WO2019062325A1 - 一种衍生自rps23rg1的多肽及其应用 - Google Patents

一种衍生自rps23rg1的多肽及其应用 Download PDF

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Publication number
WO2019062325A1
WO2019062325A1 PCT/CN2018/098264 CN2018098264W WO2019062325A1 WO 2019062325 A1 WO2019062325 A1 WO 2019062325A1 CN 2018098264 W CN2018098264 W CN 2018098264W WO 2019062325 A1 WO2019062325 A1 WO 2019062325A1
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psd
variant
sequence
seq
polypeptide
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PCT/CN2018/098264
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English (en)
French (fr)
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张云武
赵东栋
张慕娴
孟健
许华曦
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厦门大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to the field of molecular biology and disease treatment, particularly in the field of disease treatment associated with the low activity of PSD-95 and/or PSD-93.
  • the invention relates to polypeptides (or variants thereof) useful for treating diseases associated with hypoactivity of PSD-95 and/or PSD-93 (eg, neurological diseases), comprising such polypeptides (or variants thereof) Fusion proteins, as well as the use of such polypeptides (or variants thereof) and fusion proteins.
  • the invention also relates to a pharmaceutical composition useful for treating or ameliorating one or more symptoms of a disease associated with hypoactivity of PSD-95 and/or PSD-93 (eg, a neurological disorder) comprising a polypeptide of the invention (or a variant thereof) or a fusion protein.
  • AD Alzheimer's disease
  • AD is one of the most common age-related cognitively degenerative neurodegenerative diseases that seriously endangers people's health.
  • AD Alzheimer's disease
  • the proportion of people with AD will increase rapidly, bringing enormous economic and spiritual burden to individuals and society.
  • the current existing drugs can not completely cure AD, and can only improve the symptoms of patients to a small extent. It is urgent to develop a new generation of drugs with more efficient and clear mechanism of action.
  • the inventors of the present application have been subjected to extensive experiments and repeated explorations, and it has been unexpectedly discovered that a partial fragment of the RPS23RG1 protein can interact with PSD-95 and PSD-93 to inhibit ubiquitination of PSD-95 and PSD-93, thereby improving PSD- 95 and PSD-93 levels. Based on this finding, the inventors have developed a novel polypeptide drug for treating a disease associated with an activity of PSD-95 and/or PSD-93 (for example, a nervous system disease) and a therapy based on the drug.
  • a disease associated with an activity of PSD-95 and/or PSD-93 for example, a nervous system disease
  • the invention provides an isolated polypeptide or variant thereof, wherein the polypeptide consists of 5 to 50 contiguous amino acid residues of the RPS23RG1 protein and comprises a sequence selected from the group consisting of:
  • the variant differs from the polypeptide from which it is derived by only one or several (eg, 1, 2, 3, 4, 5, 6, 7, 8, or 9) a substitution of an amino acid residue (eg, a conservative substitution or a non-conservative substitution) and retains the biological function of the polypeptide from which it is derived.
  • a substitution of an amino acid residue eg, a conservative substitution or a non-conservative substitution
  • the isolated polypeptide of the invention consists of no more than 45 contiguous amino acid residues of the RPS23RG1 protein, for example, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24 , 23, 22, 21, 20, 19, 18, 17, 16, 15, 15, 14, 13, 12, 11, 10, 9, 8, 7 One, six or five consecutive amino acid residues.
  • the RPS23RG1 protein is of human or murine origin.
  • the RPS23RG1 protein is of murine origin (eg, of mouse origin) and the polypeptide comprises a sequence selected from the group consisting of:
  • the RPS23RG1 protein is of human origin, and the polypeptide comprises a sequence selected from the group consisting of:
  • polypeptide comprises the sequence set forth in SEQ ID NO:5.
  • the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NOs: 5-19, 71-73.
  • the polypeptide consists of 5 to 45 intracellular segments of the RPS23RG1 protein (eg, 45, 44, 43, 42, 42, 41, 39, 38) , 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5)
  • a contiguous amino acid residue consists of, and comprises: an amino acid residue at a position corresponding to positions 130-134 of SEQ ID NO: 1 in the RPS23RG1 protein (for example, the sequence set forth in SEQ ID NO: 5).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: SEQ ID NOs: 20-42; wherein X 1 - X 11 are each Independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 (SEQ ID NO: 21); wherein X 6 is selected from Natural amino acids.
  • X 6 is a proline (P) or a leucine (L).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 A (SEQ ID NO: 22); wherein X 6 is selected From natural amino acids.
  • X 6 is a proline (P) or a leucine (L).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 AX 7 (SEQ ID NO: 23); wherein, X 6 X 7 is each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 6 is a proline (P) or a leucine (L);
  • X 7 is valine (V) or glycine (G).
  • X 6 is a proline (P) or leucine (L)
  • X 7 is valine (V) or glycine (G).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 AX 7 X 8 (SEQ ID NO: 24); X 6 , X 7 , X 8 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A).
  • X 6 is a proline (P) or leucine (L)
  • X 7 is valine (V) or glycine (G)
  • X 8 is leucine (L) Or alanine (A).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 AX 7 X 8 X 9 (SEQ ID NO: 25); Wherein X 6 , X 7 , X 8 and X 9 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T).
  • X 6 is a proline (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or Alanine (A); and
  • X 9 is arginine (R) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 AX 7 X 8 X 9 X 10 (SEQ ID NO: 26 Wherein X 6 , X 7 , X 8 , X 9 , X 10 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T);
  • X 10 is alanine (A) or glycine (G).
  • X 6 is a proline (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or Alanine (A);
  • X 9 is arginine (R) or threonine (T); and
  • X 10 is alanine (A) or glycine (G).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: TTLAHX 6 AX 7 X 8 X 9 X 10 X 11 (SEQ ID NO :27); wherein X 6 , X 7 , X 8 , X 9 , X 10 , X 11 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T);
  • X 10 is alanine (A) or glycine (G);
  • X 11 is a proline (P).
  • X 6 is a proline (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or Alanine (A);
  • X 9 is arginine (R) or threonine (T);
  • X 10 is alanine (A) or glycine (G); and
  • X 11 is a proline (P).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: X 5 TTLAH (SEQ ID NO: 28); wherein X 5 is selected From natural amino acids.
  • X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: X 4 X 5 TTLAH (SEQ ID NO: 29); wherein, X 4 or X 5 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 4 is arginine (R) or glutamic acid (E); and X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: X 3 X 4 X 5 TTLAH (SEQ ID NO: 30); X 3 to X 5 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or Threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: SX 3 X 4 X 5 TTLAH (SEQ ID NO: 31); X 3 to X 5 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or Threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: X 2 SX 3 X 4 X 5 TTLAH (SEQ ID NO: 32) Wherein X 2 to X 5 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: PX 2 SX 3 X 4 X 5 TTLAH (SEQ ID NO: 33) Wherein X 2 to X 5 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: X 1 PX 2 SX 3 X 4 X 5 TTLAH (SEQ ID NO: 34); wherein, X 1 to X 5 are each independently selected from a natural amino acid.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence selected from the group consisting of: EX 1 PX 2 SX 3 X 4 X 5 (SEQ ID NO: 20) Wherein X 1 to X 5 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAH (SEQ ID NO: 35); wherein, X 1 to X 5 are each independently selected from a natural amino acid.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E); and
  • X 5 is serine (S) or threonine (T).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 (SEQ ID NO 36); wherein, X 1 to X 6 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or a leucine (L).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T); and
  • X 6 is a proline (P) or leucine (L).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 A (SEQ ID NO: 37); wherein, X 1 to X 6 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or a leucine (L).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T); and
  • X 6 is a proline (P) or leucine (L).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 AX 7 (SEQ ID NO: 38); wherein, X 1 to X 7 are each independently selected from a natural amino acid.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or leucine (L);
  • X 7 is valine (V) or glycine (G).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or leucine ( L); and
  • X 7 is valine (V) or glycine (G).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 AX 7 X 8 (SEQ ID NO: 39); wherein, X 1 to X 8 are each independently selected from a natural amino acid.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or leucine ( L);
  • X 7 is valine (V) or glycine (G); and
  • X 8 is leucine (L) or alanine (A).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 AX 7 X 8 X 9 (SEQ ID NO: 40); wherein X 1 to X 9 are each independently selected from a natural amino acid.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or leucine ( L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A); and
  • X 9 is arginine (R) or threonine ( T).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 AX 7 X 8 X 9 X 10 (SEQ ID NO: 41); wherein X 1 to X 10 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T);
  • X 10 is alanine (A) or glycine (G).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or leucine ( L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T)
  • X 10 is alanine (A) or glycine (G).
  • the isolated polypeptide or variant thereof comprises a sequence as shown below, or consists of the sequence shown below: EX 1 PX 2 SX 3 X 4 X 5 TTLAHX 6 AX 7 X 8 X 9 X 10 X 11 (SEQ ID NO: 42); wherein X 1 to X 11 are each independently selected from natural amino acids.
  • the isolated polypeptide or variant thereof has one or more of the following characteristics:
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a helper (P) or leucine (L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T);
  • X 10 is alanine (A) or glycine (G);
  • X 11 is a proline (P).
  • X 1 is threonine (T) or leucine (L);
  • X 2 is serine (S) or glycine (G);
  • X 3 is methionine (M) or serine (S);
  • X 4 is arginine (R) or glutamic acid (E);
  • X 5 is serine (S) or threonine (T);
  • X 6 is a proline (P) or leucine ( L);
  • X 7 is valine (V) or glycine (G);
  • X 8 is leucine (L) or alanine (A);
  • X 9 is arginine (R) or threonine (T)
  • X 10 is alanine (A) or glycine (G); and
  • X 11 is a proline (P).
  • polypeptides of the invention can be linked to other functional units.
  • it can be linked to a cell penetrating peptide (CPP) to increase the ability of the polypeptide of the invention (or variants thereof) to penetrate cell membranes.
  • CPP cell penetrating peptide
  • it can be linked to a targeting moiety such that the polypeptide of the invention (or variant thereof) is targeted.
  • it can be linked to a detectable label to facilitate detection of a polypeptide of the invention (or variants thereof).
  • it can be linked to a protein tag to facilitate expression, detection, tracing, and/or purification of a polypeptide (or variant thereof) of the invention.
  • the invention provides a conjugate comprising an isolated polypeptide (or variant thereof) and a modified portion as described above.
  • the modified moiety is selected from additional polypeptides, detectable labels, or any combination thereof.
  • the additional polypeptide is a CPP, eg, a Tat derived peptide, MPG, MPG ⁇ NLS, EB1 or Stearyl-R8.
  • the CPP is preferably a CPP that is capable of promoting the isolation of the polypeptide of the invention (or variants thereof) through the blood brain barrier.
  • Such CPPs are well known in the art and examples include, but are not limited to, Tat-derived peptides, MPG, MPG ⁇ NLS, EB1 or Stearyl-R8.
  • the CPP is a Tat derived peptide.
  • the CPP has the sequence shown below: YARAARRAARR (SEQ ID NO: 43).
  • the CPP is optionally linked to the N-terminus or C-terminus of a polypeptide of the invention (or variant thereof) via a linker. In certain exemplary embodiments, the CPP is directly linked to the N-terminus or C-terminus of a polypeptide of the invention (or a variant thereof). In certain exemplary embodiments, the CPP is directly linked to the C-terminus of a polypeptide of the invention (or a variant thereof).
  • the conjugates of the invention have an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-58, 74-76.
  • the additional polypeptide is a targeting moiety, such as a ligand, receptor or antibody.
  • the additional polypeptide is a protein tag.
  • protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to perform according to the desired purpose (eg, purification, detection) Or trace) select the appropriate protein label.
  • the detectable label is a fluorescent dye, such as FITC.
  • an isolated polypeptide (or variant thereof) of the invention is optionally coupled, conjugated or fused to the modified moiety by a linker.
  • the modified moiety is directly linked to the N-terminus or C-terminus of the polypeptide (or variant thereof).
  • the modified moiety is linked to the N-terminus or C-terminus of the polypeptide (or variant thereof) by a linker.
  • linkers are well known in the art, examples of which include, but are not limited to, one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Gly or Ser) or amino acid derivatives.
  • a linker eg, Ahx, ⁇ -Ala, GABA, or Ava
  • PEG and the like.
  • the linker is an amino acid sequence consisting of one or more (eg, 1, 2, 3, 5 or 5) Gly.
  • the linker is GGG (SEQ ID NO: 59).
  • the invention provides a fusion protein comprising an isolated polypeptide (or variant thereof) of the invention and an additional polypeptide.
  • the additional polypeptide is selected from the group consisting of a CPP, a targeting moiety, a protein tag, or any combination thereof.
  • the CPP is preferably a CPP that is capable of promoting the isolation of the polypeptide of the invention (or variants thereof) through the blood brain barrier.
  • the CPP is a Tat derived peptide.
  • the CPP has the sequence shown below: YARAARRAARR (SEQ ID NO: 43).
  • the CPP is optionally linked to the N-terminus or C-terminus of a polypeptide of the invention (or variant thereof) via a linker.
  • the CPP is directly linked to the N-terminus or C-terminus of a polypeptide of the invention (or a variant thereof).
  • the CPP is directly linked to the C-terminus of a polypeptide of the invention (or a variant thereof).
  • the fusion protein of the invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 44-58, 74-76.
  • the targeting moiety comprises a ligand, a receptor or an antibody.
  • the protein tag is well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how The appropriate protein tag is selected according to the desired purpose (eg, purification, detection or tracing).
  • the additional polypeptide is optionally linked to the N-terminus or C-terminus of the polypeptide of the invention (or variant thereof) via a linker.
  • the linker is a sequence comprising one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Gly or Ser).
  • the linker is an amino acid sequence consisting of one or more (eg, 1, 2, 3, 5 or 5) Gly.
  • the linker is GGG (SEQ ID NO: 59).
  • polypeptide of the present invention (or a variant thereof), the conjugate of the present invention or the fusion protein of the present invention is not limited by the manner in which it is produced, for example, it can be produced by genetic engineering methods (recombination techniques) or by chemical synthesis. The method is produced.
  • Nucleic acid molecule Nucleic acid molecule, vector and host cell
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide (or variant thereof) or fusion protein of the invention.
  • the invention also provides a vector comprising an isolated nucleic acid molecule as described above.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • the vector of the invention is, for example, a plasmid, a cosmid, a phage, a cosmid, and the like.
  • the vector is capable of expressing a polypeptide of the invention (or a variant thereof) or a fusion protein of the invention in a subject (e.g., a mammal, such as a human).
  • the invention also provides a host cell comprising an isolated nucleic acid molecule or vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as 293T cells.
  • the invention provides a method of making a polypeptide (or variant thereof) of the invention or a fusion protein of the invention, comprising: conditions permitting expression of the polypeptide (or variant thereof) or fusion protein
  • the host cell of the invention is cultured, and the polypeptide (or variant thereof) or fusion protein is recovered from the cultured host cell culture.
  • polypeptide (or variant thereof) or fusion protein of the invention can be used to inhibit ubiquitination of PSD-95 and/or PSD-93 in vitro or in a subject, thereby for prevention and/or treatment with PSD-95 And/or diseases in which PSD-93 is too low in activity (eg, neurological diseases).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated polypeptide (or variant thereof), fusion protein, isolated nucleic acid molecule, vector or host cell of the invention, and pharmaceutically acceptable Carrier and / or excipient.
  • the pharmaceutical composition comprises an isolated polypeptide (or variant thereof) or fusion protein of the invention.
  • the pharmaceutical composition optionally further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug having activity for treating a neurological disease (eg, a neurodegenerative disease), for example, levodopa, a dopamine receptor agonist, a monoamine oxidase inhibitor, an anticholinergic agent , a glutamate receptor antagonist, a catechol-O-methyltransferase (COMT) inhibitor, a dopa decarboxylase inhibitor, or any combination thereof.
  • the invention provides a method for treating a disease associated with a hypoactivity of PSD-95 and/or PSD-93 in a subject, or inhibiting ubiquitin of PSD-95 and/or PSD-93
  • a method of increasing the level, or increasing the expression level of PSD-95 and/or PSD-93 comprising administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide of the invention (or variant thereof), fusion
  • the methods comprise the step of administering to a subject in need thereof a therapeutically effective amount of an isolated polypeptide (or variant thereof) or fusion protein of the invention.
  • the invention also relates to an isolated polypeptide (or variant thereof), fusion protein, isolated nucleic acid molecule, vector or host cell of the invention for use in treatment with a PSD-95 and/or PSD in a subject -93 an activity that is too low in activity, or a level that inhibits ubiquitination of PSD-95 and/or PSD-93, or increases the expression level of PSD-95 and/or PSD-93, or uses in the preparation of a medicament
  • the drug is used to treat a disease associated with a hypoactivity of PSD-95 and/or PSD-93 in a subject, or to inhibit ubiquitination of PSD-95 and/or PSD-93, or to increase PSD- Expression levels of 95 and/or PSD-93.
  • the invention also relates to an isolated polypeptide (or variant thereof), fusion protein, isolated nucleic acid molecule, vector or host cell of the invention for use as a medicament.
  • the invention relates also to an isolated polypeptide (or variant thereof), fusion protein, isolated nucleic acid molecule, vector or host cell of the invention for use in treatment with PSD-95 and / in a subject
  • PSD-93 is associated with an activity that is too low in activity, or inhibits the level of ubiquitination of PSD-95 and/or PSD-93, or increases the expression level of PSD-95 and/or PSD-93.
  • the present invention also relates to a method for inhibiting the level of ubiquitination of PSD-95 and/or PSD-93, or increasing the expression level of PSD-95 and/or PSD-93 in vitro, comprising An isolated polypeptide (or variant thereof), fusion protein or conjugate of the invention is contacted with a cell in need thereof.
  • the method is for non-therapeutic purposes.
  • the cell is a neuron.
  • the disease associated with the activity of PSD-95 and/or PSD-93 is too low to refer to a disease or condition that would benefit from PSD-95 and/or PSD-93 levels. Elevation, inhibition of PSD-95 and/or PSD-93 degradation (eg, ubiquitination), or enhancement of PSD-95 and/or PSD-93 activity is thereby ameliorated or cured.
  • PSD-95 expression by epigenetic targeting and modification of the PSD-95 locus can repair cognition in Alzheimer's disease (AD) model mice (APP/PS1 mice) (Bustos el al., 2017). Repair of PSD-93 expression in the hippocampus of APP/PS1AD mice by lentiviral transfection also reduced their spatial learning and memory impairment (Yu et al., 2017). In addition, in other neurodegenerative diseases, such as in the striatum region of Huntington's disease (HD) patients, a decrease in PSD-95 levels was also observed (Fourie et al., 2014).
