Targeted degradation TMEM59 polypeptide and application thereof

Abstract

The invention discloses a polypeptide for targeted degradation of TMEM59 and application of the polypeptide, the polypeptide is composed of 17-33 continuous amino acids of ATG16L1 protein and contains HVVSVDKGXXAVLWAQX (SEQ ID NO.09), and X is C, K, S, R, Y or P. The invention further discloses a preparation method of the polypeptide for targeted degradation of TMEM59. The targeted degradation TMEM59 polypeptide and the fusion protein can obviously reduce the expression of TMEM59, so that the targeted degradation TMEM59 polypeptide and the fusion protein can be used for treating diseases (such as Alzheimer's disease and other nervous system diseases) related to abnormal expression of TMEM59, and have great clinical value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a targeted degradation TMEM59 polypeptide and an application thereof. Background Art

[0002] TMEM59 is a protein related to autophagy, which is widely expressed in various tissues and organs. It can interact with ATG16L1 to promote the activation of LC3 and the autophagic process. Studies have shown that in the frontal cortex of patients with late-onset Alzheimer's disease (AD), the methylation level of the promoter region of the TMEM59 gene is significantly lower than that of the control group, and the methylation of this site is correlated with the mRNA and protein expression levels of TMEM59. Gene chip analysis also showed that the expression of the TMEM59 gene in AD patients was higher than that in the control group. Our previous study found that TMEM59 can regulate the activity and neuroinflammatory response of microglia by interacting with the AD risk factor TREM2, and downregulating TMEM59 can improve the abnormal microglial function caused by the loss of TREM2. In addition, we found that in the 5xFAD AD model mouse, the expression of TMEM59 increased with the aggravation of the pathology, and the heterozygous deletion of TMEM59 can significantly improve the learning and memory function and pathological characteristics of 5xFAD mice. Research by another research group also found that TMEM59 deficiency can reduce the deposition of amyloid plaques and alleviate cognitive dysfunction in APP / PS1 AD model mice.

[0003] Inducing protein degradation is an emerging drug development strategy used to regulate the expression of "undruggable" target proteins in cells. In eukaryotic cells, damaged proteins or organelles can be cleared by proteasomes or lysosomes. The concept of protein degradation targeting chimeras (PROTACs) was first proposed by Crews et al. in 2001. It uses the naturally existing protein cleaning system in the body to induce ubiquitination of target proteins and degradation through the ubiquitin-proteasome system, thereby playing a role in treating diseases. PROTACs mainly target intracellular proteins, while lysosome-targeting chimeras (LYTACs) complement PROTACs to induce degradation of extracellular proteins and membrane proteins through the endosomal-lysosomal pathway. LYTACs molecules can simultaneously bind to the binding domain (protein binding domain, PBD) of the target protein and the lysosome-targeting receptor (lysosome-targeting receptor, LTR) located on the cell surface, and then the target protein is degraded by the lysosome.

[0004] In summary, reducing the expression of TMEM59 may become a new strategy for treating a variety of neurological diseases and other diseases. This strategy may provide a new perspective for the treatment of neurodegenerative diseases such as AD by regulating pathways related to autophagy and neuroinflammation. Summary of the invention

[0005] The purpose of the present invention is to provide a method for targeted degradation of TMEM59 polypeptide.

[0006] Another object of the present invention is to provide the use of the above-mentioned targeted degradation of TMEM59 polypeptide.

[0007] The technical solution of the present invention is as follows:

[0008] A polypeptide targeting degradation of TMEM59, which consists of 17-33 consecutive amino acids of ATG16L1 protein and comprises HVVSVDKGXXAVLWAQX (SEQ ID NO.09), wherein X is C, K, S, R, Y or P.

[0009] Preferably, the targeted degradation TMEM59 polypeptide consists of 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18 or 17 consecutive amino acid residues.

[0010] In a preferred embodiment of the present invention, the amino acid sequence comprises HVVSVDKGCKAVLWAQY (human origin: SEQ ID NO. 07) or HVVSVDKGSRAVLWAQP (mouse origin: SEQ ID NO. 08).

[0011] A conjugate, characterized in that it has the above-mentioned targeted degradation TMEM59 polypeptide and a modified portion, the modified portion is selected from another polypeptide, a detectable label (to make it useful for detection) or any combination thereof, the other polypeptide is selected from CPP (to improve its ability to penetrate the cell membrane), a targeting portion (to make it targeted) and / or a protein tag (to facilitate its expression, detection, tracing and / or purification).

