCATTCCTGATCCGGAAGTGACGGCTGTGAAGAGAGTAAAAGAAGATGATTGTCTGATTTTGGCGAGTGACGGGGTTTGGGATGTAATGACGGATGAAGAAGCGTGTGAGATGGCAAGGAAGCGGATTCTCTTGTGGCACAAGAAAAACGCGGTGGCTGGGGATGCATCGTTGCTCGCGGATGAGCGGAGAAAGGAAGGGAAAGATCCTGCGGCGATGTCCGCGGCTGAGTATTTGTCAAAGCTGGCGATACAGAGAGGAAGCAAAGACAACATAAGTGTGGTGGTGGTTGATTTGAAG (SEQ ID NO:10) The amino acid sequence of the PYLcs protein (abscisic acid receptor PYL1) is shown below: TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO:11) The nucleotide sequence of the PYLcs protein (abscisic acid receptor PYL1) is shown as follows: ACACAGGACGAATTCACCCAGCTGAGCCAGAGCATCGCCGAATTCCACACCTACCAGCTGGGCAACGGCAGATGCAGCAGCCTGCTGGCTCAGAGAATCCACGCCCCACCAGAAACCGTGTGGAGCGTGGTGAGAAGATTCGACAGACCCCAGATCTACAAGCACTTCATTAAAAGCTGTAACGTGAGCGAGGACTTCGAGATGAGAGTGGGATGCACCAGAGACGTGAACGTGATCAGCGGACTGCCTGCCAACACCAGCAGAGAGAGACTGGACCTGCTGGACGACGACAGAAGAGTGACCGGATTTAGCATTACCGGCGGCGAGCACAGGCTGAGAAACTATAAGAGCGTGACCACCGTGCATAGATTCGAGAAGGAGGAGGAAGAGGAGAGAATCTGGACAGTGGTGCTGGAGTCATATGTGGTGGACGTGCCAGAGGGAAATAGCGAAGAGGACACCAGACTGTTTGCCGACACCGTGATCAGGCTGAACCTGCAGAAGCTGGCCAGCATCACCGAGGCTATGAAC (SEQ ID NO:12) Example 2 The plasmids constructed in Example 1 (i.e., plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI) were transiently transfected using PEI25K reagent. Plated 24 hours before transfection (200,000 Hela cells per well), transfected when the cell density reached 80%. 1-2 hours before transfection, each well was replaced with 1 mL of fresh growth medium containing 10% serum. In the PEI25K-DNA transfection complex, DNA: PEI25K = 1 μg: 3μL. Cells were cultured in a 37°C, 5% CO2 incubator. 12 hours after transfection, the culture medium containing the PEI25K-DNA complex was removed and replaced with fresh growth medium. Photos were taken 24 hours after transfection.

[0049] The results are as follows Figure 1 As shown, plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI were successfully expressed.

[0050] Embodiment 3 The plasmids constructed in Example 1 (i.e., plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI) were transiently transfected using PEI25K reagent. Plated 24 hours before transfection (200,000 Hela cells per well), transfected when the cell density reached 80%. 1-2 hours before transfection, each well was replaced with 1 mL of fresh growth medium containing 10% serum. In the PEI25K-DNA transfection complex, DNA: PEI25K = 1 μg: 3μL, the mass ratio of plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI was 2:1. Cells were cultured in a 37°C, 5% CO2 incubator. 12 hours after transfection, the culture medium containing the PEI25K-DNA complex was removed and replaced with fresh growth medium. Photos were taken 24 hours after transfection.

[0051] The results are as follows Figure 2 As shown, plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI can be successfully expressed in one cell.

[0052] Embodiment 4 Before plating, place a cell slide in the well plate. Each well contains about 200,000 (2×10 5)Hela cells were inoculated in a twelve-well plate for culture (5% CO2, 37℃), and co-transfection was performed when the cell density reached 80%. The mass ratio of plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI was 2:1; in the PEI25K-DNA transfection complex, DNA: PEI25K = 1 μg: 3μL. 24 hours after transfection, drug ABA or control group PBS was added. 1.5 hours after drug addition, the solution was changed to wash out the drug; 22.5 hours later, the cell slide was taken out, a slide was placed in a humidified box, 35μL of anti-fluorescence quenching sealing medium (containing DAPI) was aspirated, and dropped on the center of the slide in a water drop shape, and the cell slide was covered on the sealing medium (cell side down). After the sealing was completed, the humidified box was placed in a 4℃ refrigerator to store the slices. (After drying at room temperature for 20 minutes, the slices were stored at -20 degrees.) After the slices were completely dried, the results were observed using a confocal microscope.