  • the disease associated with an activity that is too low in PSD-95 and/or PSD-93 is preferably a neurological disease.
  • the nervous system disorder is characterized by a cognitive dysfunction.
  • the neurological disease is a neurodegenerative disease.
  • the neurological condition is selected from the group consisting of Alzheimer's disease, autism, Louis dementia, frontotemporal dementia, vascular dementia, Huntington's disease, progressive supranuclear palsy, Cortical basal dementia, post-traumatic neurodegenerative diseases, chronic traumatic encephalopathy, and stroke.
  • polypeptide (or variant thereof), fusion protein or pharmaceutical composition of the invention may be formulated into any dosage form known in the medical art, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, Powder, granules, elixirs, lozenges, suppositories, injections (including injections, lyophilized powders), and the like.
  • a polypeptide (or variant thereof), fusion protein or pharmaceutical composition of the invention may be formulated as an injectable solution or as a lyophilized powder.
  • polypeptide (or variant thereof) or fusion protein of the invention may be present in a pharmaceutical composition in unit dosage form for ease of administration.
  • the polypeptide (or variant thereof), fusion protein or pharmaceutical composition of the invention can be administered by any suitable method known in the art including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, In the rectum, in the sheath, in the inner cytoplasmic trough, in the groin, intravesical, local (eg, powder, ointment or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the polypeptide (or variant thereof), fusion protein or pharmaceutical composition of the invention is administered by intravenous infusion or injection.
  • polypeptide (or variant thereof), fusion protein or pharmaceutical composition provided by the present invention may be used singly or in combination, or may be used in combination with another pharmaceutically active agent such as a drug having therapeutic activity for treating nervous system diseases.
  • a polypeptide (or variant thereof) or fusion protein of the invention is used in combination with other drugs having therapeutic activity for treating nervous system disorders for prevention and/or treatment with PSD-95 and/or PSD -93 Diseases that are too low in activity (eg, neurological diseases).
  • additional pharmaceutically active agents can be administered prior to, concurrently with, or subsequent to administration of the polypeptide (or variant thereof), fusion protein or pharmaceutical composition of the invention.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of a polypeptide of the invention (or variant thereof) or a fusion protein.
  • prophylactically effective amount is meant an amount sufficient to prevent, arrest, or delay the onset of a disease, such as a disease associated with a low activity of PSD-95 and/or PSD-93.
  • therapeutically effective amount is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
  • the therapeutically effective amount of a polypeptide (or variant thereof) or fusion protein of the invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight, and Gender, the mode of administration of the drug, and other treatments administered simultaneously.
  • the dosage regimen can be adjusted to achieve the best purpose response (e.g., therapeutic or prophylactic response).
  • the best purpose response e.g., therapeutic or prophylactic response
  • it may be administered in a single administration, may be administered multiple times over a period of time, or may be proportionally reduced or increased depending on the urgency of the treatment.
  • a typical non-limiting range of therapeutically or prophylactically effective amounts of a polypeptide (or variant thereof) or fusion protein of the invention is from 0.001 to 100 mg/kg body weight, such as from 0.01 to 50 mg/kg body weight, from 0.1 to 25 mg/kg body weight. It should be noted that the dosage may vary depending on the type and severity of the condition to be treated. Moreover, those skilled in the art understand that for any particular patient, the particular dosage regimen should be adjusted over time according to the needs of the patient and the professional evaluation of the physician; the dosage ranges given herein are for illustrative purposes only and are not limiting Use or range of the pharmaceutical compositions of the invention.
  • the subject may be a mammal, such as a human.
  • RPS23RG1 refers to a protein expressed by the ribosomal protein S23 mRNA recurrent gene Rps23rg1, which is well known to those skilled in the art and described in Zhang et al., 2009; Yan et al. , 2016, and US patent application US 2010/0286252.
  • RPS23RG1 may be from any source, for example, may be of human or non-human origin, such as a source of non-human mammals, such as a murine source (e.g., of mouse origin).
  • amino acid sequence of RPS23RG1 when the amino acid sequence of RPS23RG1 is mentioned, it is described using the sequence shown in SEQ ID NO: 1.
  • amino acid residue at positions 130-134 of RPS23RG1 means the amino acid residues 130 to 134 of the polypeptide represented by SEQ ID NO: 1.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as RPS23RG1 from different species sources
  • substitutions, deletions and/or additions such as RPS23RG1 from different species sources
  • RPS23RG1 shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 1 as well as natural or artificial variants thereof. Also, when describing a sequence fragment of RPS23RG1, it includes not only the sequence fragment shown in SEQ ID NO: 1, but also the corresponding sequence fragment in its natural or artificial variant.
  • amino acid residues 130-134 of RPS23RG1 includes amino acid residues 130-134 of SEQ ID NO: 1, and corresponding fragments thereof (natural or artificial), such as SEQ ID NO : amino acid residues 163-167 of 2;
  • corresponding segment means a segment located at an equivalent position in the sequence to be compared when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • corresponding position refers to the equivalent position in the sequence to be compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity.
  • natural amino acid refers to an amino acid genetically encoded by an organism itself.
  • the expression "natural amino acid” refers to alanine (A), arginine (R), aspartic acid (D), cysteine (C), glutamine. (Q), glutamic acid (E), histidine (H), isoleucine (I), glycine (G), aspartic acid (N), leucine (L), lysine ( K), methionine (M), phenylalanine (F), pros (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) And a group consisting of valine (V).
  • cell penetrating peptide is also known as “cell penetrating peptide", “protein translocation domain (PTD), "Trojan horse peptides” or “Transduction peptide” or the like refers to a polypeptide capable of promoting cellular uptake of various molecules (for example, various macromolecules including proteins or nucleic acids; for example, the polypeptide of the present invention or a variant thereof).
  • Polypeptides are well known in the art and are described, for example, in Stewart, et al., 2008, and Chinese Patent Application No. CN101490081A, which is hereby incorporated by reference herein in its entirety herein in its entirety herein in The method of US Patent Application US 2008/0234183, which is incorporated herein in its entirety by reference.
  • examples of the CPP include, but are not limited to: (i) protein derived CPPs: sequences derived from a gene that controls the antennae (Antennapedia), such as pAntp (43-58); The sequence of HIV-1, such as a Tat-derived peptide, such as amino acid residues 37-72 from TAT, amino acid residues 37-60, amino acid residues 48-60 or amino acid residues 49-57 hCT(9-32); pVEC; plSL; mouse PRP (1-28); E ms (194-220); or Restricocin L3 (60-73), etc.; (ii) model peptides : for example VT5; MAP; or arginine stretch, etc.; (iii) designed CPPs: eg MPG; Transportan; Transportan 10; Pep-1; peptides selected from KALA; Peptides from Bulforin2, and so on.
  • protein derived CPPs sequences derived from a gene that controls the antennae
  • the CPP used in the conjugate of the invention may also be selected from polypeptide sequences having about 60, 70, 80, 90, 95, 99% or 100% sequence identity to any of the polypeptide sequences described above, As long as the polypeptide sequence retains its biological activity, i.e., promotes cellular uptake of the isolated polypeptide (or variant thereof) of the invention and/or promotes the isolated polypeptide (or variant thereof) of the invention to cross the blood brain barrier .
  • targeting moiety refers to a domain capable of directing a polypeptide of the invention (or a variant thereof) to a desired location, which may be a particular tissue, specific Cells, even specific intracellular locations (eg, nucleus, ribosomes, endoplasmic reticulum, lysosomes, or peroxisomes). It is known to those skilled in the art how to design corresponding targeting domains by the properties of the desired position.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or PAM250.
  • the matrix and the gap weight of 16, 14, 12, 10, 8, 6, or 4 and the length weights of 1, 2, 3, 4, 5 or 6 are used to determine the percent identity between the two amino acid sequences.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • isolated means that a target (eg, a polypeptide) has been purified from contaminants present in a sample, such as a sample containing a target obtained from a natural source. .
  • a sample such as a sample containing a target obtained from a natural source.
  • separation does not necessarily exclude the presence of other components that are intended to function in conjunction with the isolate.
  • a polypeptide of the invention can be described as isolated, although it can be linked to a cell penetrating peptide.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient.
  • a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like.
  • Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvant (e.g., complete Freund's adjuvant), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art to stabilize the desired activity of the active ingredient in the drug (eg, inhibitory activity against PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, Sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation Products such as lactalbumin hydrolysate and the like.
  • treating refers to treating or curing a disease (eg, a nervous system disease), delaying the onset of symptoms of a disease (eg, a nervous system disease), and/or delaying a disease (eg, a nervous system disease). development of.
  • a disease eg, a nervous system disease
  • delaying the onset of symptoms of a disease eg, a nervous system disease
  • delaying a disease eg, a nervous system disease
  • prevention refers to preventing, inhibiting or delaying the onset of a disease, such as a neurological disease.
  • a therapeutically effective amount refers to an amount that can effectively achieve the intended purpose.
  • a therapeutically effective amount can be an amount effective or sufficient to treat or cure a disease (eg, a nervous system disorder), delay the onset of symptoms of a disease (eg, a nervous system disorder), and/or delay the progression of a disease (eg, a nervous system disorder).
  • a prophylactically effective amount can be an amount effective or sufficient to prevent, inhibit or delay the onset of a disease, such as a neurological disorder.
  • Such an effective amount can be readily determined by one skilled in the art or by a physician, and can be associated with the intended purpose (eg, treatment or prevention), the general health of the subject, age, sex, weight, severity of the condition being treated, concurrent Symptoms, methods of administration, etc. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, a human
  • has a disease associated with a PSD-95 and/or PSD-93 activity that is too low eg, a neurological disease
  • polypeptide of the invention include, but are not limited to, one or more selected from the group consisting of:
  • polypeptides of the invention (or variants thereof) and fusion proteins comprising the polypeptides (or variants thereof) have significant advantages compared to the prior art.
  • the polypeptides (or variants thereof) and fusion proteins of the invention can significantly inhibit ubiquitination of PSD-95 and/or PSD-93, thereby increasing the levels of PSD-95 and/or PSD-93.
  • the polypeptides (or variants thereof) and fusion proteins of the invention can be used to treat diseases associated with hypoactivity of PSD-95 and/or PSD-93 (eg, neurological diseases, such as neurodegenerative diseases), with significant Clinical value.
  • Figure 1 Construction of Rps23rg1 knockout mice.
  • Figure 1a Schematic diagram of the Rps23rg1 gene structure and TALEN targeting.
  • the CDS sequence of RPS23RG1 ends from 674 bases in the 5th exon to 1099 bases in the 6th exon; the solid box indicates the left and right arms of the TALEN target, and the dotted box indicates the TALEN target deletion.
  • the base is ACTTC.
  • Figure 1b Comparison of amino acid sequences of WT and KO mice. The complete sequence of RPS23RG1 is 141 amino acids. KO mice contained 39 amino acid residues after TALEN targeting.
  • Figure 2 Validation of Rps23rg1 knockout mice.
  • Figure 2a Genotype identification of WT and KO mice. WT and KO mouse DNA were extracted separately and analyzed by SDS-PAGE.
  • Figure 2b Sequencing peaks of WT and KO mice. The WT and KO mouse DNAs were extracted separately and the peak map was sequenced.
  • Figure 2d Analysis of RPS23RG1 protein expression levels in WT and KO mice.
  • RPS23RG1 was overexpressed in HEK293T cells as a positive control (PC), empty plasmid was used as a negative control (NC), brain tissues of WT and KO mice were extracted, and IP-WB was used to detect RPS23RG1 (RR1) protein levels.
  • KO Rps23rg1 knockout mouse; Hetero: Rps23rg1 gene knockout heterozygous mouse; WT: wild type C57BL/6 mouse.
  • Figures 3A-3D Analysis of physiological status of Rps23rg1 knockout mice.
  • Figure 3A Hybrid knockout mice were analyzed as parental hybrid progeny, and the offspring met Mendelian inheritance laws.
  • Figure 3C WT and KO were taken on the first day after birth (P0), and the mouse brain was frozen and sectioned for Nissil staining.
  • MZ edge layer
  • CP cortical plate
  • IZ intermediate zone
  • VZ /SVZ ventricle area / subventricular zone
  • LV lateral ventricle.
  • Figures 4A-4H Behavioral analysis of Rps23rg1 knockout mice.
  • Figure 4A Mine experiment, analyzing the total active distance of KO and WT mice in the mine and the time spent in the middle.
  • Figure 4B Y-maze experiment, analyzing the total active distance of KO and WT mice in the maze and the sequence of the correct order of exploration in the three active arms (Spontaneous alternation, spontaneous spatial alternating behavior).
  • Figure 4C New object recognition experiment, in which A and B are two objects in the training phase, and in the test phase, the B object is replaced by a C object, and the time at which the mouse explores the C object is tested.
  • Figure 4D Conditional panic test, KO and WT mice were placed in a conditional fear box for environmental conflagration and cued analysis.
  • Figure 4E Water maze test, KO and WT mice were placed in a water maze for 7 days, and the platform of the labyrinth was removed on day 8 to test the number of crossings of the mouse in the platform and the time spent in the quadrant of the platform. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figures 5A-5C Analysis of PSD-95 and PSD-93 expression levels in Rps23rg1 knockout mice.
  • Figure 5A WB analysis of WT and KO mouse brain tissue lysed extracts at 3 month old (3-mo) and 5 months old (5-mo), respectively.
  • On the right side is a statistical analysis chart, using the Image J software to perform grayscale analysis of the protein levels in the graph. Results are expressed as mean ⁇ standard error (S.E.M), 4 mice per group.
  • Figure 5B After 3 months of WT and KO mouse hippocampus were isolated and synaptosomes were isolated, the extracted proteins were lysed for WB analysis.
  • S1 represents total lysate in hippocampus
  • P2 and P3 represent synaptosomes.
  • S1 represents total lysate in hippocampus
  • P2 and P3 represent synaptosomes.
  • S1 represents total lysate in hippocampus
  • P2 and P3 represent synaptosomes.
  • Figure 5C Primary neurons of WT and KO mice were obtained and cultured in vitro, and then observed and photographed by confocal microscopy.
  • S.E.M Standard error
  • Figure 5C Primary neurons of WT and KO mice were obtained and cultured in vitro, and then observed and photographed by confocal microscopy.
  • S.E.M Standard error
  • 3 mice per group 17-19 neurons. Ruler: 10 ⁇ m.
  • FIG. 6 Analysis of PSD-95 and PSD-93 ubiquitination levels in Rps23rg1 knockout mice. Three-month WT and KO mouse brain tissue lysates were extracted for IP-WB analysis.
  • Figure 7 Effect of RPS23RG1 on ubiquitination levels of PSD-95 and PSD-93.
  • HA-ubiquitin and GST-PSD-93 (a) or HA-ubiquitin and PSD-95-Flag (b) were overexpressed in HEK293T cells, and divided into 3 dishes after 6 hours, overexpressing HA-MDM2 and HA-RR1, respectively.
  • full length human RPS23RG1 or HA-RR1- ⁇ C intracellular domain deleted humanized RPS23RG1 plasmid. Lysis cells, IP-WB analysis.
  • Figures 8A-8E Evaluation of the interaction of RPS23RG1 with PSD-95 and PSD-93.
  • Figure 8A Schematic representation of the full length sequence of murine RPS23RG1 and its truncation.
  • the murine full-length RPS23RG1 consists of 141 amino acids. Among them, 96 to 116 are the TM domain.
  • Figure 8B Immunoprecipitation analysis of murine RPS23RG1 and its different truncations with PSD-93 and PSD-95 with IgG as a negative control.
  • Figure 8C Amino acid sequence alignment of murine RPS23RG1 and human RPS23RG1. "*" indicates the same amino acid.
  • FIG 8D Schematic representation of the full length sequence of human RPS23RG1 and its truncation.
  • Human full-length RPS23RG1 consists of 173 amino acids. Among them, 131 to 154 are the TM domain, and ⁇ 163-167 is the truncation of the amino acids 163 to 167.
  • Figure 8E Human RPS23RG1 and its different truncations were co-immunoprecipitated with PSD-93 and PSD-95, and IgG was used as a negative control.
  • Figure 9 Evaluation of the binding levels of ubiquitin ligase MDM2 to PSD-95 and PSD-93 in Rps23rg1 knockout mice.
  • Three-month-old WT and KO mouse brain tissues were taken and synaptosomes were isolated, and the same amount of lysate was lysed for IP-WB analysis. Among them, S1 represents total lysate and P2 represents synaptosome.
  • S1 represents total lysate
  • P2 represents synaptosome.
  • Figure 10 Effect of different truncations of the intracellular domain of RPS23RG1 on the binding of PSD-95 to MDM2.
  • the full-length polypeptide (EP-20) and different truncations (TH-5, TA-7 and TP-12) of the intracellular domain of murine RPS23RG1 were used in cells co-expressing PSD-95-Flag and GST-MDM2, respectively.
  • GST-MDM2 and its bound PSD-95 were captured with GST beads, and the level of PSD-95 was detected by immunoblot analysis. The results of the long exposure and short exposure of the glue map are shown.
  • FIG 11 In vivo localization of the RPS23RG1 intracellular sequence in Rps23rg1 knockout mice. On the left is the localization of the human RPS23RG1 intracellular sequence (hRR1-ICD) in the cortex and hippocampus (Hippo), and on the right is the control amino acid sequence (scramble) in the cerebral cortex (Cortex) and hippocampus (Hippo). ) positioning results. Two months old KO mice were injected with hRR1-ICD and control polypeptides in the peritoneal cavity for 3 consecutive days. On the 8th day, brain tissue was taken for frozen section staining to observe fluorescence. Ruler: 100 ⁇ m (large image) or 10 ⁇ m (insert diagram).
  • Figure 12A-12C Evaluation of the efficacy of the RPS23RG1 intracellular sequence in Rps23rg1 knockout mice.
  • Figure 12A Experimental analysis of the T-maze. The total number of times the mouse shuttled 3 arms and the percentage of correct shuttles of 3 arms (%SPA) were analyzed in the T-maze test. There were 10 mice in each group. "ns" indicates no significant difference, *P ⁇ 0.05, **P ⁇ 0.01 (2-tailed Student's t test).
  • Figure 12B Experimental analysis of new object recognition. In the new object location location (OLM) experiment, the time of exploring the object A in the test phase and the time of the object B after the replacement position are analyzed respectively; the object recognition memory (ORM) is recognized in the new object.