[0012] A fusion protein having the above-mentioned targeted degradation TMEM59 polypeptide and another polypeptide, wherein the other polypeptide is selected from CPP (to improve its ability to penetrate the cell membrane), a targeting portion (to make it targeted), a protein tag (to facilitate its expression, detection, tracing and / or purification) or any combination thereof.

[0013] Preferably, the modification portion is optionally connected to the N-terminus or C-terminus of the TMEM59 polypeptide targeted for degradation via a linker.

[0014] Further preferably, the CPP is a TAT-derived peptide; for example, the CPP has a sequence as shown in SEQ ID NO.11.

[0015] Further preferably, the targeting moiety is a ligand, a receptor or an antibody.

[0016] Further preferably, the detectable label is a fluorescent dye, such as FITC.

[0017] Further preferably, the protein tag is HA, myc, GFP or biotin.

[0018] An isolated nucleic acid molecule has the nucleotide sequence of the above-mentioned TMEM59 polypeptide targeted for degradation, or the above-mentioned fusion protein.

[0019] A vector having the isolated nucleic acid molecule.

[0020] Preferably, the vector includes plasmid, cosmid, phage and cosmid.

[0021] Further preferably, the vector is capable of expressing the above-mentioned targeted degradation TMEM59 polypeptide or the above-mentioned fusion protein in a subject (eg, a mammal, such as a human).

[0022] A host cell having the isolated nucleic acid molecule or the vector.

[0023] Preferably, the host cell is selected from prokaryotic cells and eukaryotic cells.

[0024] Prokaryotic cells include E. coli cells.

[0025] Eukaryotic cells include yeast cells, insect cells, plant cells and animal cells. Animal cells are preferably mammalian cells, preferably mouse cells and human cells.

[0026] The host cell may also be a cell line, such as 293T cells.

[0027] The method for preparing the above-mentioned targeted degradation TMEM59 polypeptide or the above-mentioned fusion protein comprises: culturing the above-mentioned host cell under suitable conditions, and recovering the targeted degradation TMEM59 polypeptide or the above-mentioned fusion protein from the culture of the host cell.

[0028] A pharmaceutical composition comprising the above-mentioned targeted degradation TMEM59 polypeptide, the above-mentioned conjugate, the above-mentioned fusion protein, the above-mentioned isolated nucleic acid molecule, the above-mentioned vector or the above-mentioned host cell, and a pharmaceutically acceptable carrier and / or excipient.

[0029] Use of the above-mentioned targeted degradation TMEM59 polypeptide, the above-mentioned conjugate, the above-mentioned fusion protein, the above-mentioned isolated nucleic acid molecule, the above-mentioned vector or the above-mentioned host cell in the preparation of a pharmaceutical composition, which is used to treat diseases related to excessive expression of TMEM59, or the expression of TMEM59.

[0030] The above-mentioned diseases associated with excessive expression of TMEM59 include nervous system diseases.

[0031] The above-mentioned nervous system disease is characterized by abnormal activation of the TMEM59 signaling pathway, preferably Alzheimer's disease.

[0032] Preferably, the pharmaceutical composition may further comprise another pharmaceutically active component; the other pharmaceutically active component is a drug having therapeutic activity for nervous system diseases (e.g., ischemic stroke). Or the other pharmaceutically active component is selected from ω-3 fatty acids, vitamin B12, intravenous immunoglobulin, hyperbaric oxygen therapy, glutamate receptor antagonists, catechol-O-methyltransferase (COMT) inhibitors, dopa decarboxylase inhibitors or any combination thereof.

[0033] A method for reducing the expression of TMEM59 in vitro for non-diagnostic therapeutic purposes, comprising contacting the above-mentioned targeted degradation TMEM59 polypeptide, the above-mentioned conjugate or the above-mentioned fusion protein with a cell in need thereof.

[0034] Preferably, the cells are neuronal cells or glial cells (the glial cells are preferably microglial cells).

[0035] The above-mentioned targeted degradation TMEM59 polypeptide, fusion protein or pharmaceutical composition can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), etc. In some embodiments, the above-mentioned targeted degradation TMEM59 polypeptide, fusion protein or pharmaceutical composition can be formulated into injection solutions or lyophilized powders.

[0036] In addition, the above-mentioned TMEM59 polypeptide or fusion protein targeted for degradation can be present in a pharmaceutical composition in the form of a unit dosage for easy administration.

[0037] The above-mentioned targeted degradation TMEM59 polypeptide, fusion protein or pharmaceutical composition can be administered by any suitable method known in the art, including oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The technician will understand that the route and / or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the above-mentioned targeted degradation TMEM59 polypeptide, fusion protein or pharmaceutical composition is administered by intravenous infusion or injection.

