Use of the technology for targeted degradation of amyloid deposits in the treatment of neurodegenerative diseases

Abstract

The present invention relates to the field of lysosome-targeted degradation, and particularly relates to the use of a technology for targeted degradation of intracellular amyloid deposits in the preparation of drugs for neurodegenerative diseases. The present invention provides the use of a nanochimera in the preparation of a drug for treating neurodegenerative diseases, wherein 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, amyloid precursor protein, huntingtin protein, and TAR DNA-binding protein 43. The nanochimera provided by the present invention can not only target intracellular amyloid aggregates, but also the amyloid aggregates inserted into the nanochimera can be efficiently degraded through the lysosomal pathway, with high safety.

Description

Technical Field

[0001] The present invention relates to the field of lysosomal targeted degradation, and specifically relates to the use of a technology for targeted degradation of intracellular amyloid deposits in the preparation of drugs for neurodegenerative diseases. Background Art

[0002] Targeted protein degradation technology (TPD) includes proteasome-based degradation technology and lysosome-based degradation technology. The proteasome-based degradation technology relies on the ubiquitin-proteasome pathway, which requires the target protein to be degraded to be ubiquitinated 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 depends on the formed autophagosome to enclose the target protein to be degraded, and after fusing with the lysosome, an autolysosome is formed to achieve the degradation of these target proteins. Autophagy-based degradation technologies include AUTAC, AUTOTAC, and ATTEC, all of which use small chemical molecules to induce the degradation of target proteins. However, these small molecules have problems such as complex chemical structures, difficult synthesis and preparation, off-target effects, and none of them can target and degrade intracellular amyloid deposits.

[0003] Chinese Patent Application CN116836295A discloses an autophagy-targeted nanobody chimera, which is composed of a nanobody linked to an autophagy marker. It uses the nanobody instead of a small chemical molecule to achieve targeted recognition of intracellular targets, but it also cannot target and degrade intracellular amyloid deposits. Summary of the Invention

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

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

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

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

[0008] In some embodiments, the first time includes 1 to 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 nano - chimera is used to target amyloid aggregates, which are formed by the aggregation of a second protein, and the second protein is the same as the first protein.

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

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

[0016] As used herein, "amyloid aggregate" or "amyloid deposit" refers to an insoluble aggregate formed by the pathological misfolding and aggregation of a specific amyloid protein. In some embodiments, the amyloid protein includes α - synuclein, tau protein, amyloid - β precursor protein, huntingtin, or TAR DNA - binding protein 43.

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

[0018] In some embodiments, the amyloid aggregate is insoluble.

[0019] In some embodiments, 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, Prusiner - Willke 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 Parkinsonism; when the first protein is amyloid precursor protein (Aβ), the neurodegenerative disease is Alzheimer's disease; when the first protein is huntingtin, the neurodegenerative disease is Huntington's disease; when the first protein is TAR DNA-binding protein 43 (TDP-43), the neurodegenerative disease is amyotrophic lateral sclerosis.

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

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

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

[0024] In a second aspect, the present invention provides a system for targeting and degrading amyloid aggregates, which includes:

[0025] a) A nanochimera, which includes a first protein and an ABIcs protein tag, and the first protein is selected from the group consisting of α-synuclein, tau protein, amyloid precursor protein (Aβ), huntingtin, and TAR DNA-binding protein 43 (TDP-43);

[0026] b) An autophagy promoter, which includes an LC3B autophagy marker and a PYLcs protein tag;

[0027] c) Abscisic acid.

[0028] In some embodiments, the amyloid aggregate is formed by the aggregation of a second protein, and the second protein is the same as the first protein.

[0029] In some embodiments, the nanochimera is used to target amyloid aggregates, and the amyloid aggregates are formed by the aggregation of α-synuclein, tau protein, amyloid precursor protein (Aβ), huntingtin, or TAR DNA-binding protein 43 (TDP-43).