  • OLM new object location location
  • Figure 13A-13C Effect of intracellular sequence of RPS23RG1 on PSD-95 and PSD-93 levels in Rps23rg1 knockout mice.
  • Figure 13A Effect of ICD-TAT on the interaction of MDM2 and PSD-93 and PSD-95.
  • mice treated with ICD-TAT or Scb-TAT the extracted brain tissue was isolated and the mouse brain synaptosomes were isolated, the protein was cleaved, immunoprecipitation was performed using IgG or MDM2 antibody, and then PSD-95 or PSD was used.
  • -93 antibody was subjected to immunoblot analysis. Below is a statistical analysis chart, using the Image J software to perform grayscale analysis of the protein levels in the graph.
  • FIG. 13B-3C Effect of ICD-TAT on ubiquitination levels of PSD-95 and PSD-93.
  • mice treated with ICD-TAT or Scb-TAT the rat brain synaptosome cleavage protein was isolated and used anti-PSD-93 antibody (Fig. 13B) or anti-PSD-95 antibody (Fig. 13C). Immunoprecipitation was performed, and then the anti-ubiquitin antibody was used for immunoblotting to analyze the level of related protein ubiquitination.
  • Figures 14A-14C Evaluation of the efficacy of the RPS23RG1 intracellular sequence in AD model mice.
  • Figure 14A After treatment with ICD-TAT or Scb-TAT, hippocampal synaptosomes of WT and APP/PS1 mice were taken, and proteins were lysed and extracted for IP-WB analysis.
  • Figure 14B Effect of ICD-TAT on behavior of AD model mice.
  • T-maze experimental analysis The correct shuttle percentage (% SPA) of the three arms was analyzed in the T-maze experiment. There were 10 mice in each group. "ns" indicates no significant difference, *P ⁇ 0.05, **P ⁇ 0.01 (2-tailed Student's t test).
  • New Object Recognition Location (OLM) Experiment: The time at which the mouse explored the object A during the test phase and the time after the replacement of the object B were separately analyzed.
  • New Object Recognition Memory (ORM) Experiment: The time at which the mouse explored object A and the time of new object C during the test phase were analyzed separately. For each group of 10 mice, "ns" indicates no significant difference, *P ⁇ 0.05 (2-tailed Student's t test).
  • FIG 14C Long-term potentiation (LTP) changes in KO and WT mice were recorded in electrophysiological experiments.
  • HFS High frequency stimulation. 4 mice per group, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 (2-tailed Student’s t test). Ruler: 100ms, 0.2mv.
  • the molecular biology experimental methods and immunoassays used in the present invention are basically referred to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Guide to Editing Molecular Biology, Third Edition, John Wiley & Sons, Inc., 1995; restriction endonucleases are used according to the conditions recommended by the product manufacturer.
  • the reagents from which the source is not indicated in the examples are conventional reagents or commercially available reagents in the art. The invention is described by way of example, and is not intended to limit the scope of the invention.
  • mice brain tissue was taken out, and 10 volumes of pre-cooled sucrose buffer (0.32 M sucrose, 25 mM HEPES, pH 7.4) was added, and homogenized on ice;
  • synaptosome components were used to separate and purify the postsynaptic density (PSD) component, plus Triton X-100 (in HBS) to a final concentration of 1%, and cleavage on ice for 30 min;
  • PSD postsynaptic density
  • HBS Triton X-100
  • Cell collection discard the culture solution, wash the cells with PBS 3 times; discard the PBS, scrape the cells with a cell scraper, collect the cells in a centrifuge tube; centrifuge at 1000 rpm for 5 min, collect the cells at the bottom of the tube, discard the supernatant. liquid.
  • Fresh tissue was removed and immediately placed in liquid nitrogen for freezing, and then stored in a -80 ° C ultra-low temperature freezer for use.
  • the tissue was cut with a surgical scissors before homogenization, and then homogenized by polytron in RIPA lysate, centrifuged at 12000 rpm for 15 min at 4 ° C, and the supernatant was collected and centrifuged once to measure the protein concentration.
  • the OD562 was measured using a microplate reader, and the obtained value was substituted into the protein standard curve to calculate the protein sample concentration.
  • Protein electrophoresis take 30-40 ⁇ g protein sample, add 1/4 volume of 5 ⁇ SDS loading buffer, boil at 100°C for 3-5 minutes, and electrophores in Tris-Glycine running buffer with SDS-PAGE protein gel. .
  • Electrophoresis transfer protein pre-cool the electrophoresis transfer buffer at 4 ° C, cut off the PVDF membrane of appropriate size, soak it with methanol, soak it in the electrophoresis transfer buffer for 10 min with the filter paper, and simultaneously gel the gel. After cutting, immerse in electrophoresis transfer buffer for 5 min. After that, the PVDF film is attached to the glue, and the filter paper is covered on both sides, and the bubbles are exhausted. The film is placed in the electrorotation tank in the order of the positive electrode, and is rotated at a constant current of 4 ° C (300 mA, 90 min).
  • Blocking 5% skim milk powder is blocked at room temperature for 1 h;
  • Secondary antibody reaction Wash PBST three times for 10 min each time, dilute the primary antibody in blocking solution in an appropriate ratio, and incubate for 1 h at room temperature.
  • IP Immunoprecipitation
  • CoIP co-immunoprecipitation
  • step (3) Repeat step (3) three times. The last time, carefully wash the supernatant with a micro-syringe, then add 20 ⁇ L of 2 ⁇ loading buffer, mix and cook at 100 ° C for 5 min. The resulting samples were used for immunoblot analysis.
  • the treated coverslips were placed in a 24-well plate (one per well), and cells of a certain density (density 5% to 10%) were evenly distributed and cultured for 24 hours.
  • Triton X100-PBS penetrated at room temperature for 5-10 min (the time should be strictly controlled).
  • Biotin (EZ-Link Sulfo-NHS-SS-Biotin) was dissolved in freshly prepared PBS/CM buffer to a final concentration of 0.5 mg/mL and placed on ice for storage; cells were removed from the incubator and placed on ice. The medium was aspirated, and the cells were washed three times with an appropriate amount of ice-cold PBS/CM buffer, the buffer was aspirated, the biotin solution was added to the cell culture tray, and the cells were incubated for 20 min in an ice bath to absorb the biotin solution.
  • the brain of 3 month old Rps23rg1 knockout mice and wild type mice were sliced (0.40 mm) in the lateral hippocampus, and fEPSPs were induced in the CA1 region with different intensity stimulation, and the synaptic transmission intensity was compared.
  • LTP was induced by high frequency stimulation, and the attenuation of fEPSPs was compared.
  • mice behavioral experiments were performed using Smart Video Tracking Software (Panlab, Harvard Apparatus) for data acquisition and analysis. Animal behavioral experiments were conducted between 9:00 a.m. and 18:00 p.m. per day, with a light intensity of 650 lux in the laboratory.
  • mice On the day of the experiment, the experimental mice were transferred to the preparation room before the experiment, and the mice were allowed to acclimate for 60 minutes. Prepare the box and labyrinth for the experiment with 70% alcohol before starting the experiment. At the end of each experiment, the cabinet and maze were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment, and the residual odor of the mice was excluded from the experiment.
  • the open field experiment was used to study the autonomous motor ability and anxiety behavior of mice, mainly based on the avoidance of light and open space in mice.
  • the mice were placed in the center of the open field box (40 cm (L) ⁇ 40 cm (W) ⁇ 40 cm (H)), and the mice were freely explored for 10 min in the open field, and the mice were recorded in the open field.
  • mice The Y/T maze test was used to evaluate spontaneous spatial alternation behavior and working memory in mice.
  • the mice were placed in the center of a Y/T labyrinth (30 cm (L) x 6 cm (W) x 15 cm (H)), and then the mice were allowed to freely explore for 5 min in the maze.
  • the limbs of the mice entered the labyrinth arm as the standard for entering the labyrinth arm, and the mouse entered the labyrinth arm three times in succession for a correct autonomous triplet, recording the total distance of the mouse in the maze (Total Distance) and Total arm entries and Alternation triplet.
  • the Novel object recognition test is a learning memory test method established by the principle that rodents innately explore new objects.
  • the mice were placed in the center of the open field box (length 40 cm ⁇ width 40 cm ⁇ height 40 cm), and each mouse was placed in the same position (same grid, same direction), and the mice were adapted for 10 min;
  • Two days two identical objects were placed on the side of the inner space of the market box, and the mouse was placed in the open space box.
  • the placement position was the same as the distance between the two objects, allowing the mice to freely explore for 5 minutes; Days, keep the position of the object unchanged, replace one of the old objects with a new object, put the mouse facing the box arm into the open space box, put the position and the distance between the two objects the same, let the mouse freely explore for 5 min; the mouse
  • the nose pointing at an object or nose touching the object within 2 cm from the object is considered an exploration behavior; the camera system is used to record the time at which the mouse explores the familiar object (TF) and explores the new object (TN).
  • the experiment is similar to the new object recognition experiment, but in the test it is to move one of the objects to another position in the open field box, and then let the mouse explore for 5 minutes freely; the mouse nose points to the object or the nose touches the object within 2 cm from the object. It is regarded as an exploratory behavior; the camera system is used to record the time when the mouse explores the unmoved object and the position where the moving object is moved.
  • the Morris water maze test experiment is to find rodents in the water to find hidden platforms, and to study the spatial learning and memory ability of rodents by analyzing their time and swimming path.
  • the Morris water maze is a round pale blue pool with a diameter of 180 cm and a height of 100 cm and a water depth of 30 cm. The water temperature is maintained at 24 °C ⁇ 1 °C.
  • Different markers were placed in the field of view of the mouse (pool arm), the pool wall marked 4 water inlet points (E, S, W, N), the labyrinth was divided into four quadrants, and a platform was installed in the ES quadrant with a diameter of 6 cm. , so that the water surface is 2cm above the platform.
  • mice were trained four times a day, and placed into the water from the water inlet points of E, S, W and N, respectively, and the movement trajectories of the mice were captured by the camera, and the time from the entry of the water to the climbing of the platform was recorded. Escape latency.
  • the system sets the test time of 60s, and climbs the platform for 10s to automatically shut down the system. If the mouse fails to find the platform within 60s, guide it to find the platform, and stay on the platform for 10sec, escape the incubation period by 60s; locate the navigation test for 7d, and remove the platform on the 8th, on the opposite side of the platform to the pool wall
  • the mice were placed in a space exploration experiment.
  • the camera recorded the number of passages of the mouse in the area where the original platform was located and the time in quadrants of the mouse in the target quadrant of the platform and the other three different quadrants.
  • the Fear conditioning test experiment is a closed box made of wire mesh and capable of generating electric shock and sound.
  • the experimental mice are placed in a box, and the mice are panicked by electric shock.
  • the horror time generated by the sound and the cabinet environment is to study the spatial learning and memory ability of animals.
  • each mouse was placed in a box and allowed to freely explore for 120 seconds to record the baseline of the mouse panic.
  • the mice were given 30S, 80 decibels of sound, and 2S, 0.5mA electric shock was given at 28S.
  • the stimulation was repeated 3 times, each stimulation interval was 30S, and the mice were allowed to stay in the box for 60S after 3 stimulations.
  • TALEN transcription activator-like effector nucleases
  • TALE transcription-like activator effector
  • DFB double-strand breaks
  • NHEJ non-homologous end joining
  • a gene knockout is achieved by forming a random insertion or deletion of a plurality of bases under the repair mechanism.
  • Rps23rg1 knockout mice were constructed using the TALEN gene targeting technique.
  • TALEN-L targeting 5'-TGACCTTTTCGACGAAATCCAG-3'; SEQ ID NO: 60
  • TALEN-R targeting 5'-TGCCTGAACTTCATTGAGGAAA-3'; SEQ ID NO: 61
  • Figure 1 the mouse deleted the "ACTTC” 5 nucleotide residues in the CDS region Base, resulting in frameshift mutation and early termination of protein translation; using the Golden gate TALEN and TAL Effector kit to construct the correct TALENs expression vector, then in vitro transcription into stable mRNA purification and recovery and dilution with microinjection buffer.
  • the TALS23rg1 TALEN-L and TALEN-R mRNA were mixed together and injected into the fertilized eggs of C57BL/6 mice under the microscope using a microscopic operating system, and transplanted into recipient female mice to obtain Rps23rg1 gene knockout.
  • Mouse herein referred to as KO mouse for short.
  • Rps23rg1 knockout mice were crossed with C57BL/6 wild type mice to obtain progeny mice, and Rps23rg1 knockout heterozygous mice (herein referred to as Hetero mice) were obtained by genotypic identification.
  • FIG. Figure 2a shows the results of genotypic identification, extracting DNA from wild-type C57BL/6 mice (herein referred to as WT mice), heterozygous mice (Hetero), and knockout mice (KO), respectively.
  • Design primers 5'-TTCGACGAAATCCAGCAACC-3' (SEQ ID NO: 62) and 5'-GTTCGTGCCCAATGATGGC-3' (SEQ ID NO: 63) for PCR amplification; PCR products were performed by 15% SDS-PAGE gel electrophoresis Genotyping, in which wild-type mouse (WT) PCR product has only one band, length is 74 bp; knockout mouse (KO) PCR product has only one band, length is 69 bp; and gene knockout heterozygotes
  • WT wild-type mouse
  • KO knockout mouse
  • the mouse (Hetero) PCR product contains the two bands described above.
  • Figure 2b shows the sequencing peaks of WT and KO mice, and the PCR products of the above WT and KO mouse DNAs were sent to Ingramec Sequencing for sequencing. The above results indicate that KO mice were successfully constructed at the DNA level.
  • Figure 2c shows RNA expression in WT and KO mice, extracting brain tissue RNA from wild-type mice (WT), heterozygous mice (Hetero), and knockout mice (KO), respectively, by reverse transcription and The level of RPS23RG1 was detected by real-time PCR and normalized by ⁇ -actin level.
  • FIG. 2d shows the expression of RPS23RG1 protein in WT and KO mice.
  • the brain tissues of WT and KO mice were extracted, and the RPS23RG1 protein level was detected by immunoprecipitation and western blot.
  • the plasmid expressing RPS23RG1 (pcDNA3.1/) HEC293T cells were transfected with myk-His A) to obtain HEK293T overexpressing RPS23RG1 and used as a positive control (PC), HEK293T transfected with empty plasmid as a negative control (NC); the results showed that there was no brain tissue in KO mice.
  • the expression of RPS23RG1 protein fully demonstrated that KO mice were successfully constructed at the protein level. Taken together, the results in Figure 2 show that the Rps23rg1 knockout mouse was successfully constructed.
  • FIGS 3A-3D The results of the analysis of the basic physiological conditions of the Rps23rg1 knockout mice are shown in Figures 3A-3D.
  • hybrid knockout mice are mated as parents, and the obtained offspring are cut off from the tail, DNA is extracted, and then PCR amplification is performed using specific primers, and the obtained PCR product is further subjected to SDS-PAGE analysis, and hybrid progeny analysis is performed.
  • Figure 3A The results showed that the offspring met the Mendelian law of inheritance, indicating that the reproductive status of KO mice was normal.
  • Figure 3B shows changes in body weight of WT and KO mice. WT and KO mice of different age groups were used to measure body weight.
  • FIG. 3C shows the results of Nissil staining in mouse brain tissue.
  • the results show that the marginal layer (MZ), cortical plate (CP), intermediate zone (IZ), ventricle/intraventricular region of the mouse brain of KO mice ( The structures of VZ/SVZ) and lateral ventricle (LV) were not significantly different from those of WT mice.
  • the relative thickness of cortical plate (CP) and intermediate zone (IZ) was also not significantly different from that of WT mice.
  • Fig. 3D shows the results of immunofluorescence staining of mouse brain tissue neurons, and the results showed that there was no significant difference between KO mice and WT mice in the number of neurons.
  • Figure 5A shows the results of immunoblotting of synaptic-related protein expression in mouse brain tissue.
  • the results showed that the expression levels of PSD-95 and PSD-93 protein in brain tissue of KO mice were significantly lower than those in WT mice.
  • Figure 5B shows the results of immunoblotting of protein expression in mouse synaptosomes. The results showed that the expression levels of PSD-95 and PSD-93 protein in synaptosomes of KO mice were significantly lower than those in WT mice.
  • Figure 5C shows the results of immunofluorescence staining of primary neurons of WT and KO mice, and the results showed that PSD-95 and PSD-93 were significantly reduced in primary neurons of KO mice compared to WT mice.
  • the above results fully demonstrate that knocking out the Rps23rg1 gene significantly reduced the protein content of PSD-95 and PSD-93.
  • the investigator of the present invention analyzed the ubiquitination levels of PSD-95 and PSD-93 in the brain tissue of KO mice, and the results are shown in Fig. 6.
  • Brain tissue lysate extracted proteins from WT and KO mice of 3 months were subjected to immunoprecipitation (ubiquitin antibody)-immunoblotting (PSD-95 and PSD-93 antibody) analysis.
  • the results showed that the ubiquitination levels of PSD-95 and PSD-93 in brain tissue of KO mice were significantly higher than those in WT mice.
  • the above results further indicated that the levels of PSD-95 and PSD-93 ubiquitination were elevated in KO mice, and the protein content was significantly decreased.
  • the investigator of the present invention co-transfects a plasmid expressing HA-ubiquitin and a plasmid expressing PSD-95-Flag or GST-PSD-93 into HEK293T cells to overexpress ubiquitin and PSD-95 or PSD-93,6 after 3 hours into the flat, and were transfected with the expression of HA-MDM2, HA-RR1 (full-length humanized RPS23RG1) or HA-RR1- ⁇ C (intracellular domain deleted human source RPS23RG1) respectively plasmids overexpress MDM2 , full-length RPS23RG1 or deletion of intracellular domain of RPS23RG1, and then lysed cells, extracted proteins, and analyzed the ubiquitination levels of PSD-95 and PSD-93 by immunoprecipitation-immunoblotting.
  • RPS23RG1 is a typical Ib transmembrane protein. Based on the experimental results obtained in Example 2, the intracellular domain of RPS23RG1 may have interaction with PSD-95 and PSD-93 to affect the ubiquitination of PSD-95 and PSD-93. . Therefore, the investigators of the present invention analyzed the interaction of RPS23RG1 with PSD-95 and PSD-93 by immunoprecipitation-immunoblotting.
  • the investigators of the present invention constructed HA-modified murine full-length RPS23RG1 (1-141) and different truncations (1-134, 1-130, 1-116), see Table 2 and Figure 8A.