[0038] The targeted degradation TMEM59 polypeptide, fusion protein or pharmaceutical composition provided by the present invention can be used alone or in combination, or can be used in combination with another pharmaceutically active component (e.g., a drug active for treating a nervous system disease). In certain preferred embodiments, the above-mentioned targeted degradation TMEM59 polypeptide or fusion protein is used in combination with other drugs active for treating a nervous system disease to prevent and / or treat diseases associated with excessive expression of TMEM59 (e.g., nervous system diseases). Such another pharmaceutically active component can be administered before, simultaneously with, or after the administration of the above-mentioned targeted degradation TMEM59 polypeptide, fusion protein or pharmaceutical composition.

[0039] The above-mentioned pharmaceutical composition may include a "therapeutically effective amount" or a "prophylactically effective amount" of the above-mentioned targeted degradation TMEM59 polypeptide or fusion protein. "Prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a disease associated with excessive expression of TMEM59). "Therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. The therapeutically effective amount of the above-mentioned targeted degradation TMEM59 polypeptide or fusion protein may vary depending on the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the method of administration of the drug, and other treatments administered simultaneously, etc.

[0040] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0041] A typical non-limiting range of the therapeutically or preventively effective amount of the above-mentioned targeted degradation TMEM59 polypeptide or fusion protein is 0.001-100 mg / kg body weight, such as 0.01-50 mg / kg body weight, 0.1-25 mg / kg body weight.

[0042] It should be noted that dosage may vary with the type and severity of the condition requiring treatment.

[0043] In addition, those skilled in the art will appreciate that for any particular patient, the specific dosing regimen should be adjusted over time based on the patient's needs and the physician's professional evaluation; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical composition of the present invention.

[0044] In the present invention, the subject may be a mammal, such as a human.

[0045] Definition of terms

[0046] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation steps used in the present invention are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0047] As used in the present invention, the term "Proteolysis targeting chimeras (PROTACs)", "Lysosome-Targeting Chimaeras (LYTACs)", "Cell penetrating peptide (CPP)" is also called "cell penetrating peptide", "lysosome-targeting receptor (lysosome-targeting receptor, LTR)", "protein binding domain (protein binding domain, PBD)", etc., which refers to a polypeptide that can promote the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids; for example, the targeted degradation of TMEM59 polypeptides or variants thereof of the present invention). Such polypeptides are well known in the art, or can be obtained by methods known in the art, and are described in, for example, Fan, et al., 2014 and Tong, et al., 2023 (all of which are incorporated herein by reference).

[0048] In the present invention, examples of the CPP include: (i) protein derived CPPs: such as sequences derived from the gene controlling antennae (Antennapedia), such as pAntp (43-58); sequences derived from HIV-1, such as TAT ​​derived peptides, such as amino acid residues 37-72, 37-60, 47-60 or 47-57 from TAT; 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 peptides: for example MPG; Transportan; Transportan 10; Pep-1; a peptide selected from KALA; or a peptide selected from Bulforin2, etc.

[0049] In addition, the CPP used in the conjugate of the present invention can also be selected from a polypeptide sequence having about 60, 70, 80, 90, 95, 99% or 100% sequence identity with any polypeptide sequence as described above, as long as the polypeptide sequence still retains its biological activity, i.e., promoting the cellular uptake of the isolated polypeptide of the present invention (or its variant) and / or promoting the isolated polypeptide of the present invention (or its variant) to pass through the blood-brain barrier.

[0050] As used in the present invention, the term "targeting moiety" refers to a domain that can guide the targeted degradation TMEM59 polypeptide (or its variant) of the present invention to a desired location, which can be a specific tissue, a specific cell, or even a specific intracellular location (e.g., a nucleus, a ribosome, an endoplasmic reticulum, a lysosome, or a peroxisome). Those skilled in the art know how to design a corresponding targeting domain based on the characteristics of the desired location.

[0051] As used in the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0052] As used in the present invention, the term "host cell" refers to a cell that can be used to introduce a vector, including prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0053] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, 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.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0054] As used in the present invention, the term "isolated" refers to that the target (e.g., polypeptide) has been purified from contaminants present in a sample, such as a sample containing the target obtained from a natural source. The term isolated need not exclude the presence of other components intended to work in conjunction with the isolate. For example, the targeted degradation TMEM59 polypeptide of the present invention can be described as isolated, although it can be connected to a cell-penetrating peptide.

[0055] As used in the present invention, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes: pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH regulators include phosphate buffers. Surfactants include cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include sodium chloride. Agents that maintain osmotic pressure include sugars, NaCl, and the like. Agents that delay absorption include monostearate and gelatin. Diluents include water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Adjuvants include aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug (e.g., reduced expression of TMEM59), including sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc.