[0030] In some embodiments, the amyloid aggregate is located inside the cell.

[0031] In some embodiments, the amyloid aggregate is insoluble.

[0032] 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:

[0033] S101 Co-transfect the first expression vector and the second expression vector into cells;

[0034] S102 After the first co-transfection time, add abscisic acid to the cells to achieve targeted degradation of the intracellular amyloid aggregates;

[0035] Wherein, the first expression vector includes a gene encoding a nanochimeric body, and the second expression vector includes a gene encoding an autophagy promoter; the nanochimeric body includes a first protein and an ABIcs protein tag, and the first protein is selected from the group consisting of α-synuclein, tau protein, amyloid precursor protein, huntingtin protein, and TAR DNA-binding protein 43; the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag.

[0036] In some embodiments, the first time includes 24-48 hours.

[0037] In some embodiments, the first expression vector and the second expression vector are plasmids.

[0038] In some embodiments, the mass ratio of the first expression vector to the second expression vector includes 1:1-3.

[0039] In some embodiments, the mass ratio of the first expression vector to the second expression vector is 1:2.

[0040] In some embodiments, the cells include Hela cells or neuroblastoma cells.

[0041] Compared with the prior art, the beneficial effects of the present invention at least include the following aspects:

[0042] Existing methods for degrading target proteins such as α-synuclein based on autophagy mainly use specific antibodies or chemical small molecules to induce the degradation of soluble α-synuclein. Chinese Patent Application CN116836295A discloses an autophagy-targeted nanobody chimera, which uses nanobodies for targeted recognition of target proteins inside cells. The inventors found that the above-mentioned existing technologies can only degrade some soluble proteins or prevent the accumulation of toxic proteins, and cannot degrade the already formed amyloid aggregates. For nanobodies, since they can only recognize specific target sites (i.e., fixed antigenic determinants), and the true structure of amyloid aggregates in cells varies greatly, it is difficult for them to effectively target and degrade amyloid aggregates inside cells, and thus it is difficult to apply them to the treatment of neurodegenerative diseases. Existing technologies have not reported how to use autophagy to degrade amyloid aggregates inside cells.

[0043] The present invention breaks through the existing conventional idea of using autophagy-targeted nanobody chimeras to target and recognize soluble proteins, and creatively replaces nanobodies with exogenous amyloid proteins. Unexpectedly, the present invention finds that the nanobody chimera including exogenous amyloid proteins and ABIcs protein tags can not only target amyloid aggregates inside cells, but also does not affect the normal physiological activities of soluble amyloid proteins inside cells. In addition, the present invention can control the addition time of abscisic acid, so that after the exogenous amyloid proteins of the nanobody chimera are fully inserted into the amyloid aggregates inside cells, abscisic acid is added, and then the autophagy promoter will recognize the chimera and then carry out efficient degradation, as Figure 9 shown. Finally, the amyloid aggregates inserted into the nanobody chimera can be efficiently degraded through the lysosomal pathway. The degradation products of the amyloid aggregates degraded by lysosomes are non-toxic and will not cause amyloid protein aggregation again, with high safety. Therefore, the present invention shows broad application scenarios in the treatment of neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0045] Figure 1 It is the result diagram of plasmid transfection alone (BF: bright field, DF: dark field);

[0046] Figure 2 It is the result diagram of plasmid co-transfection (BF: bright field);

[0047] Figure 3 It is the intracellular fluorescence localization result map without ABA;

[0048] Figure 4 It is the intracellular fluorescence localization result map without ABA;

[0049] Figure 5 It is the intracellular fluorescence localization result map with ABA added;

[0050] Figure 6 It is the result map of the chase experiment after transfection;

[0051] Figure 7 It is the schematic structural diagram of the plasmid α-syn-GFP-ABI;

[0052] Figure 8 It is the schematic structural diagram of the plasmid LC3B-mCherry-PYL;

[0053] Figure 9 It is the schematic diagram of the mechanism of the nanochimer to degrade amyloid aggregates. Detailed implementation manners

[0054] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0055] In this article, the orientation or positional relationship indicated by terms such as "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0056] In this article, "and / or" includes any and all combinations of one or more of the listed related items.