  • the nucleic acid sequences encoding the above polypeptides were inserted into the pCMVHA plasmid, respectively, to obtain a plasmid expressing each of the above polypeptides.
  • the above plasmids were transfected into HEK 293T cells, respectively, to overexpress full-length and different truncated RPS23RG1, and overexpress PSD-95-Flag or Myc-PSD-93 in the above cells for immunoprecipitation-immunoblot analysis .
  • the present inventors conducted a comparative analysis of the protein sequences of human and murine RPS23RG1, and found that there was a large similarity between the two (Fig. 8C). Therefore, the investigator of the present invention further examined the interaction of human RPS23RG1 with PSD-95 and PSD-93.
  • the investigators of the present invention constructed expression plasmids of HA-modified human full-length RPS23RG1 (1-173) or different truncations (1-167, 1-162, 1-154, and ⁇ 163-167), see Table 3 and Figure 8D.
  • the nucleic acid sequences encoding the above polypeptides were inserted into the pCMVHA plasmid, respectively, to obtain a plasmid expressing each of the above polypeptides.
  • the above plasmids were transfected into HEK 293T cells, respectively, to overexpress full-length and different truncated RPS23RG1, and overexpress PSD-95-Flag or Myc-PSD-93 in the above cells for immunoprecipitation-immunoblot analysis .
  • the full-length RPS23RG1 (1-173) and the truncated body (1-167) both showed a significant interaction with PSD-95, while the truncated body lacking the intracellular segment "TTLAH".
  • Neither 1-162, 1-154, and ⁇ 163-167 could interact with PSD-95 and PSD-93.
  • the investigators of the present invention synthesized the full-length polypeptide (EP-20) and different truncations (TH-5, TA-7 and TP-) of the intracellular domain of murine RPS23RG1. 12), see Table 4, the above polypeptides were all synthesized by Jill Biochemical Company. The above polypeptides were separately incubated with cells co-expressing PSD-95 and MDM2, and then the cells were lysed to extract proteins, GST-MDM2 and its bound PSD-95 were captured with GST beads, and the level of PSD-95 was detected. The results show that as shown in Figure 10, the above polypeptides can significantly reduce the binding of PSD-95 to MDM2.
  • the core sequence in which RPS23RG1 interacts with PSD-95 and PSD-93 to inhibit its ubiquitination is TTLAH (amino acid residues 163-167 of human RPS23RG1 protein, and 130th of mouse RPS23RG1 protein). -134 amino acid residues), the RPS23RG1 fragment containing this core sequence is able to interact with PSD-95 and PSD-93 to inhibit ubiquitination, increase PSD-95 and PSD-93 levels, thereby improving PSD-95 And neurological diseases caused by a decrease in PSD-93 (such as neurodegenerative diseases).
  • TTLAH amino acid residues 163-167 of human RPS23RG1 protein, and 130th of mouse RPS23RG1 protein.
  • Example 5 Evaluation of the efficacy of polypeptides based on the intracellular domain of RPS23RG1 in Rps23rg1 knockout mice
  • the investigator of the present invention artificially synthesized the human RPS23RG1 intracellular polypeptide hRR1-ICD (ETPSSMRSTTLAHPAVLRA; SEQ ID NO: 15) and the scrambled control polypeptide Scramble (STRMSSPTEARLVAPHALT; SEQ ID NO: 67), and at the amino terminus of the above polypeptide.
  • a FITC fluorescent label and linker (GGG; SEQ ID NO: 59) were added, and a cell-transmembrane peptide derived from TAT (YARAARRAARR; SEQ ID NO: 43) was added to the carboxy terminus, respectively. Called ICD-TAT and Scb-TAT.
  • the above polypeptides were all synthesized by Jill Biochemical Company.
  • the above polypeptide was intraperitoneally injected at a dose of 40 mg/kg/d into 2 month old KO mice or wild type (WT) C57BL/6 mice (obtained from Xiamen University Laboratory Animal Center) for 3 consecutive days.
  • WT wild type
  • mouse brain tissues were taken out and subjected to frozen section staining to observe fluorescence.
  • ICD-TAT was highly expressed in both the cortex (Hippo) and the hippocampus (Hippo) of mice.
  • the T-maze test Fig. 12A
  • the working memory of KO mice treated with ICD-TAT was significantly improved.
  • KO mice after ICD-TAT treatment were compared with untreated KO mice by IP-WB analysis of brain tissues of WT mice and KO mice treated as described above.
  • the levels of PSD-95 and PSD-93 bound to MDM2 in brain tissue were significantly decreased, PSD-95 and PSD-93 protein levels were significantly elevated, and were close to WT mice (Fig. 13A); and, treated with ICD-TAT
  • the levels of PSD-95 ubiquitination (Fig. 13C) and PSD-93 ubiquitination (Fig. 13B) in the brain tissue of KO mice were significantly decreased.
  • the above results indicate that the polypeptide of the present invention can significantly improve synaptic and cognitive impairment in Rps23rg1 knockout mice.
  • Example 6 Evaluation of the efficacy of polypeptides based on the intracellular domain of RPS23RG1 in Alzheimer's disease (AD) model mice
  • This example evaluates the ability of a polypeptide based on the intracellular domain of RPS23RG1 to repair synaptic and cognitive impairment in AD model mice (APP/PS1 mice).
  • the ICD-TAT or Scb-TAT described in Example 4 was intraperitoneally injected at a dose of 40 mg/kg/d to 7-month-old APP/PS1 mice (obtained from Nanjing University Model Animal Research Center) and wild type (WT). In C57BL/6 mice, continuous treatment for 3 days. On the 8th day, the hippocampal synaptosomes were taken out and the PSD-95 and PSD-93 protein levels were detected by western blot. The results are shown in Figure 14A. The results show that ICD-TAT can significantly restore the levels of PSD-95 and PSD-93 in APP/PS1 mice.
  • the mouse behavioral test results (Fig.
  • the polypeptide of the present invention capable of inhibiting the ubiquitination of PSD-95/PSD-93 to increase its expression level is also capable of repairing synaptic and cognitive defects caused by other neurodegenerative diseases, and reversing neuronal damage, thereby being particularly suitable.
  • neurological diseases such as neurodegenerative diseases).

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Abstract

提供了一种RPS23RG1蛋白的特定多肽片段或其变体，其包含Thr Thr Leu Ala His片段。还提供了包含该多肽片段或其变体的缀合物、融合蛋白、药物组合物及其用于制备治疗与PSD-95和/或PSD-93活性过低相关疾病的药物中的用途。

Description

一种衍生自RPS23RG1的多肽及其应用 技术领域
本发明涉及分子生物学和疾病治疗领域，特别是与PSD-95和/或PSD-93活性过低相关的疾病治疗领域。具体而言，本发明涉及可用于治疗与PSD-95和/或PSD-93活性过低相关的疾病(例如，神经系统疾病)的多肽(或其变体)，包含此类多肽(或其变体)的融合蛋白，以及此类多肽(或其变体)和融合蛋白的用途。本发明还涉及可用于治疗或减轻与PSD-95和/或PSD-93活性过低相关的疾病(例如，神经系统疾病)的一种或多种症状的药物组合物，其包含本发明的多肽(或其变体)或融合蛋白。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)又称老年性痴呆症,是一种最普遍的、与年龄密切相关的认知障碍性的神经退行性疾病，严重危害人民健康。随着我国人口平均寿命的延长和社会结构的老龄化，患有AD的人口比率将迅速增加，给个人和社会带来巨大的经济及精神负担。但目前现有的药物都不能完全治愈AD，只能在很小程度上改善患者的症状，迫切需要开发更高效地、具有明确作用机理的新一代药物。
之前已报道，起源于核糖体蛋白S23 mRNA返座的小鼠Rps23rg1基因在细胞系中的过表达能够缓解AD的疾病相关症状。过量表达RPS23RG1的转基因小鼠在体内也发挥作用，并且将此转基因小鼠与三重转基因AD小鼠杂交后，AD小鼠的病理特征改善，突触标志物蛋白水平提高。在AD小鼠中，Rps23rg1的mRNA水平随着小鼠年龄的增加而逐渐减少，提示Rps23rg1在AD中具有保护作用。然而，RPS23RG1蛋白在AD中的具体分子作用机理依然是未知的，因而难以发展出基于RPS23RG1蛋白的可有效用于神经退行性疾病治疗的药物。
因此，针对神经系统疾病(例如神经退行性疾病)患者发展创新的、能更为有效地治疗方法和药物是迫切而必要的。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料的发现，RPS23RG1蛋白的部分片段可以与PSD-95和PSD-93相互作用，抑制PSD-95和PSD-93的泛素化，从而提高PSD-95和PSD-93水平。基于这一发现，本发明人开发了新的用于治疗与PSD-95和/或PSD-93活性过低相关的疾病(例如，神经系统疾病)的多肽药物以及基于该药物的疗法。
多肽或其变体
因此，在一个方面，本发明提供了一种分离的多肽或其变体，其中，所述多肽由RPS23RG1蛋白的5～50个连续氨基酸残基组成，且包含选自下列的序列：
(1)RPS23RG1蛋白中位于与SEQ ID NO:1的第130-134位相对应的位置的氨基酸残基；
(2)RPS23RG1蛋白中位于与SEQ ID NO:1的第122-129位相对应的位置的氨基酸残基；
(3)RPS23RG1蛋白中位于与SEQ ID NO:1的第135-140位相对应的位置的氨基酸残基；和
(4)RPS23RG1蛋白中位于与SEQ ID NO:1的第135-141位相对应的位置的氨基酸残基；
其中，所述变体与其所源自的多肽相异仅在于1个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的置换(例如，保守置换或非保守置换)，且保留了其所源自的多肽的生物学功能。
在某些优选的实施方案中，本发明的分离的多肽由RPS23RG1蛋白的不多于45个的连续氨基酸残基组成，例如，其由45个，44个，43个，42个，41个，40个，39个，38个，37个，36个，35个，34个，33个，32个，31个，30个，29个，28个，27个，26个，25个，24个，23个，22个，21个，20个，19个，18个，17个，16个，15个，14个，13个，12个，11个，10个，9个，8个，7个，6个或5个的连续氨基酸残基组成。
在某些优选的实施方案中，所述RPS23RG1蛋白为人源或鼠源。
在某些优选的实施方案中，所述RPS23RG1蛋白为鼠源(例如，小鼠来源)，并且所述多肽包含选自下列的序列：
(1)鼠源RPS23RG1蛋白的第130-134位氨基酸残基；
(2)鼠源RPS23RG1蛋白的第122-129位氨基酸残基；
(3)鼠源RPS23RG1蛋白的第135-140位氨基酸残基；和
(4)鼠源RPS23RG1蛋白的第135-141位氨基酸残基。
在某些优选的实施方案中，所述RPS23RG1蛋白为人源，并且所述多肽包含选自下列的序列：
(1)人源RPS23RG1蛋白的第163-167位氨基酸残基；
(2)人源RPS23RG1蛋白的第155-162位氨基酸残基；和
(3)人源RPS23RG1蛋白的第168-173位氨基酸残基。
在某些优选的实施方案中，所述多肽包含如SEQ ID NO:5所示的序列。
在某些优选的实施方案中，所述多肽包含选自下列的序列，或由选自下列的序列组成：SEQ ID NO：5～19、71～73。
在某些优选的实施方案中，所述多肽由RPS23RG1蛋白的胞内段的5～45个(例如，45个，44个，43个，42个，41个，40个，39个，38个，37个，36个，35个，34个，33个，32个，31个，30个，29个，28个，27个，26个，25个，24个，23个，22个，21个，20个，19个，18个，17个，16个，15个，14个，13个，12个，11个，10个，9个，8个，7个，6个或5个)连续氨基酸残基组成，且包含：RPS23RG1蛋白中位于与SEQ ID NO:1的第130-134位相对应的位置的氨基酸残基(例如，如SEQ ID NO:5所示的序列)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：SEQ ID NO：20～42；其中，X 1～X 11各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的 序列，或由如下所示的序列组成：TTLAHX 6(SEQ ID NO：21)；其中，X 6选自天然氨基酸。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6A(SEQ ID NO：22)；其中，X 6选自天然氨基酸。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7(SEQ ID NO：23)；其中，X 6、X 7各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
(i)X 6为辅氨酸(P)或亮氨酸(L)；和
(ii)X 7为缬氨酸(V)或甘氨酸(G)。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)，并且X 7为缬氨酸(V)或甘氨酸(G)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8(SEQ ID NO：24)；其中，X 6、X 7、X 8各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
(i)X 6为辅氨酸(P)或亮氨酸(L)；
(ii)X 7为缬氨酸(V)或甘氨酸(G)；和