[0053] The results are as follows Figure 3 , Figure 4 and Figure 5 As shown, regardless of whether the drug ABA is added or not, the fusion protein LC3B-mCherry-PYL can be localized to the autophagosome, and each red spot represents an autophagosome.

[0054] Figure 3 The green fluorescence showed that the exogenous α-synuclein in the nanochimera was evenly distributed in the cell and could not be localized in the autophagosome, that is, the exogenous α-synuclein could not be localized in the lysosome in the absence of ABA.

[0055] Figure 4 It shows that when the drug ABA is not added, although some a-synuclein forms amyloid aggregates due to overexpression under the action of a strong promoter, the two fluorescent spots do not completely overlap. This indicates that without the addition of the drug ABA, amyloid aggregates will not be targeted to the autophagosome by the autophagy promoter LC3B-mCherry-PYL.

[0056] Figure 5 It was shown that when the drug ABA was added, the exogenous α-synuclein in the nanochimera was targeted to the autophagosome by the autophagy promoter LC3B-mCherry-PYL due to the action of the drug ABA, co-localized with the autophagosome, and the two fluorescences overlapped.

[0057] Embodiment 5 About 400,000 (4×10 5)Hela cells were inoculated in six-well plates for culture (5% CO2, 37℃), and co-transfection was performed when the cell density reached 80%. Transfection conditions: the mass ratio of plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI was 2:1; in the PEI25K-DNA transfection complex, DNA: PEI25K = 1 μg: 3μL. Drug ABA was added 24h after transfection. After 1.5h of drug addition, the solution was changed to wash out ABA; after 22.5h of washing out ABA, cycloheximide (CHX) was added to block protein expression (CHX final concentration 100μg / ml). Samples were collected and proteins were extracted at time intervals (0h, 4h, 8h). Western blot was used to detect the degradation of the target protein (sample loading: 20μg).

[0058] The results are as follows Figure 6 As shown, after adding the drug cycloheximide to block protein expression, α-synuclein undergoes time-dependent degradation under the action of ABA. After adding cycloheximide for 8 hours, α-synuclein can be basically completely degraded. The above experimental results show that the nanochimera provided by the present invention can successfully target and degrade amyloid aggregates formed by α-synuclein.

[0059] 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 modes, which are merely illustrative rather than 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 within the protection of the present invention.

Claims

1. Use of a nanochimera in the preparation of a drug for treating neurodegenerative diseases, characterized in that: The nanochimera comprises a first protein and an ABIcs protein tag, wherein the first protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43.

2. The use according to claim 1, characterized in that The nano chimera is used in combination with an autophagy promoter and abscisic acid, wherein the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag.

3. The use according to claim 1, characterized in that The nanochimera is used to target amyloid aggregates, which are formed by aggregation of a second protein, which is identical to the first protein.

4. The use according to claim 1, characterized in that The amyloid aggregates are located inside cells.

5. The use according to claim 1, characterized in that The neurodegenerative disease includes one or more of Parkinson's disease, Lewy body disease, multiple system atrophy, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Prussner-Wilk's disease and Pick's disease.

6. The use according to claim 3, characterized in that The nano chimera is used to insert into the amyloid aggregates. Under the action of the autophagy promoter and abscisic acid, the amyloid aggregates inserted into the nano chimera are degraded through the lysosomal pathway.

7. A system for targeted degradation of amyloid aggregates, comprising: a) a nanochimera comprising a first protein and an ABIcs protein tag, wherein the first protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43; b) autophagy promoters, which include LC3B autophagy marker and PYLcs protein tag; c) Abscisic acid.

8. The system according to claim 7, characterized in that The amyloid aggregates are formed by aggregation of a second protein, which is identical to the first protein.

9. A method for non-therapeutic targeted degradation of intracellular amyloid aggregates, characterized in that: The specific steps are as follows: S101 co-transfecting the first expression vector and the second expression vector into cells; S102: adding abscisic acid to the cells after the first co-transfection time, thereby achieving targeted degradation of amyloid aggregates in the cells; Among them, the first expression vector includes a gene encoding a nanochimera, and the second expression vector includes a gene encoding an autophagy promoter; the nanochimera includes a first protein and an ABIcs protein tag, and the first protein is selected from the group consisting of α-synuclein, tau protein, Aβ protein precursor, huntingtin protein and TAR DNA binding protein 43; the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag.

10. The method according to claim 9, characterized in that The first time period includes 24-48 hours.

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