[0057] In this article, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0058] It should be noted that in this text, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising such element.

[0059] 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.

[0060] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that such a description of "within a certain range" is merely for convenience and brevity and should not be construed 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 the individual numerical values within that 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 the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0061] Example 1

[0062] Construction of a System for Targeted Degradation of Amyloid Aggregates

[0063] a) A nanochimera, comprising a first protein and an ABIcs protein tag, wherein the first protein can be α-synuclein, tau protein, amyloid precursor protein, huntingtin or TAR DNA-binding protein 43;

[0064] b) An autophagy promoter, comprising an LC3B autophagy marker and a PYLcs protein tag;

[0065] c) Abscisic acid.

[0066] As Figure 9 shown, in this example, α-synuclein was selected as the first protein, and plasmids expressing the nanochimera and the autophagy promoter were constructed, namely plasmid α-syn-GFP-ABI (as Figure 7 shown) and plasmid LC3B-mCherry-PYL (as Figure 8 shown).

[0067] The amino acid sequence of α-synuclein is shown below:

[0068] DVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO:1)

[0069] The nucleotide sequence of α-synuclein is shown below:

[0070] GATGTGTTCATGAAAGGTCTGAGCAAAGCGAAAGAAGGCGTAGTTGCTGCTGCTGAGAAAACCAAACAGGGCGTTGCTGAGGCAGCGGGTAAAACCAAAGAAGGCGTGCTGTACGTTGGCTCTAAGACTAAAGAAGGCGTTGTTCACGGTGTTGCCACTGTGGCGGAGAAAACTAAGGAGCAGGTAACCAATGTTGGTGGTGCGGTTGTAACCGGTGTAACCGCAGTTGCGCAGAAGACTGTTGAAGGTGCTGGTTCCATTGCAGCGGCGACTGGTTTCGTTAAGAAGGACCAGCTGGGTAAGAACGAGGAGGGTGCGCCGCAGGAAGGCATTCTGGAAGATATGCCGGTTGATCCAGACAACGAGGCTTATGAAATGCCGTCCGAGGAGGGTTACCAAGACTACGAGCCGGAAGCA (SEQ ID NO:2)

[0071] The amino acid sequence of eGFP protein is shown below:

[0072] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:3)

[0073] The nucleotide sequence of the eGFP protein is shown below:

[0074] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA (SEQ ID NO:4)

[0075] The amino acid sequence of the mCherry protein is shown below:

[0076] VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO:5)

[0077] The nucleotide sequence of the mCherry protein is shown below:

[0078] ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAG (SEQ ID NO:6)

[0079] The amino acid sequence of LC3B protein (microtubule-associated protein 1 light chain 3 beta) is shown below:

[0080] MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKDEDGFLYMVYASQETFGMKLSV (SEQ ID NO:7)

[0081] The nucleotide sequence of LC3B protein is shown below:

[0082] ATGCCGTCGGAGAAGACCTTCAAGCAGCGCCGCACCTTCGAACAAAGAGTAGAAGATGTCCGACTTATTCGAGAGCAGCATCCAACCAAAATCCCGGTGATAATAGAACGATACAAGGGTGAGAAGCAGCTTCCTGTTCTGGATAAAACAAAGTTCCTTGTACCTGACCATGTCAACATGAGTGAGCTCATCAAGATAATTAGAAGGCGCTTACAGCTCAATGCTAATCAGGCCTTCTTCCTGTTGGTGAACGGACACAGCATGGTCAGCGTCTCCACACCAATCTCAGAGGTGTATGAGAGTGAGAAAGATGAAGATGGATTCCTGTACATGGTCTATGCCTCCCAGGAGACGTTCGGGATGAAATTGTCAGTGA (SEQ ID NO:8)