(iii)X 8为亮氨酸(L)或丙氨酸(A)。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)，X 7为缬氨酸(V)或甘氨酸(G)，并且X 8为亮氨酸(L)或丙氨酸(A)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8X 9(SEQ ID NO：25)；其中，X 6、X 7、X 8、X 9各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
(i)X 6为辅氨酸(P)或亮氨酸(L)；
(ii)X 7为缬氨酸(V)或甘氨酸(G)；
(iii)X 8为亮氨酸(L)或丙氨酸(A)；和
(iv)X 9为精氨酸(R)或苏氨酸(T)。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；X 8为亮氨酸(L)或丙氨酸(A)；并且X 9为精氨酸(R)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8X 9X 10(SEQ ID NO：26)；其中，X 6、X 7、X 8、X 9、X 10各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
(i)X 6为辅氨酸(P)或亮氨酸(L)；
(ii)X 7为缬氨酸(V)或甘氨酸(G)；
(iii)X 8为亮氨酸(L)或丙氨酸(A)；
(iv)X 9为精氨酸(R)或苏氨酸(T)；和
(v)X 10为丙氨酸(A)或甘氨酸(G)。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；X 8为亮氨酸(L)或丙氨酸(A)；X 9为精氨酸(R)或苏氨酸(T)；并且X 10为丙氨酸(A)或甘氨酸(G)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8X 9X 10X 11(SEQ ID NO：27)；其中，X 6、X 7、X 8、X 9、X 10、X 11各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
(i)X 6为辅氨酸(P)或亮氨酸(L)；
(ii)X 7为缬氨酸(V)或甘氨酸(G)；
(iii)X 8为亮氨酸(L)或丙氨酸(A)；
(iv)X 9为精氨酸(R)或苏氨酸(T)；
(v)X 10为丙氨酸(A)或甘氨酸(G)；和
(vi)X 11为辅氨酸(P)。
在一个优选的实施方案中，X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；X 8为亮氨酸(L)或丙氨酸(A)；X 9为精氨酸(R)或苏氨酸(T)；X 10为丙氨酸(A)或甘氨酸(G)；并且X 11为辅氨酸(P)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 5TTLAH(SEQ ID NO：28)；其中，X 5选自天然氨基酸。
在一个优选的实施方案中，X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 4X 5TTLAH(SEQ ID NO：29)；其中，X 4或X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 4为精氨酸(R)或谷氨酸(E)；和
(ii)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 3X 4X 5TTLAH(SEQ ID NO：30)； 其中，X 3～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 3为甲硫氨酸(M)或丝氨酸(S)；
(ii)X 4为精氨酸(R)或谷氨酸(E)；和
(iii)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：SX 3X 4X 5TTLAH(SEQ ID NO：31)；其中，X 3～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 3为甲硫氨酸(M)或丝氨酸(S)；
(ii)X 4为精氨酸(R)或谷氨酸(E)；和
(iii)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 2SX 3X 4X 5TTLAH(SEQ ID NO：32)；其中，X 2～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 2为丝氨酸(S)或甘氨酸(G)；
(ii)X 3为甲硫氨酸(M)或丝氨酸(S)；
(iii)X 4为精氨酸(R)或谷氨酸(E)；和
(iv)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 2为丝氨酸(S)或甘氨酸(G)；X 3为 甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：PX 2SX 3X 4X 5TTLAH(SEQ ID NO：33)；其中，X 2～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 2为丝氨酸(S)或甘氨酸(G)；
(ii)X 3为甲硫氨酸(M)或丝氨酸(S)；
(iii)X 4为精氨酸(R)或谷氨酸(E)；和
(iv)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 1PX 2SX 3X 4X 5TTLAH(SEQ ID NO：34)；其中，X 1～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 1为苏氨酸(T)或亮氨酸(L)；
(ii)X 2为丝氨酸(S)或甘氨酸(G)；
(iii)X 3为甲硫氨酸(M)或丝氨酸(S)；
(iv)X 4为精氨酸(R)或谷氨酸(E)；和
(v)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：EX 1PX 2SX 3X 4X 5(SEQ ID NO：20)；其中，X 1～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有选自下列的特征中的一项或多项：
(i)X 1为苏氨酸(T)或亮氨酸(L)；
(ii)X 2为丝氨酸(S)或甘氨酸(G)；
(iii)X 3为甲硫氨酸(M)或丝氨酸(S)；
(iv)X 4为精氨酸(R)或谷氨酸(E)；和
(v)X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAH(SEQ ID NO：35)；其中，X 1～X 5各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；和
X 5为丝氨酸(S)或苏氨酸(T)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；并且X 5为丝氨酸(S)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6(SEQ ID NO： 36)；其中，X 1～X 6各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；和
X 6为辅氨酸(P)或亮氨酸(L)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；并且X 6为辅氨酸(P)或亮氨酸(L)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6A(SEQ ID NO：37)；其中，X 1～X 6各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；和
X 6为辅氨酸(P)或亮氨酸(L)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；并且X 6为辅氨酸(P)或亮氨酸(L)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7(SEQ ID NO：38)；其中，X 1～X 7各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；
X 6为辅氨酸(P)或亮氨酸(L)；和
X 7为缬氨酸(V)或甘氨酸(G)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；X 6为辅氨酸(P)或亮氨酸(L)；并且X 7为缬氨酸(V)或甘氨酸(G)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8(SEQ ID NO：39)；其中，X 1～X 8各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；
X 6为辅氨酸(P)或亮氨酸(L)；
X 7为缬氨酸(V)或甘氨酸(G)；和
X 8为亮氨酸(L)或丙氨酸(A)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；并且X 8为亮氨酸(L)或丙氨酸(A)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8X 9(SEQ ID NO：40)；其中，X 1～X 9各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；
X 6为辅氨酸(P)或亮氨酸(L)；
X 7为缬氨酸(V)或甘氨酸(G)；
X 8为亮氨酸(L)或丙氨酸(A)；和
X 9为精氨酸(R)或苏氨酸(T)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；X 8为亮氨酸(L)或丙氨酸(A)；并且X 9为精氨酸(R)或苏氨酸(T)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8X 9X 10(SEQ ID NO：41)；其中，X 1～X 10各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中 的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；
X 6为辅氨酸(P)或亮氨酸(L)；
X 7为缬氨酸(V)或甘氨酸(G)；
X 8为亮氨酸(L)或丙氨酸(A)；
X 9为精氨酸(R)或苏氨酸(T)；和
X 10为丙氨酸(A)或甘氨酸(G)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；X 8为亮氨酸(L)或丙氨酸(A)；X 9为精氨酸(R)或苏氨酸(T)；并且X 10为丙氨酸(A)或甘氨酸(G)。
在某些优选的实施方案中，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8X 9X 10X 11(SEQ ID NO：42)；其中，X 1～X 11各自独立地选自天然氨基酸。
在某些优选的实施方案中，所述分离的多肽或其变体具有以下特征中的一项或多项：
X 1为苏氨酸(T)或亮氨酸(L)；
X 2为丝氨酸(S)或甘氨酸(G)；
X 3为甲硫氨酸(M)或丝氨酸(S)；
X 4为精氨酸(R)或谷氨酸(E)；
X 5为丝氨酸(S)或苏氨酸(T)；
X 6为辅氨酸(P)或亮氨酸(L)；
X 7为缬氨酸(V)或甘氨酸(G)；
X 8为亮氨酸(L)或丙氨酸(A)；
X 9为精氨酸(R)或苏氨酸(T)；
X 10为丙氨酸(A)或甘氨酸(G)；和
X 11为辅氨酸(P)。
在一个优选的实施方案中，X 1为苏氨酸(T)或亮氨酸(L)；X 2为丝氨酸(S)或甘氨酸(G)；X 3为甲硫氨酸(M)或丝氨酸(S)；X 4为精氨酸(R)或谷氨酸(E)；X 5为丝氨酸(S)或苏氨酸(T)；X 6为辅氨酸(P)或亮氨酸(L)；X 7为缬氨酸(V)或甘氨酸(G)；X 8为亮氨酸(L)或丙氨酸(A)；X 9为精氨酸(R)或苏氨酸(T)；X 10为丙氨酸(A)或甘氨酸(G)；并且X 11为辅氨酸(P)。
缀合物
特别地，可以将本发明的多肽(或其变体)连接其他功能性单元。例如，可以将其与细胞穿膜肽(CPP)连接，以提高本发明的多肽(或其变体)的穿透细胞膜的能力。例如，可以将其与靶向部分连接，以使得本发明的多肽(或其变体)具有靶向性。例如，可以将其与可检测的标记连接，以便于对本发明的多肽(或其变体)进行检测。例如，可以将其与蛋白标签连接，以便于本发明的多肽(或其变体)的表达、检测、示踪和/或纯化。
因此，在另一个方面，本发明提供了一种缀合物，其包含如上所述的分离的多肽(或其变体)和修饰部分。
在某些优选的实施方案中，所述修饰部分选自另外的多肽、可检测的标记或其任意组合。
在某些优选的实施方案中，所述另外的多肽为CPP，例如，Tat衍生肽、MPG、MPGΔNLS、EB1或Stearyl-R8。在某些优选的实施方案中，所述CPP优选为那些能够促进本发明的分离的多肽(或其变体)透过血脑屏障的CPP。这类CPP是本领域熟知的，其实例包括但不限于Tat衍生肽、MPG、MPGΔNLS、EB1或Stearyl-R8。在一个优选的实施方案中，所述CPP为Tat衍生肽。在一个优选的实施方案中，所述CPP具有如下所示的序列：YARAARRAARR(SEQ ID NO：43)。在某些优选的实施 方案中，所述CPP任选地通过接头连接于本发明的多肽(或其变体)的N端或C端。在某些示例性实施方案中，所述CPP直接连接于本发明的多肽(或其变体)的N端或C端。在某些示例性实施方案中，所述CPP直接连接于本发明的多肽(或其变体)的C端。
在一个优选的实施方案中，本发明的缀合物具有选自下列的氨基酸序列：SEQ ID NO：44～58、74～76。
在某些优选的实施方案中，所述另外的多肽为靶向部分，例如配体、受体或抗体。
在某些优选的实施方案中，所述另外的多肽为蛋白标签(protein tag)。这类蛋白标签是本领域熟知的，其实例包括但不限于His、Flag、GST、MBP、HA、Myc、GFP或生物素，并且本领域技术人员已知如何根据期望目的(例如，纯化、检测或示踪)选择合适的蛋白标签。
在某些优选的实施方案中，所述可检测的标记为荧光染料，例如FITC。
在某些优选的实施方案中，本发明的分离的多肽(或其变体)任选地通过接头与所述修饰部分偶联、缀合或融合。
在某些优选的实施方案中，所述修饰部分直接连接至所述多肽(或其变体)的N端或C端。
在某些优选的实施方案中，所述修饰部分通过接头连接至所述多肽(或其变体)的N端或C端。这类接头是本领域熟知的，其实例包括但不限于包含一个或多个(例如，1个，2个，3个，4个或5个)氨基酸(如，Gly或Ser)或氨基酸衍生物(如，Ahx、β-Ala、GABA或Ava)的接头，或PEG等。在一个优选的实施方案中，所述接头为由一个或多个(例如，1个，2个，3个，4个或5个)Gly组成的氨基酸序列。在一个具体的实施方案中，所述接头为GGG(SEQ ID NO：59)。
融合蛋白
在另一个方面，本发明提供了一种融合蛋白，其包含本发明的分离的多肽(或其变体)和另外的多肽。
在某些优选的实施方案中，所述另外的多肽选自CPP、靶向部分、蛋白标签或其任意组合。
在某些优选的实施方案中，所述CPP优选为那些能够促进本发明的分离的多肽(或其变体)透过血脑屏障的CPP。在一个优选的实施方案中，所述CPP为Tat衍生肽。在一个优选的实施方案中，所述CPP具有如下所示的序列：YARAARRAARR(SEQ ID NO：43)。在某些优选的实施方案中，所述CPP任选地通过接头连接于本发明的多肽(或其变体)的N端或C端。在某些示例性实施方案中，所述CPP直接连接于本发明的多肽(或其变体)的N端或C端。在某些示例性实施方案中，所述CPP直接连接于本发明的多肽(或其变体)的C端。
在一个优选的实施方案中，本发明的融合蛋白具有选自下列的氨基酸序列：SEQ ID NO：44～58、74～76。
在某些优选的实施方案中，所述靶向部分包括配体、受体或抗体。
在某些优选的实施方案中，所述蛋白标签是本领域熟知的，其实例包括但不限于His、Flag、GST、MBP、HA、Myc、GFP或生物素，并且本领域技术人员已知如何根据期望目的(例如，纯化、检测或示踪)选择合适的蛋白标签。
在某些优选的实施方案中，所述另外的多肽任选地通过接头连接至本发明的多肽(或其变体)的N端或C端。在某些优选的实施方案中，所述接头为包含一个或多个(例如，1个，2个，3个，4个或5个)氨基酸(如，Gly或Ser)的序列。在一个优选的实施方案中，所述接头为由一个或多个(例如，1个，2个，3个，4个或5个)Gly组成的氨基酸序列。在一个具体的实施方案中，所述接头为GGG(SEQ ID NO：59)。
本发明的多肽(或其变体)、本发明的缀合物或本发明的融合蛋白不受其产生方式的限定，例如，其可以通过基因工程方法(重组技术)产生，也可以通过化学合成方法产生。
核酸分子、载体及宿主细胞
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的多肽(或其变体)或融合蛋白的核苷酸序列。
在另一个方面，本发明还提供了一种载体，其包含如上所述的分离的 核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。在一个优选实施方案中，本发明的载体是例如质粒，粘粒，噬菌体，柯斯质粒等等。在一个优选实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的多肽(或其变体)或本发明的融合蛋白。
在另一个方面，本发明还提供了包含本发明的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。
在另一个方面，本发明还提供了制备本发明的多肽(或其变体)或本发明的融合蛋白的方法，其包括，在允许所述多肽(或其变体)或融合蛋白表达的条件下，培养本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述多肽(或其变体)或融合蛋白。
治疗方法和药物组合物
本发明的多肽(或其变体)或融合蛋白可用于体外或在受试者体内中抑制PSD-95和/或PSD-93的泛素化，从而用于预防和/或治疗与PSD-95和/或PSD-93活性过低相关的疾病(例如，神经系统疾病)。
因此，在另一个方面，本发明提供了一种药物组合物，其包含本发明的分离的多肽(或其变体)、融合蛋白、分离的核酸分子、载体或宿主细胞，以及药学上可接受的载体和/或赋形剂。
在某些优选的实施方案中，所述药物组合物包含本发明的分离的多肽(或其变体)或融合蛋白。
在某些优选的实施方案中，所述药物组合物任选地还包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是具有治疗神经系统疾病(例如，神经退行性疾病)活性的药物，例如，左旋多巴、多巴胺受体激动剂、单胺氧化酶抑制剂、抗胆碱药、谷氨酸受体拮抗剂、儿茶酚-O-甲基转移酶(COMT)抑制剂、多巴脱羧酶抑制剂或其任意组合。
在另一个方面，本发明提供了一种用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制PSD-95和/或PSD-93的泛素化水 平、或提高PSD-95和/或PSD-93的表达水平的方法，其包括，给有此需要的受试者施用治疗有效量的本发明的分离的多肽(或其变体)、融合蛋白、分离的核酸分子、载体或宿主细胞的步骤。在某些优选的实施方案中，所述方法包括，给有此需要的受试者施用治疗有效量的本发明的分离的多肽(或其变体)或融合蛋白的步骤。
在另一个方面，本发明还涉及本发明的分离的多肽(或其变体)、融合蛋白、分离的核酸分子、载体或宿主细胞用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93的表达水平的用途，或者在制备药物中的用途，所述药物用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93的表达水平。
在另一个方面，本发明还涉及本发明的分离的多肽(或其变体)、融合蛋白、分离的核酸分子、载体或宿主细胞，其用作药物。
在另一个方面，本发明还涉及本发明的分离的多肽(或其变体)、融合蛋白、分离的核酸分子、载体或宿主细胞，其用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93的表达水平。
在另一个方面，本发明还涉及一种用于体外抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93的表达水平的方法，其包括，将本发明的分离的多肽(或其变体)、融合蛋白或缀合物与有此需要的细胞接触。在某些优选的实施方案中，所述方法用于非治疗目的。在某些优选的实施方案中，所述细胞是神经元。
在本发明中，所述与PSD-95和/或PSD-93活性过低相关的疾病是指这样一类疾病或病症，该疾病或病症将受益于PSD-95和/或PSD-93水平的升高、PSD-95和/或PSD-93降解(例如泛素化)的抑制、或PSD-95和/或PSD-93活性的增强从而得到缓解或治愈。