[0056] As used herein, the term "treat" 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 the progression of a disease (eg, a nervous system disease).

[0057] As used in the present invention, the term "prevention" refers to preventing, inhibiting or delaying the occurrence of a disease (eg, a nervous system disease).

[0058] As used in the present invention, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, a therapeutically effective amount can be an amount that is effective or sufficient to treat or cure a disease (e.g., a nervous system disease), delay the onset of symptoms of a disease (e.g., a nervous system disease) and / or delay the development of a disease (e.g., a nervous system disease). A preventive effective amount can be an amount that is effective or sufficient to prevent, inhibit or delay the occurrence of a disease (e.g., a nervous system disease). Such an effective amount can be easily determined by a person skilled in the art or a doctor, and can be related to the intended purpose (e.g., treatment or prevention), the general health of the subject, age, sex, weight, severity of the disease to be treated, complications, mode of administration, etc. The determination of such an effective amount is fully within the capabilities of those skilled in the art.

[0059] As used in the present invention, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a disease associated with excessive expression of TMEM59 (e.g., a nervous system disease), or has a risk of suffering from a disease associated with excessive expression of TMEM59 (e.g., a nervous system disease).

[0060] As used in the present invention, the biological functions of the above-mentioned targeted degradation of TMEM59 polypeptide include one or more selected from the following:

[0061] 1) Ability to specifically bind to TMEM59;

[0062] 2) the ability to reduce TMEM59 protein expression;

[0063] 3) the ability to reduce TMEM59 protein expression in a subject (optionally after conjugating the polypeptide to a CPP);

[0064] 4) the ability to improve synaptic dysfunction and / or learning and memory dysfunction caused by excessive levels of TMEM59 in a subject (optionally, after conjugating the polypeptide to a CPP);

[0065] 5) The ability to treat a disease associated with abnormal expression of TMEM59 (eg, a neurological disease) in a subject (optionally after conjugating the polypeptide to a CPP).

[0066] The beneficial effect of the present invention is that the targeted degradation of TMEM59 polypeptide and fusion protein in the present invention can significantly reduce the expression of TMEM59, and thus can be used to treat diseases related to abnormal expression of TMEM59 (such as neurological diseases such as Alzheimer's disease), and has great clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1AF: Screening for the fragments of ATG16L1 that have the strongest interaction with TMEM59-CTF. Among them: Figure 1 A and B: Co-IP analysis of the interaction of TMEM59-CTF with each WD of ATG16L1. Figure 1 CE: Co-IP analysis of the interaction between TMEM59-CTF and three fragments of WD7. Figure 1 F: Co-IP analysis of the interaction between mouse TMEM59 (m59) and WD7-D3 fragment. n = 3, one-way ANOVA with Tukey's post hoc test. Data represent mean ± standard error. **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0068] Figure 2 AC: Construction of LYTACs peptides targeting degradation of TMEM59. Figure 2 A: Construction strategy of LYTACs polypeptide, wherein the PBD sequence is SEQ ID NO.07, the LTR sequence is SEQ ID NO.10, and the CPP sequence is SEQ ID NO.11. Figure 2 B and C: Two-photon microscopy was used to detect the efficiency of LYTACs peptides in passing through the blood-brain barrier. Data represent mean ± standard error.

[0069] Figure 3 AI: Using LYTACs peptides to degrade TMEM59 significantly improves the learning and memory abilities of 5xFAD mice. Figure 3 AC: Western blot analysis of the changes in TMEM59 protein expression levels after tail vein injection of LYTACs peptides in mice. n = 6, one-way ANOVA with Tukey's post hoc test. Figure 3 D and E: The percentage of autonomous alternation behavior (D) and movement distance (E) of mice in the Y-maze test. Figure 3 F and G: In the Morris water maze test, the escape latency of mice during training (F) and the percentage of time that mice stayed in the target quadrant during testing (G). Figure 3H and I: The percentage of time that mice showed freezing behavior in the context memory test (H) and the cued memory test (I) in the conditioned fear test. WT-Scr group n = 12, 5xFAD-Scr group n = 11, 5xFAD-CTM group n = 11, D, E, GI used one-way ANOVAwith Tukey's posthoc test, F used two-way ANOVAwith Tukey's post hoc test. Data represent mean ± standard error. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0070] Figure 4 AI: Using LYTACs peptides to degrade TMEM59 improves neurological impairment and motor-related behaviors in MCAO mice. Figure 4 A and B: Immunoblotting analysis of TMEM59 protein expression before and after MCAO modeling. n = 3, unpaired t test. Figure 4 C and D: Immunoblotting analysis of the changes in TMEM59 protein expression levels after tail vein injection of LYTACs peptides in mice. n = 3, one-way ANOVA with Tukey's post hoc test. Figure 4 E and F: TTC staining analysis of cerebral infarction area in MCAO mice. n = 5, one-way ANOVA with Tukey's post hoc test. Figure 4 GI: mNSS score (G), rotarod test (H), adhesion removal test (I). Sham group n = 14, MCAO group n = 15, MCAO-Scr group n = 14, MCAO-CTM group n = 15, one-way ANOVA with Tukey's post hoc test. Data represent mean ± standard error. *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0071] The above technical solution is further illustrated and described below through specific implementation methods in combination with the accompanying drawings.