[0083] The amino acid sequence of the ABIcs protein (protein phosphatase 2C 56) is shown below:

[0084] VPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK (SEQ ID NO:9)

[0085] The nucleotide sequence of the ABIcs protein (protein phosphatase 2C 56) is shown below:

[0086] GTGCCTTTGTATGGTTTTACTTCGATTTGTGGAAGAAGACCTGAGATGGAAGATGCTGTTTCGACTATACCAAGATTCCTTCAATCTTCCTCTGGTTCGATGTTAGATGGTCGGTTTGATCCTCAATCCGCCGCTCATTTCTTCGGTGTTTACGACGGCCATGGCGGTTCTCAGGTAGCGAACTATTGTAGAGAGAGGATGCATTTGGCTTTGGCGGAGGAGATAGCTAAGGAGAAACCGATGCTCTGCGATGGTGATACGTGGCTGGAGAAGTGGAAGAAAGCTCTTTTCAACTCGTTCCTGAGAGTTGACTCGGAGATTGAGTCAGTTGCGCCGGAGACGGTTGGGTCAACGTCGGTGGTTGCCGTTGTTTTCCCGTCTCACATCTTCGTCGCTAACTGCGGTGACTCTAGAGCCGTTCTTTGCCGCGGCAAAACTGCACTTCCATTATCCGTTGACCATAAACCGGATAGAGAAGATGAAGCTGCGAGGATTGAAGCCGCAGGAGGGAAAGTGATTCAGTGGAATGGAGCTCGTGTTTTCGGTGTTCTCGCCATGTCGAGATCCATTGGCGATAGATACTTGAAACCATCCATCATTCCTGATCCGGAAGTGACGGCTGTGAAGAGAGTAAAAGAAGATGATTGTCTGATTTTGGCGAGTGACGGGGTTTGGGATGTAATGACGGATGAAGAAGCGTGTGAGATGGCAAGGAAGCGGATTCTCTTGTGGCACAAGAAAAACGCGGTGGCTGGGGATGCATCGTTGCTCGCGGATGAGCGGAGAAAGGAAGGGAAAGATCCTGCGGCGATGTCCGCGGCTGAGTATTTGTCAAAGCTGGCGATACAGAGAGGAAGCAAAGACAACATAAGTGTGGTGGTGGTTGATTTGAAG (SEQ ID NO:10)

[0087] The amino acid sequence of the PYLcs protein (abscisic acid receptor PYL1) is shown below:

[0088] TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN (SEQ ID NO:11)

[0089] The nucleotide sequence of the PYLcs protein (abscisic acid receptor PYL1) is shown below:

[0090] ACACAGGACGAATTCACCCAGCTGAGCCAGAGCATCGCCGAATTCCACACCTACCAGCTGGGCAACGGCAGATGCAGCAGCCTGCTGGCTCAGAGAATCCACGCCCCACCAGAAACCGTGTGGAGCGTGGTGAGAAGATTCGACAGACCCCAGATCTACAAGCACTTCATTAAAAGCTGTAACGTGAGCGAGGACTTCGAGATGAGAGTGGGATGCACCAGAGACGTGAACGTGATCAGCGGACTGCCTGCCAACACCAGCAGAGAGAGACTGGACCTGCTGGACGACGACAGAAGAGTGACCGGATTTAGCATTACCGGCGGCGAGCACAGGCTGAGAAACTATAAGAGCGTGACCACCGTGCATAGATTCGAGAAGGAGGAGGAAGAGGAGAGAATCTGGACAGTGGTGCTGGAGTCATATGTGGTGGACGTGCCAGAGGGAAATAGCGAAGAGGACACCAGACTGTTTGCCGACACCGTGATCAGGCTGAACCTGCAGAAGCTGGCCAGCATCACCGAGGCTATGAAC (SEQ ID NO:12)

[0091] Example 2

[0092] Plasmids constructed in Example 1 (i.e., LC3B-mCherry-PYL and α-syn-GFP-ABI) were transiently transfected using PEI25K reagent. 200,000 HeLa cells were plated 24 hours before transfection and transfection was performed 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. The PEI25K-DNA transfection complex consisted of 1 μg DNA: PEI25K = 3 μL. Cells were cultured at 37°C in a 5% CO2 incubator. 12 hours after transfection, the culture medium containing the PEI25K-DNA complex was removed and replaced with fresh growth medium. Images were taken 24 hours after transfection.