之前已报道，PSD-95及PSD-93的删除或失调会损伤突触功能，进而 影响学习和记忆(Carlisle et al.,2008；Migaud et al.,1998；Nithianantharajah et al.,2013；Tao et al.,2003)。特别地，之前的研究已发现在阿尔兹海默病(AD)患者的大脑及AD小鼠模型中均存在PSD-95水平的降低，并且与增强的病理严重程度和痴呆程度相关(Almeida et al.,2005；Gylys et al.,2004；Proctor et al.,2010；Roselli et al.,2005；Sultana et al.,2010)。另外，通过表观遗传靶向并修饰PSD-95基因座来上调PSD-95表达能够修复阿尔茨海默病(AD)模型小鼠(APP/PS1小鼠)的认知(Bustos el al.,2017)。通过慢病毒转染修复APP/PS1AD小鼠海马区的PSD-93表达水平也能够减少其在空间学习和记忆上的损伤(Yu et al.,2017)。另外，在其他神经退行性疾病中，例如在亨廷顿舞蹈症(HD)患者的纹状体区域中，也观察到PSD-95水平降低(Fourie et al.,2014)。
因此，在某些优选的实施方案中，所述与PSD-95和/或PSD-93活性过低相关的疾病优选为神经系统疾病。在某些优选的实施方案中，所述神经系统疾病的特征在于认知功能障碍(cognitive dysfunction)。在某些优选的实施方案中，所述神经系统疾病为神经退行性疾病(neurodegenerative disease)。在某些优选的实施方案中，所述神经系统疾病选自阿尔兹海默症、自闭症、路易氏痴呆、额颞叶痴呆、血管性痴呆、亨廷顿舞蹈病、进行性核上性麻痹、皮质基底痴呆、创伤后神经退行性疾病、慢性创伤性脑病和脑卒中。
本发明的多肽(或其变体)、融合蛋白或药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，本发明的多肽(或其变体)、融合蛋白或药物组合物可以配制成注射液或冻干粉剂。
此外，本发明的多肽(或其变体)或融合蛋白可以以单位剂量形式存在于药物组合物中，以便于施用。
本发明的多肽(或其变体)、融合蛋白或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如， 粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中，本发明的多肽(或其变体)、融合蛋白或药物组合物通过静脉输注或注射给予。
本发明所提供的多肽(或其变体)、融合蛋白或药物组合物可以单独使用或联合使用，也可以与另外的药学活性剂(例如具有治疗神经系统疾病活性的药物)联合使用。在某些优选的实施方案中，将本发明的多肽(或其变体)或融合蛋白与其它具有治疗神经系统疾病活性的药物联合使用，以预防和/或治疗与PSD-95和/或PSD-93活性过低相关的疾病(例如神经系统疾病)。这种另外的药学活性剂可以在施用本发明的多肽(或其变体)、融合蛋白或药物组合物之前、同时或之后施用。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明的多肽(或其变体)或融合蛋白。“预防有效量”是指，足以预防，阻止，或延迟疾病(例如与PSD-95和/或PSD-93活性过低相关的疾病)的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明的多肽(或其变体)或融合蛋白的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在本发明中，可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
本发明的多肽(或其变体)或融合蛋白的治疗或预防有效量的典型非极限范围是0.001～100mg/kg体重，例如0.01～50mg/kg体重，0.1～25mg/kg体重。应注意的是，剂量可随需要治疗的症状的类型和严重性不同而发生变化。此外，本领域技术人员理解，对于任一特定患者，特定的给药方案应根据患者需要和医生的专业评价而随时间调整；此处给出的剂量范围只用于举例说明目的，而不限定本发明药物组合物的使用或范围。
在本发明中，所述受试者可以为哺乳动物，例如人。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“RPS23RG1”是指，由核糖体蛋白S23mRNA返座基因Rps23rg1表达的蛋白，其是本领域技术人员公知的，并描述于Zhang et al.,2009；Yan et al.,2016，以及美国专利申请US 2010/0286252。在本发明中，RPS23RG1可以来自任何来源，例如可以为人源或非人源，例如非人哺乳动物来源，例如鼠源(如小鼠来源)。
在本发明中，当提及RPS23RG1的氨基酸序列时，其使用SEQ ID NO：1所示的序列来进行描述。例如，表述“RPS23RG1的第130-134位氨基酸残基”是指，SEQ ID NO：1所示的多肽的第130-134位氨基酸残基。然而，本领域技术人员理解，在RPS23RG1的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同物种来源的RPS23RG1)，而不影响其生物学功能。因此，在本发明中，术语“RPS23RG1”应包括所有此类序列，包括例如SEQ ID NO：1所示的序列以及其天然或人工的变体。并且，当描述RPS23RG1的序列片段时，其不仅包括SEQ ID NO：1所示的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“RPS23RG1的第130-134位氨基酸残基”包括，SEQ ID NO：1的第130-134位氨基酸残基，以及其变体(天然或人工)中的相应片段，例如SEQ ID NO：2的第163-167位氨基酸残基。
在本发明中，表述“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。表述“相对应的位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中的等同位置。
如本文中所使用的，术语“天然氨基酸”是指，由生物体自身遗传编码的氨基酸。在某些优选的实施方案中，表述“天然氨基酸”是指由丙氨酸(A)、精氨酸(R)、天冬氨酸(D)、半胱氨酸(C)、谷氨酰胺(Q)、 谷氨酸(E)、组氨酸(H)、异亮氨酸(I)、甘氨酸(G)、天冬氨酸(N)、亮氨酸(L)、赖氨酸(K)、甲硫氨酸(M)、苯丙氨酸(F)、辅氨酸(P)、丝氨酸(S)、苏氨酸(T)、色氨酸(W)、酪氨酸(Y)和缬氨酸(V)组成的组。
如本文中所使用的，术语“细胞穿膜肽(cell penetrating peptide，CPP)”又称为“细胞穿透肽”、“蛋白质转导域(protein translocation domain，PTD)、“Trojan horse peptides”或“转导肽(transduction peptide)”等，其是指，能够促进各种分子(例如，各种大分子包括蛋白或核酸；例如，本发明的多肽或其变体)的细胞摄取的多肽。这类多肽是本领域熟知的，并描述于例如，Stewart,et al.,2008以及中国专利申请CN101490081A(其全部通过引用并入本文)；或者可以通过本领域已知的方法获得，例如详细描述于美国专利申请US 2008/0234183中的方法，其全部通过引用并入本文。
在本发明中，所述CPP的实例包括但不限于：(i)蛋白衍生肽(protein derived CPPs)：如源于控制触角的基因(Antennapedia)的序列，例如pAntp(43-58)；源于HIV-1的序列，例如Tat衍生肽，例如来自TAT的第37-72位氨基酸残基、第37-60位氨基酸残基、第48-60位氨基酸残基或第49-57位氨基酸残基；hCT(9-32)；pVEC；plSL；小鼠PRP(1-28)；E ms(194-220)；或Restricocin L3(60-73)，等等；(ii)模型肽(model peptides)：例如VT5；MAP；或精氨酸伸长序列(arginine stretch)，等等；(iii)设计肽(designed CPPs)：例如MPG；Transportan；Transportan10；Pep-1；选自KALA的肽；或选自Bulforin2的肽，等等。
此外，用作本发明的缀合物中的CPP还可以选自与如上所述的任何多肽序列具有大约60、70、80、90、95、99％或100％的序列同一性的多肽序列，只要该多肽序列仍然保留其生物学活性，即，促进本发明的分离的多肽(或其变体)的细胞摄取和/或促进本发明的分离的多肽(或其变体)透过血脑屏障。
如本文中所使用的，术语“靶向部分”是指，能够将本发明的多肽(或其变体)引导至所期望的位置的结构域，所述期望的位置可以为特定的组织、特定的细胞、甚至特定的细胞内位置(例如细胞核、核糖体、内质网、 溶酶体或过氧化物酶体)。本领域技术人员已知如何通过期望位置的特性设计相应的靶向结构域。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol. Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“分离的”是指，目标物(例如，多肽)已经从存在于样品中的污染物中纯化出来，所述样品例如是从天然来源获得的包含目标物的样品。术语分离的不必排除旨在与分离物联合起作用的其它组 分的存在。例如，本发明的多肽可以被描述为分离的，尽管其可以与细胞穿膜肽连接。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂，稳定剂等。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性(例如对PSD-95泛素化的抑制活性)，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“治疗”是指，治疗或治愈疾病(例如神经系统疾病)，延缓疾病(例如神经系统疾病)的症状的发作，和/或延缓疾病(例如神经系统疾病)的发展。
如本文中所使用的，术语“预防”是指，预防、抑制或延缓疾病(例如神经系统疾病)的发生。
如本文中所使用的，术语“有效量”是指，可以有效实现预期目的的量。例如，治疗有效量可以是有效地或足以治疗或治愈疾病(例如神经系统疾病)，延缓疾病(例如神经系统疾病)症状的发作和/或延缓疾病(例如神经系统疾病)发展的量。预防有效量可以是有效地或足以预防、抑制或延 缓疾病(例如神经系统疾病)发生的量。这样的有效量可以由本领域技术人员或医生容易地确定，并且可以与预期目的(例如治疗或预防)、受试者的一般健康状况、年龄、性别、体重、待治疗的疾病的严重程度、并发症、施用方式等相关。这样的有效量的确定完全在本领域技术人员的能力范围内。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有与PSD-95和/或PSD-93活性过低相关的疾病(例如，神经系统疾病)，或者，具有患有与PSD-95和/或PSD-93活性过低相关的疾病(例如，神经系统疾病)风险。
如本文中所使用的，本发明的多肽的生物学功能包括但不限于选自下列的一种或多种：
1)与PSD-95和/或PSD-93特异性结合的能力；
2)抑制泛素化连接酶MDM2结合PSD-95和/或PSD-93的能力；
3)抑制PSD-95和/或PSD-93泛素化的能力；
4)在受试者体内提高脑组织中PSD-95和/或PSD-93水平的能力(任选地，在将所述多肽与CPP缀合后)；
5)在受试者体内改善由PSD-95和/或PSD-93水平过低而引起的突触功能障碍和/或认知功能障碍的能力(任选地，在将所述多肽与CPP缀合后)；
6)在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病(例如神经系统疾病)的能力(任选地，在将所述多肽与CPP缀合后)。
发明的有益效果
与现有技术相比，本发明的多肽(或其变体)和含有所述多肽(或其变体)的融合蛋白具有显著的有利方面。特别地，本发明的多肽(或其变体)和融合蛋白可显著抑制PSD-95和/或PSD-93的泛素化，从而提高PSD-95和/或PSD-93的水平。因此，本发明的多肽(或其变体)和融合蛋白能够用于治疗与PSD-95和/或PSD-93活性过低相关的疾病(例如神经系统疾病，例如神经退行性疾病)，具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1：Rps23rg1基因敲除小鼠的构建。图1a：Rps23rg1基因结构以及TALEN打靶示意图。RPS23RG1的CDS序列从第5个外显子的674个碱基到第6个外显子的1099个碱基结束；实线方框表示的是TALEN打靶的左右臂，虚线方框表示TALEN打靶缺失碱基为ACTTC。图1b：WT和KO小鼠氨基酸序列比较。RPS23RG1完整序列为141个氨基酸。TALEN打靶后KO小鼠含有39个氨基酸残基。
图2：Rps23rg1基因敲除小鼠的验证。图2a：WT和KO小鼠基因型鉴定。分别提取WT和KO小鼠DNA，SDS-PAGE跑胶分析。图2b：WT和KO小鼠测序峰图。分别提取WT和KO小鼠DNA，测序峰图。图2c：WT和KO小鼠中Rps23rg1的RNA表达水平分析。分别提取WT和KO小鼠脑组织RNA，经反转录和荧光定量PCR检测Rps23rg1水平，再用β-actin水平标准化后进行比较。n＝3。图2d：WT和KO小鼠中RPS23RG1蛋白表达水平分析。在HEK293T细胞中过表达RPS23RG1作为阳性对照(PC)，空载质粒作为阴性对照(NC)，分别提取WT和KO小鼠脑组织，IP-WB检测RPS23RG1(RR1)蛋白水平。KO：Rps23rg1基因敲除小鼠；Hetero：Rps23rg1基因敲除杂合子小鼠；WT：野生型C57BL/6小鼠。
图3A-3D：Rps23rg1基因敲除小鼠的生理状况分析。图3A：杂合敲除小鼠作为亲本杂交后代分析，后代符合孟德尔遗传定律。图3B：分别取不同年龄段的WT和KO小鼠测量体重。结果表示为平均值±标准误差(S.E.M)，n＝50。图3C：分别取WT和KO出生后第一天(P0)小鼠，鼠脑冰冻切片后进行尼氏染色(Nissil staining)分析，MZ：缘层，CP：皮质板，IZ：中间带，VZ/SVZ：脑室区/脑室下区，LV：侧脑室。CP及 IZ区域的厚度由Image J软件定量(右侧图)。结果表示为平均值±标准误差(S.E.M)，n＝3，“ns”表示无显著性差异(2-tailed Student’s t test)。标尺:100μm。图3D：分别取WT和KO出生后第一天(P0)小鼠，鼠脑冰冻切片后，使用anti-NeuN抗体及DAPI进行免疫荧光染色并拍照。右侧图为利用Image J软件对图中神经元数量进行定量分析的结果。其中，Cortex：皮层，Hippo：海马区。结果表示为平均值±标准误差(S.E.M)，n＝3，“ns”表示无显著性差异(2-tailed Student’s t test)。标尺:100μm。
图4A-4H：Rps23rg1基因敲除小鼠的行为学分析。图4A：矿场实验，分析KO和WT小鼠在矿场中总的活动距离和在中间停留的时间。图4B：Y迷宫实验，分析KO和WT小鼠在迷宫中总的活动距离和在3个活动臂中探索的正确顺序的次数比(Spontaneous alternation，自发性空间交替行为)。图4C：新物体识别实验，其中A、B为训练阶段两个物体，在测试阶段把B物体换成C物体，测试小鼠探索C物体的时间。图4D：条件惊恐实验，把KO和WT小鼠分别放到条件惊恐箱中，进行环境惊恐(contex)和线索惊恐(cued)分析。图4E：水迷宫测试，把KO和WT小鼠分别放到水迷宫中训练7天，在第8天撤去迷宫的平台测试小鼠在平台中的穿越次数和在平台所在象限停留的时间。*P<0.05,**P<0.01,***P<0.001。图4F：Input-output记录分析KO和WT小鼠的差异。n＝6-8。**P<0.01(重复测量ANOVA)。图4G-4H：长时程增强(LTP)记录分析KO和WT小鼠的差异，HFS：高频刺激。n＝7-8。**P<0.01(2-tailed Student’s t test)。
图5A-5C：Rps23rg1基因敲除小鼠的PSD-95和PSD-93表达水平分析。图5A：分别取3月龄(3-mo)和5月龄(5-mo)的WT和KO小鼠脑组织裂解提取蛋白进行WB分析。右侧为统计分析图，利用Image J软件对图中蛋白水平进行灰度分析。结果表示为平均值±标准误差(S.E.M)，每组4只小鼠。图5B：分别取3个月的WT和KO小鼠海马区并分离突触小体后，裂解提取蛋白进行WB分析，其中，S1表示海马区总裂解物，P2、P3表示突触小体部分。右侧为统计分析图，利用Image J软件对图中蛋白水平进行灰度分析。结果表示为平均值±标准误差(S.E.M)，每组4只小鼠。图5C：分别获得WT和KO小鼠的原代神经元，并体外培养，而后通过共聚焦显微镜观察并拍照。右侧为利用Image J软件对图中荧光 水平进行的定量分析图。结果表示为平均值±标准误差(S.E.M)，每组3只小鼠、17-19个神经元。标尺：10μm。
图6：Rps23rg1基因敲除小鼠的PSD-95和PSD-93泛素化水平分析。分别取3个月的WT和KO小鼠脑组织裂解提取蛋白进行IP-WB分析。
图7：RPS23RG1对PSD-95和PSD-93泛素化水平的影响。在HEK293T细胞中过表达HA-ubiquitin和GST-PSD-93(a)或HA-ubiquitin和PSD-95-Flag(b)，6小时后平分成3盘，分别过表达HA-MDM2、HA-RR1(全长人源RPS23RG1)或HA-RR1- ΔC(缺失胞内段的人源RPS23RG1)的质粒。裂解细胞，IP-WB分析。
图8A-8E：RPS23RG1与PSD-95和PSD-93相互作用评价。图8A：鼠源RPS23RG1全长序列以及其截短体示意图。鼠源全长RPS23RG1由141个氨基酸组成。其中96位至116位是跨膜区(TM domain)。图8B：鼠源RPS23RG1及其不同截短体与PSD-93以及PSD-95的免疫共沉淀分析，IgG作为阴性对照。图8C：鼠源RPS23RG1和人源RPS23RG1氨基酸序列比对。“*”表示相同的氨基酸。图8D：人源RPS23RG1全长序列以及其截短体示意图。人源全长RPS23RG1由173个氨基酸组成。其中131位至154位是跨膜区(TM domain)，Δ163-167表示缺失第163-167位氨基酸的截短体。图8E：人源RPS23RG1及其不同截短体与PSD-93和PSD-95免疫共沉淀分析，IgG作为阴性对照。
图9：Rps23rg1基因敲除小鼠中泛素连接酶MDM2与PSD-95和PSD-93结合水平的评价。分别取3个月大的WT和KO小鼠脑组织并分离突触小体，裂解取等量的裂解液进行IP-WB分析，其中，S1表示总裂解物，P2表示突触小体部分。下方为统计分析图，利用Image J软件对图中蛋白水平进行灰度分析。结果表示为平均值±标准误差(S.E.M)，n＝3,*P<0.05,**P<0.01(2-tailed Student’s t test)。
图10：RPS23RG1胞内段的不同截短体对PSD-95与MDM2的结合的影响。在共表达PSD-95-Flag和GST-MDM2的细胞中分别用鼠源RPS23RG1胞内段的全长多肽(EP-20)及不同截短体(TH-5、TA-7和TP-12)处理后，用GST珠子捕获GST-MDM2及其结合的PSD-95，再通过免疫印迹分析检测PSD-95的水平。图中显示了胶图的长时间曝光(long  exposure)和短时间曝光(short exposure)的结果。
图11：RPS23RG1胞内段序列在Rps23rg1基因敲除小鼠中的体内定位。左侧为人源RPS23RG1胞内段序列(hRR1-ICD)在脑皮层(Cortex)和海马区(Hippo)的定位结果，右侧为对照氨基酸序列(scramble)在大脑皮质(Cortex)和海马区(Hippo)的定位结果。2个月的KO小鼠连续3天腹腔分别注射hRR1-ICD以及对照多肽，在第8天，取脑组织进行冰冻切片染色观察荧光。标尺：100μm(大图)or 10μm(插入图)。
图12A-12C：RPS23RG1胞内段序列在Rps23rg1基因敲除小鼠中的疗效评价。图12A：T迷宫实验分析。在T迷宫实验中分别分析小鼠穿梭3个臂的总次数以及3个臂的正确穿梭百分率(％SPA)。每组各10只小鼠。“ns”表示无显著性差异，*P<0.05，**P<0.01(2-tailed Student’s t test)。图12B：新物体识别实验分析。在新物体识别位置识别(Object location memory,OLM)实验中分别分析小鼠在测试阶段探索物体A的时间和更换位置后的物体B的时间；在新物体识别物体识别(Object recognition memory,ORM)实验中分别分析小鼠在测试阶段探索物体A的时间和新物体C的时间。每组10只小鼠，“ns”表示无显著性差异，*P<0.05(2-tailed Student’s t test)。图12C：在电生理实验实验中，记录分析KO和WT小鼠的长时程增强(LTP)变化。HFS：高频刺激。每组4只小鼠，*P<0.05,**P<0.01,***P<0.001(2-tailed Student’s t test)。标尺：5ms,2mv。
图13A-13C：RPS23RG1胞内段序列对Rps23rg1基因敲除小鼠的PSD-95和PSD-93水平的影响。图13A：ICD-TAT对MDM2和PSD-93以及PSD-95相互作用的影响。对于经ICD-TAT或Scb-TAT治疗后的小鼠，获得提取脑组织取脑后分离鼠脑突触小体，裂解提取蛋白，使用IgG或MDM2抗体进行免疫沉淀,然后使用PSD-95或PSD-93抗体进行免疫印迹分析。下方为统计分析图，利用Image J软件对图中蛋白水平进行灰度分析。结果表示为平均值±标准误差(S.E.M)，每组3只小鼠，“ns”表示无显著性差异，*P<0.05(2-tailed Student’s t test)。图13B-3C：ICD-TAT对PSD-95及PSD-93泛素化水平的影响。对于经ICD-TAT或Scb-TAT治疗后的小鼠，取脑后分离鼠脑突触小体裂解提取蛋白，使用anti-PSD-93抗体(图13B)或anti-PSD-95抗体(图13C)进行免疫沉淀,然后使用anti-泛素抗体进行 免疫印迹分析相关蛋白泛素化水平。
图14A-14C：RPS23RG1胞内段序列在AD模型小鼠中的疗效评价。图14A：经ICD-TAT或Scb-TAT治疗后，分别取WT和APP/PS1小鼠海马突触小体，裂解并提取蛋白进行IP-WB分析。右侧为统计分析图，利用Image J软件对图中蛋白水平进行灰度分析。结果表示为平均值±标准误差(S.E.M)，n＝3,*P<0.05,**P<0.01(2-tailed Student’s t test)。图14B：ICD-TAT对AD模型小鼠行为学的影响。T迷宫实验分析：在T迷宫实验中分析3个臂的正确穿梭百分率(％SPA)。每组各10只小鼠。“ns”表示无显著性差异，*P<0.05，**P<0.01(2-tailed Student’s t test)。新物体识别位置识别(Object location memory,OLM)实验：分别分析小鼠在测试阶段探索物体A的时间和更换位置后的物体B的时间。新物体识别物体识别(Object recognition memory,ORM)实验：分别分析小鼠在测试阶段探索物体A的时间和新物体C的时间。每组10只小鼠，“ns”表示无显著性差异，*P<0.05(2-tailed Student’s t test)。图14C：在电生理实验实验中，记录分析KO和WT小鼠的长时程增强(LTP)变化。HFS：高频刺激。每组4只小鼠，*P<0.05,**P<0.01,***P<0.001(2-tailed Student’s t test)。标尺：100ms,0.2mv。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2018098264-appb-000001
Figure PCTCN2018098264-appb-000002
Figure PCTCN2018098264-appb-000003
Figure PCTCN2018098264-appb-000004
Figure PCTCN2018098264-appb-000005
Figure PCTCN2018098264-appb-000006
Figure PCTCN2018098264-appb-000007
Figure PCTCN2018098264-appb-000008
Figure PCTCN2018098264-appb-000009
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明来源的试剂均是本领域的常规试剂或市售可得的试剂。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实验材料及方法
实时荧光定量PCR(qRT-PCR)
使用TRIzol试剂并根据厂家说明从细胞和组织中提取总RNA，酶标仪测定浓度后，利用试剂盒将mRNA反转录成cDNA。使用FastStart Universal SYBR Green Maste(Roche)进行qRT-PCR实验。qRT-PCR实验结束后，将PCR产物于1.5％琼脂糖凝胶电泳中进行分析，以确定PCR产物片段大小是否正确，是否为特异性扩增。
突触小体和突触后致密区(PSD)的分离
(1)取出小鼠脑组织，加10倍体积的预冷的蔗糖缓冲液(0.32M sucrose，25mM HEPES,pH7.4)，冰上匀浆；
(2)750g，4℃离心5min，去除细胞核以及大的组织碎片，分离出上清(S1)；
(3)将S1组分于10000g，4℃离心12min，分离出上清(S2，密 度较小的细胞膜和包浆组分)以及沉淀(P2，粗的突触小体(Synaptosome)组分)；
(4)将P2组分用蔗糖缓冲液洗2次，然后用预冷的HBS缓冲液(25mM HEPES,pH 7.4，150mM NaCl)重悬，即得到突触体组分，留取一部分用于分析；
(5)剩余的突触体组分全部用于分离纯化突触后致密区(Postsynaptic density，PSD)组分，加Triton X-100(in HBS)至终浓度为1％，冰上裂解30min；
(6)20000g，4℃离心30min，所得沉淀即为PSD组分，然后加入3％SDS(in HBS)溶解沉淀。
蛋白提取与Western blot
1.细胞样品蛋白的提取：
(1)细胞收集：弃去培养液，用PBS清洗细胞3次；弃去PBS，用细胞刮子刮下细胞，收集到离心管中；1000rpm离心5min，将细胞收集到管底，弃上清液。
(2)细胞裂解：向(1)中所收集到的细胞团中加入含cocktail蛋白酶抑制剂和磷酸酶抑制剂(临用前加)的TNEN细胞裂解液，冰上振荡30min，4℃，12000rpm离心10min，收集上清，测蛋白浓度。
2.组织样品蛋白的提取：
新鲜组织取出后立即置入液氮冷冻，随后存放于-80℃超低温冰箱待用。匀浆前用手术剪将组织剪碎，之后在RIPA裂解液中用polytron电动匀浆，4℃，12000rpm离心15min，收集上清，重复离心一次，测蛋白浓度。
3.蛋白浓度的测定：
(1)配制BCA蛋白测定液，将A液与B液50:1混合，备用；
(2)以BSA为标准样品，制作蛋白标准曲线；
(3)在96孔板中分别加入蛋白样品，以TNEN/RIPA裂解液作为空白对照，每组设3孔；
(4)在各孔中加入配制好的蛋白测定液200μl，混匀，37℃恒温箱 中反应30min；
(5)使用酶标仪测定OD562，所得值代入蛋白标准曲线中计算蛋白样品浓度。
4.Western blot：
(1)蛋白电泳：取30-40μg蛋白样品，加入1/4体积的5×SDS上样缓冲液，100℃煮沸3～5min，用SDS-PAGE蛋白胶，于Tris-Glycine电泳缓冲液中电泳。
(2)电泳转移蛋白质：将电泳转移缓冲液于4℃预冷，剪下适当大小的PVDF膜，用甲醇浸湿后，与滤纸一起在电泳转移缓冲液中浸泡10min，同时将电泳后的胶切好后浸泡于电泳转移缓冲液5min。之后把PVDF膜贴于胶上，两面覆盖滤纸，赶尽气泡，按膜朝正极的顺序装于电转槽中，于4℃恒流电转(300mA，90min)。
(3)抗原抗体反应：
a.封闭：5％脱脂奶粉室温封闭1h；
b.一抗反应：把一抗以适当比例稀释于封闭液中，4℃过夜；
c.二抗反应：PBST洗三次，每次10min，把一抗以适当比例稀释于封闭液中，室温孵育1h。
(4)ECL检测：
PBST洗三次，每次10～15min；将ECL试剂盒中的A液和B液以1:1(V/V)混合，于暗室中滴加ECL到膜表面，孵育5min后曝光，依照荧光强度调整曝光时间。
免疫沉淀(IP)或免疫共沉淀(CoIP)
(1)收集蛋白样品，蛋白样品中加入10μl Protein-A Sepharose，于4℃旋转孵育1-2h。
(2)4℃，5000rpm离心2min。将上清转移至新的EP管，用0.5％TNEN裂解液补齐至700ul，加入20μl Protein-G Sepharose和相应抗体，于4℃旋转孵育过夜。
(3)4℃，5000rpm离心2min，吸去上清后，加入预冷的1％TNEN溶液，4℃旋转孵育8min。
(4)重复步骤(3)三次，最后一次用微量注射器小心将上清吸干，然后加入20μL 2×上样缓冲液，混匀后100℃煮样5min。所得的样品用于免疫印迹分析。
免疫荧光
(1)将经过处理的盖玻片放入24孔板中(每孔一个)，均匀分入一定密度的细胞(密度5％～10％)，培养24h。
(2)按lip2000转染方法转入相应质粒。
(3)转染24h后，吸掉培养基，预冷的PBS洗细胞3次。吸去PBS用4％多聚甲醛室温固定10min。
(4)0.1％Triton X100-PBS室温穿透5-10min(此处应严格控制时间)。
(5)3％BSA-PBS封闭1h。
(6)以不同的一抗(1:100稀释在3％BSA-PBS中)4℃孵育过夜。
(7)PBS洗4次，每次5min，此时培养盘静置，无需摇动。
(8)用相应的荧光二抗室温孵育60min。
(9)如有需要，DAPI室温孵育3-5min。
(10)PBS洗4次，每次5min。
(11)室温封片过夜，待封片剂干后在共聚焦显微镜下观察并拍照。
高尔基染色
分别取一个月大的WT和KO小鼠，快速处死取脑，用预冷的PBS清洗脑袋多余的血液，根据FD Rapid Golgistain TM Kit(FD NeuroTechnologies)染色法进行染色，用振动切片机取150-200um厚度的脑片，中性树脂封片后用共聚焦显微镜观察。
细胞表面蛋白生物素标记
将生物素(EZ-Link Sulfo-NHS-SS-Biotin)溶于新鲜配制的PBS/CM缓冲液，终浓度为0.5mg/mL，置冰上备用；从培养箱中取出细胞，置于冰上，吸去培养基，加入适量冰预冷的PBS/CM缓冲液清洗细胞3次，吸 去缓冲液，向细胞培养盘中加入生物素溶液，在冰浴条件下培养20min，吸去生物素溶液，重复一次；加入含有50mM NH 4Cl的PBS/CM缓冲液终止反应(10min)；用TNEN裂解液(含蛋白酶抑制剂)裂解细胞，于4℃，12000rpm离心10min，转移上清液置于新的离心管中，经BCA法测定蛋白浓度后，取等量蛋白样品加入等体积2×上样缓冲液混匀，100℃煮5min，用免疫印迹分析方法检测蛋白表达；向等量剩余蛋白样品中加入链霉亲和素珠子(Streptavidin beads)，4℃过夜沉淀生物素化蛋白；次日，4℃，5000rpm离心2min，吸去上清液，链霉亲和素珠子用1％TNEN漂洗3次，每次10min，用微量进样器吸去残液，加入20μL2×上样缓冲液，100℃煮5min，用蛋白质免疫印迹分析方法测定目的蛋白含量。
电生理学实验
将3月龄Rps23rg1基因敲除小鼠和野生型小鼠的大脑进行横向海马脑片切片(0.40毫米)，用不同强度刺激在CA1区诱发fEPSPs，比较突触传递强度。此外，经高频刺激诱发LTP，比较fEPSPs的衰减。
动物行为学实验
所有的小鼠行为学实验采用Smart Video Tracking Software(Panlab，Harvard Apparatus)进行数据采集及分析处理。动物行为学实验于每天9:00a.m.-18:00p.m.之间进行，实验室内的光强度为650lux。
(1)在实验开始前7天接触抚摸小鼠，每天一次，每次抚摸一只小鼠，轻柔地抓住鼠尾拿起小鼠，让小鼠在手上停留30sec，然后用记号笔在鼠尾上划线标记，标记完小鼠后，仍将其放在手掌里，抓住尾巴，然后轻柔地将其放回鼠笼；
(2)实验当天，在实验前先将实验的小鼠转移到准备间，让小鼠适应60min。准备开始实验前，用70％酒精清洁实验所用的箱体和迷宫。每次实验结束后使用70％酒精对箱体和迷宫进行清洁，以清除实验过程中小鼠排泄的粪便和尿液，排除小鼠残留的气味对实验的干扰。
1.旷场实验
旷场实验用于研究小鼠的自主运动能力以及焦虑行为，主要基于小鼠 对于亮光以及开阔空间的趋避性。旷场实验中，将小鼠放置于旷场箱体(40cm(L)×40cm(W)×40cm(H))的中心位置，让小鼠在旷场里自由探索10min，记录小鼠在旷场中总的运动距离(Total distance)以及在旷场中间区域活动的时间(Time in center)。
2.Y/T迷宫实验(Y/T maze test)
Y/T迷宫实验用于评价小鼠的自发性空间交替行为和工作记忆。将小鼠放置于Y/T迷宫(30cm(L)×6cm(W)×15cm(H))中央，然后让小鼠在迷宫中自由探索5min。以小鼠的四肢都进入迷宫臂作为其进入迷宫臂的标准，以小鼠三次连续进入不同迷宫臂为一次正确的自主交替穿梭(Alternation triplet)，记录小鼠在迷宫中总的运动距离(Total distance)和进入迷宫臂的次数(Total arm entries)以及自主交替穿梭次数(Alternation triplet)。
3.新物体识别实验
新物体识别实验(Novel object recognition test)是利用啮齿类动物先天对新物体有探索倾向的原理而建立的学习记忆测试方法。第一天，将小鼠放入旷场箱(长40cm×宽40cm×高40cm)内正中央，每只小鼠放置位置相同(同一格、同一方向)，让小鼠适应性训练10min；第二天，在旷场箱内部场地一侧放置两个相同的物体，将小鼠面向箱臂放入旷场箱内，放置位置与两物体间的距离相同，让小鼠自由探索5min；第三天，保持物体位置不变，用新物体更换其中一个旧物体，将小鼠面向箱臂放入旷场箱内，放入位置与两物体间的距离相同，让小鼠自由探索5min；小鼠鼻子在距离物体2cm内指向物体或鼻子触及物体被视为探索行为；用摄像系统记录小鼠探索熟悉物体(TF)和探索新物体(TN)的时间。每只小鼠测试结束后，用30％酒精清理旷场箱内部场地，并用干纸巾擦干。
4.物体空间位置记忆实验
该实验与新物体识别实验类似，但是在测试时是将其中一个物体移动到旷场箱的另外一个位置，再让小鼠自由探索5min；小鼠鼻子在距离物体2cm内指向物体或鼻子触及物体被视为探索行为；用摄像系统记录小鼠分别探索未移动物体和位置发生移动物体的时间。
5.Morris水迷宫实验
Morris水迷宫(Morris water maze test)实验是在水中让啮齿类动物寻找隐藏平台，通过分析其寻找平台的时间和游泳路径来研究啮齿类动物的空间学习记忆能力。Morris水迷宫为直径180cm、高100cm的圆形淡蓝色水池，水深30cm。水温维持在24℃±1℃。在小鼠视野范围内(池臂)放置不同的标记物，池壁标记4个入水点(E、S、W、N)，把迷宫分成四个象限，在ES象限中安装一平台，直径6cm，使水面高出平台2cm。小鼠每天训练四次，分别从E、S、W和N 4个入水点面向池壁放入水中，通过摄像头捕获小鼠的运动轨迹，并记录小鼠从入水至爬上平台的时间，即逃避潜伏期(escape latency)。系统设定60s测试时间，爬上平台停留10s系统自动关闭。如果60s内小鼠未能找到平台，则引导其找到平台，并在平台上停留10sec，逃避潜伏期按60s计算；定位航行实验连续测试7d，第8天撤去平台，在平台的对侧面向池壁放入小鼠，进行空间探索实验，摄像头记录小鼠在原来平台所在区域穿梭的次数(Number of crossing)以及小鼠在平台所在目标象限以及其他三个不同象限的游泳时间(Time in quadrants)。
6.条件惊恐实验
条件惊恐实验(Fear conditioning test)实验是在有金属丝地板构成的封闭箱体并且能够产生电击和声音，将实验小鼠放到箱体中，由于电击使小鼠产生惊恐，通过分析小鼠对于声音和箱体环境的所产生的惊恐时间来研究动物的空间学习记忆能力。在训练阶段，将每只小鼠放入箱体内，允许自由探索120秒以记录小鼠惊恐基线。接着，持续给小鼠30S、80分贝的声音，在28S时候给予2S、0.5mA的电击，该刺激重复3次，每次刺激间隔30S，3次刺激结束后让小鼠在箱体内停留60S。第二天进行测试，分为海马依赖的环境惊恐记忆和杏仁核依赖的声音惊恐记忆，前者需要更换箱体环境但不给于任何刺激，后者不更换箱体环境需要给予同样的声音刺激，然后记录小鼠的惊恐时间。
材料及试剂
Figure PCTCN2018098264-appb-000010
Figure PCTCN2018098264-appb-000011
Figure PCTCN2018098264-appb-000012
实施例1:Rps23rg1基因敲除小鼠的构建及特性分析
1.1基因敲除小鼠的构建
TALEN(transcription activator-like effector nucleases)通过类转录激活因子效应物(TALE)识别DNA序列，并在特定位点对DNA进行切割，形成双链断裂(DSB)，随后在非同源末端连接(NHEJ)修复机制下形成随机的多个碱基的插入或删除，从而实现基因敲除。在本实施例中，利用TALEN基因打靶技术构建Rps23rg1基因敲除小鼠。
首先根据NCBI上Rps23rg1基因序列(GenBank ID:546049)，在Rps23rg1基因的外显子6上设计两个TALEN靶向结合序列：TALEN-L(targeting 5’-TGACCTTTTCGACGAAATCCAG-3’；SEQ ID NO：60)和TALEN-R(targeting 5’-TGCCTGAACTTCATTGAGGAAA-3’；SEQ ID NO：61)，其TALEN打靶示意图如图1所示，该小鼠在CDS区缺失了了“ACTTC”5个核苷酸残基，导致移码突变和蛋白翻译早期终止；采用Golden gate TALEN and TAL Effector kit构建提取正确的TALENs表达载体，然后体外转录成稳定的mRNA纯化回收并用显微注射缓冲液稀释备用。将Rps23rg1的TALEN-L和TALEN-R mRNA混合在一起后在显微镜下利用显微操作系统注射到C57BL/6小鼠的受精卵中，并移植到受体雌鼠中，以获得Rps23rg1基因敲除小鼠(在本文中，简称为KO小鼠)。将Rps23rg1基因敲除小鼠与C57BL/6野生型小鼠杂交得到子代小鼠，并通过基因型鉴定获得Rps23rg1基因敲除杂合子小鼠(在本文中，简称为Hetero小鼠)。
然后，在DNA、RNA、和蛋白水平验证Rps23rg1基因敲除小鼠的构建是否成功，结果如图2所示。图2a显示了基因型鉴定结果，分别提取野生型C57BL/6小鼠(在本文中，简称为WT小鼠)、杂合子小鼠(Hetero)、基因敲除小鼠(KO)的DNA，并设计引物：5’-TTCGACGAAATCCAGCAACC-3’(SEQ ID NO：62)和5’-GTTCGTGCCCAATGATGGC-3’(SEQ ID NO：63)进行PCR扩增；通过15％SDS-PAGE凝胶电泳对PCR产物进行基因型鉴定，其中野生型小鼠(WT)PCR产物仅有一条条带，长度为74bp；基因敲除小鼠(KO)PCR产物仅有一条条带， 长度为69bp；而基因敲除杂合子小鼠(Hetero)PCR产物包含上述两条条带。图2b显示了WT和KO小鼠的测序峰图，将上述WT和KO小鼠DNA的PCR产物，分别送至英潍捷基测序公司进行测序。以上结果表明，在DNA水平，KO小鼠构建成功。图2c显示了WT和KO小鼠的RNA表达情况，分别提取野生型小鼠(WT)、杂合子小鼠(Hetero)、基因敲除小鼠(KO)的脑组织RNA，经反转录和荧光定量PCR检测RPS23RG1水平，再用β-actin水平标准化后进行比较。结果显示，KO小鼠的Rps23rg1基因的mRNA水平几乎为零，也即无Rps23rg1基因的转录产物，这充分证明，在RNA水平，KO小鼠构建成功。图2d显示了WT和KO小鼠的RPS23RG1蛋白的表达情况，分别提取WT和KO小鼠脑组织，通过免疫沉淀和western blot法检测RPS23RG1蛋白水平，其中，将表达RPS23RG1的质粒(pcDNA3.1/myc-His A)转染HEK293T细胞以获得过表达RPS23RG1的HEK293T并作为阳性对照(PC)，以空载的质粒转染的HEK293T作为阴性对照(NC)；结果显示，KO小鼠脑组织中无RPS23RG1蛋白表达，充分证明，在蛋白水平，KO小鼠构建成功。综上所述，图2的结果显示Rps23rg1基因敲除小鼠构建成功。
1.2基因敲除小鼠的生理状况分析
Rps23rg1基因敲除小鼠的基本生理状况分析结果如图3A-3D所示。首先，将杂合敲除小鼠作为亲本进行交配，得到的后代剪取尾巴，提取DNA，而后用特异性引物进行PCR扩增，进一步对得到的PCR产物进行SDS-PAGE分析，杂交后代分析的结果如图3A所示。结果显示其后代符合孟德尔遗传定律，说明KO小鼠的生殖状态正常。图3B显示了WT和KO小鼠的体重变化情况，分别取不同年龄段的WT和KO小鼠测量体重，结果显示，WT和KO小鼠的体重变化无差异。图3C显示了小鼠脑组织的尼氏染色(Nissil staining)结果，结果显示KO小鼠大脑的缘层(MZ)、皮质板(CP)、中间带(IZ)、脑室区/脑室下区(VZ/SVZ)、侧脑室(LV)等区域的结构均与WT小鼠无明显差异，皮质板(CP)、中间带(IZ)的相对厚度也与WT小鼠无显著性差异。图3D显示了小鼠脑组织神经元的免疫荧光染色结果，结果显示在神经元数目上，KO小鼠与WT小鼠无 明显区别。
上述结果证明，Rps23rg1基因敲除小鼠的生理状况与野生型小鼠无明显差异。
1.3基因敲除小鼠的行为学分析
由于RPS23RG1在海马后突触小体中都有表达，暗示它可能会影响学习记忆功能。因此，本发明的研究者进行了一系列的行为学分析，结果如图4A-4H所示。在旷场实验中(图4A)，KO小鼠总的运动距离比野生小鼠低，但到中心停留的时间没有明显差异。Y迷宫实验(图4B)、新物体识别实验(图4C)、条件惊恐实验(图4D)、水迷宫实验(图4E)均显示，相比于WT小鼠，KO小鼠的学习记忆明显受损。电生理实验(图4F-4H)显示KO小鼠突触传递明显受损。
以上结果表明，Rps23rg1基因敲除小鼠的学习记忆能力明显缺陷，突触传递明显受损。
实施例2:Rps23rg1基因敲除小鼠中PSD-95和PSD-93的表达情况
首先，本发明的研究者检测Rps23rg1基因敲除小鼠中突触相关蛋白的表达情况，结果如图5A-5C所示。图5A显示了小鼠脑组织中突触相关蛋白表达情况的免疫印迹检测结果，结果显示，KO小鼠脑组织中PSD-95和PSD-93蛋白表达水平显著低于WT小鼠。图5B显示了小鼠突触小体中蛋白表达情况的免疫印迹检测结果，结果显示，KO小鼠突触小体中PSD-95和PSD-93蛋白表达水平显著低于WT小鼠。图5C显示了WT和KO小鼠的原代神经元的免疫荧光染色结果，结果显示，相比于WT小鼠，KO小鼠原代神经元中的PSD-95和PSD-93显著减少。以上结果充分证明，敲除Rps23rg1基因显著降低了PSD-95和PSD-93的蛋白含量。
然后，本发明的研究者对KO小鼠脑组织中PSD-95和PSD-93的泛素化水平进行了分析，结果如图6所示。分别对3个月的WT和KO小鼠的脑组织裂解提取蛋白进行免疫沉淀(泛素抗体)-免疫印迹(PSD-95和PSD-93抗体)分析。结果显示，KO小鼠脑组织中PSD-95和PSD-93的泛素化水平显著高于WT小鼠。上述结果进一步说明，在KO小鼠中PSD-95 和PSD-93泛素化水平升高，其蛋白含量明显下降。