[0093] The results are as follows Figure 1 As shown, plasmid LC3B-mCherry-PYL and plasmid α-syn-GFP-ABI were successfully expressed, respectively.

[0094] Embodiment 3

[0095] The plasmids constructed in Example 1 (i.e., LC3B-mCherry-PYL and α-syn-GFP-ABI) were transiently transfected using PEI25K reagent. HeLa cells were plated 24 hours before transfection (200,000 cells per well) and transfected when the cell density reached 80%. One to two hours before transfection, each well was replaced with 1 mL of fresh growth medium containing 10% serum. The PEI25K-DNA transfection complex consisted of 1 μg DNA:3 μL PEI25K, with a mass ratio of 2:1 for LC3B-mCherry-PYL and α-syn-GFP-ABI. Cells were cultured at 37°C in a 5% CO2 incubator. Twelve hours after transfection, the culture medium containing the PEI25K-DNA complex was removed and replaced with fresh growth medium. Images were taken 24 hours after transfection.

[0096] 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.

[0097] Embodiment 4

[0098] Before plating, place a cell slide in the well plate. Each well contains about 200,000 (2×10 5Hela cells were seeded in 12-well plates and cultured (5% CO2, 37 °C). Co-transfection was performed when the cell density reached 80%. The mass ratio of plasmid LC3B-mCherry-PYL to plasmid α-syn-GFP-ABI was 2:1; in the PEI25K-DNA transfection complex, DNA:PEI25K = 1 μg: 3 μL. After 24 h of transfection, drug ABA or control PBS was added. After 1.5 h of drug addition, the medium was changed to wash out the drug; after 22.5 h, the cell slides were taken out, a glass slide was placed in a wet box, 35 μL of anti-fluorescence quenching mounting medium (containing DAPI) was aspirated and dropped onto the center of the glass slide in a water droplet shape, and the cell slide was covered on the mounting medium (cell side down). After sealing the slide, the wet box was placed in a 4 °C refrigerator to store the sections. (After drying at room temperature for 20 minutes, store the sections at -20 °C.) After the sections were completely dry, the results were observed under a confocal microscope.

[0099] The results are as Figure 3 , Figure 4 and Figure 5 shown. Whether drug ABA was added or not, the fusion protein LC3B-mCherry-PYL could be localized to autophagosomes, and each red spot represented an autophagosome.

[0100] Figure 3 The green fluorescence of

[0101] Figure 4 showed that the exogenous α-synuclein in the nano-chimeras was evenly distributed in the cells and could not be localized to autophagosomes, that is, the exogenous α-synuclein could not be localized to lysosomes in the absence of ABA.

[0102] Figure 5 showed that when drug ABA was not added, although some α-synuclein formed amyloid aggregates due to overexpression under the action of a strong promoter, the two fluorescent spots did not completely overlap. This indicated that without adding drug ABA, amyloid aggregates would not be targeted to autophagosomes by the autophagy promoter LC3B-mCherry-PYL.