进一步，本发明的研究者将表达HA-ubiquitin的质粒和表达PSD-95-Flag或GST-PSD-93的质粒共转染至HEK293T细胞以过表达泛素和PSD-95或PSD-93，6小时后平分成3盘，并分别转染表达HA-MDM2、HA-RR1(全长人源RPS23RG1)或HA-RR1- ΔC(缺失胞内段的人源RPS23RG1)的质粒以分别过表达MDM2、全长RPS23RG1或缺失胞内段的RPS23RG1，而后裂解细胞、提取蛋白，通过免疫沉淀-免疫印迹分析PSD-95和PSD-93的泛素化水平。结果如图7所示，过表达全长RPS23RG1可以显著抑制PSD-95和PSD-93的泛素化，而过表达缺失胞内段的RPS23RG1则没有明显的抑制效果。上述结果提示，RPS23RG1的胞内段具有抑制PSD-95和PSD-93泛素化的潜力。
实施例3:RPS23RG1与PSD-95和PSD-93的相互作用及其对PSD-95和PSD-93降解的影响
RPS23RG1是典型的Ib跨膜蛋白，基于实施例2所获得的实验结果，RPS23RG1的胞内段可能具有与PSD-95和PSD-93相互作用从而影响PSD-95和PSD-93泛素化的潜力。因此，本发明的研究者通过免疫沉淀-免疫印迹分析了RPS23RG1与PSD-95和PSD-93的相互作用情况。
首先，本发明的研究者构建了HA修饰的鼠源全长RPS23RG1(1-141)及不同截短体(1-134、1-130、1-116)，参见表2及图8A。上述多肽的编码核酸序列分别插入pCMVHA质粒中，以获得表达上述各多肽的质粒。将上述质粒分别转染至HEK 293T细胞以过表达全长及不同截短的RPS23RG1，并且在上述细胞中，分别过表达PSD-95-Flag或Myc-PSD-93以进行免疫沉淀-免疫印迹分析。结果如图8B所示，全长RPS23RG1(1-141)及截短体1-134均显示出与PSD-95之间明显的相互作用，而缺失胞内段“TTLAH”的截短体1-130和1-116均不能与PSD-95和PSD-93相互作用。
表2：鼠源全长RPS23RG1的截短体
Figure PCTCN2018098264-appb-000013
Figure PCTCN2018098264-appb-000014
进一步，本发明的研究者对人源和鼠源RPS23RG1的蛋白序列进行了对比分析，结果发现发现两者之间具有较大的相似性(图8C)。因此，本发明的研究者进一步检测了人源RPS23RG1与PSD-95和PSD-93相互作用情况。本发明的研究者构建了HA修饰的人源全长RPS23RG1(1-173)或不同截短体(1-167、1-162、1-154及Δ163-167)的表达质粒，参见表3及图8D。上述多肽的编码核酸序列分别插入pCMVHA质粒中，以获得表达上述各多肽的质粒。将上述质粒分别转染至HEK 293T细胞以过表达全长及不同截短的RPS23RG1，并且在上述细胞中，分别过表达PSD-95-Flag或Myc-PSD-93以进行免疫沉淀-免疫印迹分析。结果如图8E所示，全长RPS23RG1(1-173)及截短体(1-167)均显示出与PSD-95之间明显的相互作用，而缺失胞内段“TTLAH”的截短体1-162、1-154及Δ163-167均不能与PSD-95和PSD-93相互作用。
表3：人源全长RPS23RG1的截短体
Figure PCTCN2018098264-appb-000015
Figure PCTCN2018098264-appb-000016
由于本领域已知PSD-95及PSD-93的泛素化是由泛素连接酶MDM2介导的，并根据上述实验结果，为明确RPS23RG1对PSD-95及PSD-93的泛素化及降解的影响，本发明的研究者进一步通过免疫共沉淀研究了WT小鼠及Rps23rg1基因敲除小鼠(KO小鼠)的突触小体中MDM2与PSD-95及PSD-93的相互作用情况。结果如图9所示，结果发现在KO小鼠中，与WT小鼠相比，有显著更多的MDM2与PSD-95及PSD-93结合。上述结果表明，RPS23RG1能够通过与MDM2竞争性结合PSD-95及PSD-93抑制由MDM2介导的PSD-95及PSD-93泛素化和降解。
实施例4:RPS23RG1胞内段的核心序列的确定
为进一步确定RPS23RG1胞内段的核心序列，本发明的研究者人工合成了鼠源RPS23RG1胞内段的全长多肽(EP-20)及不同截短体(TH-5、TA-7和TP-12)，参见表4，上述多肽均由吉尔生化公司合成。将上述多肽分别与共表达PSD-95和MDM2的细胞孵育，然后裂解细胞提取蛋白，用GST珠子捕获GST-MDM2及其结合的PSD-95，再检测PSD-95的水平。结果显示如图10所示，上述多肽均可以明显降低PSD-95与MDM2的结合。这一结果进一步确认，RPS23RG1与PSD-95和PSD-93相互作用从而抑制其泛素化的核心序列是TTLAH(人源RPS23RG1蛋白的第163-167位氨基酸残基、鼠源RPS23RG1蛋白的第130-134位氨基酸残基)，包含该核心序列的RPS23RG1片段均能够与PSD-95和PSD-93相互作用从而抑制其泛素化，提高PSD-95和PSD-93水平，从而改善由PSD-95及PSD-93减少而引起的神经系统疾病(例如神经退行性疾病)。
表4：鼠源RPS23RG1胞内段的截短体
Figure PCTCN2018098264-appb-000017
Figure PCTCN2018098264-appb-000018
实施例5:基于RPS23RG1胞内段的多肽在Rps23rg1基因敲除小鼠中的疗效评价
上述实验结果已显示鼠源和人源RPS23RG1胞内段可以直接与PSD-95及PSD-93相互作用，这提示RPS23RG1的胞内段可能可以干预MDM2与PSD-95及PSD-93的相互作用，影响PSD-95及PSD-93的泛素化水平，从而改善由PSD-95及PSD-93减少而引起的神经系统疾病(例如神经退行性疾病)。因此，本实施例进一步评价了基于RPS23RG1胞内段的多肽对Rps23rg1基因敲除小鼠的治疗效果。
本发明的研究者人工合成了人源RPS23RG1胞内段多肽hRR1-ICD(ETPSSMRSTTLAHPAVLRA；SEQ ID NO：15)及乱序对照多肽Scramble(STRMSSPTEARLVAPHALT；SEQ ID NO：67)，并在上述多肽的氨基端分别添加了一个FITC荧光标记和接头(GGG；SEQ ID NO：59)，在其羧基端分别添加了一段衍生自TAT的细胞穿膜肽(YARAARRAARR；SEQ ID NO：43)，所获得的多肽分别称为ICD-TAT和Scb-TAT。上述多肽均由吉尔生化公司合成。将上述多肽以40mg/kg/d的剂量腹腔注射至2月龄的KO小鼠或野生型(WT)C57BL/6小鼠(获得自厦门大学实验室动物中心)中，连续治疗3天。在第8天，分别取出小鼠脑组织进行冰冻切片染色观察荧光。结果如图11所示，ICD-TAT在小鼠的皮层(Cortex)和海马区域(Hippo)都有高表达。在T迷宫实验(图12A)中，经ICD-TAT治疗后的KO小鼠的工作记忆有明显提高。在物体位置识别(OLM)和新物体识别(ORM)实验(图12B)中，经ICD-TAT治疗后的KO小鼠的空间记忆有了明显改善。在电生理实验(图12C)中， 经ICD-TAT治疗后的KO小鼠的长时程增强(LTP)也有了明显的回升。
进一步，本发明的研究者通过对WT小鼠和经过上述治疗的KO小鼠脑组织进行IP-WB分析发现，与未经治疗的KO小鼠相比，经ICD-TAT治疗后的KO小鼠脑组织中的PSD-95及PSD-93与MDM2结合的水平明显降低，PSD-95及PSD-93蛋白水平明显升高，且与WT小鼠接近(图13A)；并且，经ICD-TAT治疗后的KO小鼠脑组织中的PSD-95泛素化水平(图13C)及PSD-93泛素化水平(图13B)均明显下降。以上结果表明，本发明的多肽能够明显改善Rps23rg1基因敲除小鼠的突触及认知功能损伤。
实施例6:基于RPS23RG1胞内段的多肽在阿尔兹海默病(AD)模型小鼠中的疗效评价
本实施例评价了基于RPS23RG1胞内段的多肽对AD模型小鼠(APP/PS1小鼠)的突触及认知功能损伤的修复能力。
将实施例4中所述的ICD-TAT或Scb-TAT以40mg/kg/d的剂量腹腔注射至7月龄的APP/PS1小鼠(获得自南京大学模型动物研究中心)及野生型(WT)C57BL/6小鼠中，连续治疗3天。在第8天，分别取出小鼠海马突触小体对PSD-95及PSD-93蛋白水平进行western blot检测。结果如图14A所示，结果显示，ICD-TAT能够明显恢复APP/PS1小鼠中的PSD-95及PSD-93的水平。小鼠行为学检测结果(图14B)显示，在T迷宫实验中，经ICD-TAT治疗的APP/PS1小鼠的自发性空间交替行为有明显提高；在物体位置识别(OLM)和新物体识别(ORM)实验中，经ICD-TAT治疗的APP/PS1小鼠的空间记忆有了显著改善。在电生理实验(图14C)中，经ICD-TAT治疗的APP/PS1小鼠的长时程增强(LTP)明显回升。以上结果表明，本发明的多肽能够明显修复AD所造成的突触及认知功能损伤。
除AD之外，突触稳定性及稳态、以及突触后密度蛋白的缺陷也存在于其他神经退行性疾病中。例如，之前已报道在亨廷顿舞蹈症(HD)患者的纹状体区域观察到PSD-95水平降低(Fourie et al.,2014)，并且发现HD的致病蛋白(HTT蛋白)与PSD-95存在相互作用(Shirasaki et al.,2012)， 而非致病性HTT能够在神经元中聚集PSD-95(Parsons et al.,2014)。另外，在帕金森病和肌萎缩侧索硬化症(ALS)中分别观测到了树突棘密度的降低(Stephens et al.,2005)和突触丢失(Sunico et al.,2011)。因此，本发明的能够抑制PSD-95/PSD-93泛素化从而提高其表达水平的多肽也能够修复由其他神经退行性疾病导致的突触和认知缺陷，逆转神经元损伤，从而特别适用于治疗神经系统疾病(例如神经退行性疾病)。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
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Claims (12)

  1. 一种分离的多肽或其变体，其中，所述多肽由RPS23RG1蛋白的5～50个连续氨基酸残基组成，且包含：RPS23RG1蛋白中位于与SEQ ID NO:1的第130-134位相对应的位置的氨基酸残基；
    其中，所述变体与其所源自的多肽相异仅在于1个或几个(例如，1个、2个、3个、4个、5个、6个、7个、8个或9个)氨基酸残基的置换(例如，保守置换或非保守置换)，且保留了其所源自的多肽的生物学功能；
    例如，所述分离的多肽由RPS23RG1蛋白的不多于45个(例如，45个，44个，43个，42个，41个，40个，39个，38个，37个，36个，35个，34个，33个，32个，31个，30个，29个，28个，27个，26个，25个，24个，23个，22个，21个，20个，19个，18个，17个，16个，15个，14个，13个，12个，11个，10个，9个，8个，7个，6个或5个)的连续氨基酸残基组成；
    例如，所述RPS23RG1蛋白为人源或鼠源；
    例如，所述RPS23RG1蛋白为鼠源，并且所述多肽包含：鼠源RPS23RG1蛋白的第130-134位氨基酸残基；例如，所述鼠源RPS23RG1蛋白的第130-134位氨基酸残基具有如SEQ ID NO:5所示的序列；
    例如，所述RPS23RG1蛋白为人源，并且所述多肽包含：人源RPS23RG1蛋白的第163-167位氨基酸残基；例如，所述人源RPS23RG1蛋白的第163-167位氨基酸残基具有如SEQ ID NO:5所示的序列；
    例如，所述多肽包含选自下列的序列，或由选自下列的序列组成：SEQ ID NO：5～17。
  2. 权利要求1的分离的多肽或其变体，其中，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：SEQ ID NO：21～42；其中，X 1～X 11各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6(SEQ ID NO：21)；其中，X 6选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6A(SEQ ID NO：22)；其中，X 6选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7(SEQ ID NO：23)；其中，X 6、X 7各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8(SEQ ID NO：24)；其中，X 6～X 8各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8X 9(SEQ ID NO：25)；其中，X 6～X 9各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8X 9X 10(SEQ ID NO：26)；其中，X 6～X 10各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：TTLAHX 6AX 7X 8X 9X 10X 11(SEQ ID NO：27)；其中，X 6～X 11各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 5TTLAH(SEQ ID NO：28)；其中，X 5选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 4X 5TTLAH(SEQ ID NO：29)；其中，X 4或X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 3X 4X 5TTLAH(SEQ ID NO：30)；其中，X 3～X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：SX 3X 4X 5TTLAH(SEQ ID NO：31)；其中，X 3～X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列 的序列组成：X 2SX 3X 4X 5TTLAH(SEQ ID NO：32)；其中，X 2～X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：PX 2SX 3X 4X 5TTLAH(SEQ ID NO：33)；其中，X 2～X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含选自下列的序列，或由选自下列的序列组成：X 1PX 2SX 3X 4X 5TTLAH(SEQ ID NO：34)；其中，X 1～X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAH(SEQ ID NO：35)；其中，X 1～X 5各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6(SEQ ID NO：36)；其中，X 1～X 6各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6A(SEQ ID NO：37)；其中，X 1～X 6各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7(SEQ ID NO：38)；其中，X 1～X 7各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8(SEQ ID NO：39)；其中，X 1～X 8各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8X 9(SEQ ID NO：40)；其中，X 1～X 9各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8X 9X 10(SEQ ID NO：41)；其中，X 1～X 10各自独立地选自天然氨基酸；
    例如，所述分离的多肽或其变体包含如下所示的序列，或由如下所示 的序列组成：EX 1PX 2SX 3X 4X 5TTLAHX 6AX 7X 8X 9X 10X 11(SEQ ID NO：42)；其中，X 1～X 11各自独立地选自天然氨基酸。
  3. 一种缀合物，其包含权利要求1或2的分离的多肽或其变体和修饰部分；
    例如，所述修饰部分任选地通过接头连接至所述多肽或其变体的N端或C端；
    例如，所述修饰部分选自另外的多肽、可检测的标记或其任意组合；
    例如，所述另外的多肽为CPP；例如，所述CPP为Tat衍生肽；例如，所述CPP具有SEQ ID NO：43所示的序列；
    例如，所述另外的多肽为靶向部分，例如配体、受体或抗体；
    例如，所述可检测的标记为荧光染料，例如FITC；
    例如，所述另外的多肽为蛋白标签，例如His、Flag、GST、MBP、HA、Myc、GFP或生物素；
    例如，所述缀合物具有选自下列的氨基酸序列：SEQ ID NO：44～56。
  4. 一种融合蛋白，其包含权利要求1或2的分离的多肽或其变体和另外的多肽；
    例如，所述另外的多肽任选地通过接头连接至所述多肽或其变体的N端或C端；
    例如，所述另外的多肽选自CPP、靶向部分、蛋白标签或其任意组合；
    例如，所述另外的多肽为CPP；例如，所述CPP为Tat衍生肽；例如，所述CPP具有SEQ ID NO：43所示的序列；
    例如，所述另外的多肽为靶向部分，例如配体、受体或抗体；
    例如，所述另外的多肽为蛋白标签，例如His、Flag、GST、MBP、HA、Myc、GFP或生物素；
    例如，所述融合蛋白具有选自下列的氨基酸序列：SEQ ID NO：44～56。
  5. 一种分离的核酸分子，其包含编码权利要求1或2的分离的多肽或 其变体，或权利要求4的融合蛋白的核苷酸序列。
  6. 一种载体，其包含权利要求5的分离的核酸分子。
  7. 一种宿主细胞，其包含权利要求5的分离的核酸分子或权利要求6的载体。
  8. 制备权利要求1或2的分离的多肽或其变体、或者权利要求4的融合蛋白的方法，其包括，在合适的条件下培养权利要求7的宿主细胞，和从细胞培养物中回收所述分离的多肽或其变体或者融合蛋白。
  9. 一种药物组合物，其包含权利要求1或2的分离的多肽或其变体、权利要求3的缀合物、权利要求4的融合蛋白、权利要求5的分离的核酸分子、权利要求6的载体或权利要求7的宿主细胞，以及药学上可接受的载体和/或赋形剂；
    例如，所述药物组合物包含权利要求1或2的分离的多肽或其变体或权利要求4的融合蛋白；
    例如，所述药物组合物任选地还包含另外的药学活性剂；例如，所述另外的药学活性剂为具有治疗神经系统疾病(例如，神经退行性疾病)活性的药物；
    例如，所述另外的药学活性剂选自左旋多巴、多巴胺受体激动剂、单胺氧化酶抑制剂、抗胆碱药、谷氨酸受体拮抗剂、儿茶酚-O-甲基转移酶(COMT)抑制剂、多巴脱羧酶抑制剂或其任意组合。
  10. 权利要求1或2的分离的多肽或其变体、权利要求3的缀合物、权利要求4的融合蛋白、权利要求5的分离的核酸分子、权利要求6的载体或权利要求7的宿主细胞用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93水平的用途，或者在制备药物中的用途，所述药物用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制 PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93水平；
    例如，所述与PSD-95和/或PSD-93活性过低相关的疾病为神经系统疾病；
    例如，所述神经系统疾病的特征在于认知功能障碍；
    例如，所述神经系统疾病为神经退行性疾病；
    例如，所述神经系统疾病选自阿尔兹海默症、自闭症、路易氏痴呆、额颞叶痴呆、血管性痴呆、亨廷顿舞蹈病、进行性核上性麻痹、皮质基底痴呆、创伤后神经退行性疾病、慢性创伤性脑病和脑卒中。
  11. 一种用于在受试者中治疗与PSD-95和/或PSD-93活性过低相关的疾病、或抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93水平的方法，其包括，给有此需要的受试者施用治疗有效量的权利要求1或2的分离的多肽或其变体、权利要求3的缀合物、权利要求4的融合蛋白、权利要求5的分离的核酸分子、权利要求6的载体或权利要求7的宿主细胞的步骤；
    例如，所述方法包括给所述受试者施用治疗有效量的权利要求1或2的分离的多肽或其变体、权利要求3的缀合物或权利要求4的融合蛋白；
    例如，所述与PSD-95和/或PSD-93活性过低相关的疾病为神经系统疾病；
    例如，所述神经系统疾病的特征在于认知功能障碍；
    例如，所述神经系统疾病为神经退行性疾病；
    例如，所述神经系统疾病选自阿尔兹海默症、自闭症、路易氏痴呆、额颞叶痴呆、血管性痴呆、亨廷顿舞蹈病、进行性核上性麻痹、皮质基底痴呆、创伤后神经退行性疾病、慢性创伤性脑病和脑卒中。
  12. 一种用于体外抑制PSD-95和/或PSD-93的泛素化水平、或提高PSD-95和/或PSD-93水平的方法，其包括，将权利要求1或2的分离的多肽或其变体、权利要求3的缀合物或权利要求4的融合蛋白与有此需要的细胞接触；
    例如，所述细胞是神经元。
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