[0103] Example 5

[0104] Approximately 400,000 per well (4×10 5)Hela cells were seeded in six-well plates and cultured (5% CO2, 37 °C). Co-transfection was performed when the cell density reached 80%. Transfection conditions: The mass ratio of plasmid LC3B-mCherry-PYL to plasmid α-syn-GFP-ABI was 2:1; in the PEI25K-DNA transfection complex, DNA:PEI25K = 1 μg: 3 μL. After 24 h of transfection, drug ABA was added. After 1.5 h of adding the drug, the medium was changed to wash out ABA; 22.5 h after washing out ABA, cycloheximide (CHX) was added to block protein expression (final concentration of CHX was 100 μg / ml). Samples were collected at time intervals (0 h, 4 h, 8 h) to extract proteins. Western blot was used to detect the degradation of the target protein (loading amount: 20 μg).

[0105] The results are as Figure 6 shown. After adding cycloheximide to block protein expression, α-synuclein showed time-dependent degradation under the action of ABA. After 8 h of adding cycloheximide, α-synuclein could be basically completely degraded. The above experimental results indicate that the nano-chimeras provided by the present invention can successfully target and degrade the amyloid aggregates formed by α-synuclein.

[0106] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. Use of a nanochimera in the preparation of a medicament for treating neurodegenerative diseases, characterized in that, The nano-chimera includes a first protein and an ABIcs protein tag, and the first protein is α-synuclein; the nano-chimera is used in combination with an autophagy promoter and abscisic acid, and the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag; the use is to target the degradation of amyloid aggregates through the lysosomal pathway; wherein, the amyloid aggregates are formed by the aggregation of α-synuclein, and the abscisic acid is added after the first time when the nano-chimera and the autophagy promoter are added; the first time is 1-7 days; wherein, the amino acid sequence of α-synuclein is as shown in SEQ ID NO:1, the amino acid sequence of the ABIcs protein tag is as shown in SEQ ID NO:9, the amino acid sequence of the LC3B autophagy marker is as shown in SEQ ID NO:7, and the amino acid sequence of the PYLcs protein tag is as shown in SEQ ID NO:11; the neurodegenerative disease is Parkinson's disease.

2. A system for targeting the degradation of amyloid aggregates, comprising: a) A nano-chimera, which includes a first protein and an ABIcs protein tag, and the first protein is α-synuclein; b) An autophagy promoter, which includes an LC3B autophagy marker and a PYLcs protein tag; c) Abscisic acid; wherein the amyloid aggregates are formed by the aggregation of α-synuclein; the system targets the degradation of amyloid aggregates through the lysosomal pathway, and the abscisic acid is added after the first time when the nano-chimera and the autophagy promoter are added; the first time is 1-7 days; wherein, the amino acid sequence of α-synuclein is as shown in SEQ ID NO:1, the amino acid sequence of the ABIcs protein tag is as shown in SEQ ID NO:9, the amino acid sequence of the LC3B autophagy marker is as shown in SEQ ID NO:7, and the amino acid sequence of the PYLcs protein tag is as shown in SEQ ID NO:

11.

3. A method for targeted degradation of intracellular amyloid aggregates for non-therapeutic purposes, characterized in that, The specific steps are as follows: S101 Co-transfect a first expression vector and a second expression vector into cells; S102 After the first time of co-transfection, add abscisic acid to the cells, thereby achieving the targeted degradation of amyloid aggregates in the cells; Wherein, the first expression vector includes a gene encoding a nano-chimera, and the second expression vector includes a gene encoding an autophagy promoter; the nano-chimera includes a first protein and an ABIcs protein tag, and the first protein is α-synuclein; the autophagy promoter includes an LC3B autophagy marker and a PYLcs protein tag; wherein, the amyloid aggregates are formed by the aggregation of α-synuclein; the first time is 1-7 days; wherein, the amino acid sequence of α-synuclein is as shown in SEQ ID NO:1, the amino acid sequence of the ABIcs protein tag is as shown in SEQ ID NO:9, the amino acid sequence of the LC3B autophagy marker is as shown in SEQ ID NO:7, and the amino acid sequence of the PYLcs protein tag is as shown in SEQ ID NO:

11.

4. The method according to claim 3, characterized in that, The first time period is 24 - 48 hours.

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