[0072] Sequence information

[0073] The information of the sequences involved in the present invention is provided in Table 1 below.

[0074] Table 1: Description of sequences

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compendium of Molecular Biology Experiments, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturers. The reagents whose sources are not indicated in the examples are all conventional reagents in the art or commercially available reagents.

[0081] 1. Experimental Materials and Methods

[0082] A. Protein extraction and Western blotting (WB)

[0083] 1. Preparation of Protein Samples

[0084] (1) Cells: Aspirate the cell culture medium, add 1 mL of pre-cooled 1× PBS to wash three times, then add an appropriate amount of 1% TNEN lysis buffer, scrape the cells with a cell scraper and place them in a 1.5 mL EP tube. Lyse on ice for 30 min, and shake with a vortex shaker every 10 min.

[0085] Tissue: Use a dissecting instrument to remove the mouse brain tissue, place it in pre-cooled 1× PBS buffer, carefully peel off the required brain tissue part, place it in a 1.5mL EP tube and weigh it, add an appropriate amount of RIPA lysis buffer according to the weight of the tissue, and grind the tissue into a homogenized state with a tissue homogenizer. Lyse on ice for 1 hour, and shake it with a vortex oscillator every 10 minutes.

[0086] (2) After lysis, centrifuge at 13,000 × g for 15 min at 4°C and carefully pipette the supernatant into a new 1.5 mL EP tube.

[0087] (3) Determine the protein concentration by BCA method (refer to BCA Protein Assay Kit manual). Calculate the required sample and loading buffer volumes based on the measured protein concentration and prepare the sample. Take 30-40 μg of protein sample, add the corresponding volume of 5× loading buffer, and make up with 1× loading buffer. After mixing, place the sample on a sample cooker at 100°C for 5 minutes. The prepared sample is used for immunoblotting analysis.

[0088] 2. SDS-polyacrylamide gel (PAGE) electrophoresis

[0089] (1) Prepare SDS-polyacrylamide gels of different concentrations according to the molecular weight of the protein to be detected.

[0090] (2) Add electrophoresis buffer to the electrophoresis tank, slowly add the prepared sample into the gel wells using a pipette, and adjust the voltage to 80V; when the sample enters the separation gel, adjust the voltage to 120V.

[0091] (3) The position of the protein to be detected can be estimated based on the position of the protein marker electrophoresis, and the electrophoresis can be stopped at the appropriate time.

[0092] 3. Protein Electrotransfer

[0093] (1) Pre-cool the transfer buffer in a 4°C refrigerator. Soak the PVDF membrane in methanol and then place it in the pre-cooled transfer buffer. Soak the filter paper in the transfer buffer for a few minutes.

[0094] (2) Place the transfer clip in the order of 4 filter papers, protein gel, PDVF membrane, and 4 filter papers from the negative electrode to the positive electrode. Avoid the generation of bubbles during this process. Then place the transfer clip into the electrotransfer tank accordingly. Perform electrotransfer at a constant current of 300 mA for 90 minutes in an ice bath.

[0095] (3) After the protein electrotransfer was completed, the PVDF membrane was placed in a blocking solution containing 5% skimmed milk powder and blocked for 1 h at room temperature.

[0096] 4. Antigen-antibody reaction

[0097] (1) Dilute the primary antibody at an appropriate ratio with 5% BSA (prepared in PBST) according to the antibody instructions, place it in an antibody incubation box together with the PVDF membrane, and incubate it on a shaker at 4°C overnight.

[0098] (2) Recover the primary antibody and then wash three times with PBST, each time for 10 min.

[0099] (3) Dilute the secondary antibody at a ratio of 1:3000 to 1:5000 with 5% skim milk (prepared in PBST) and incubate the membrane for 1 h at room temperature.

[0100] (4) Aspirate and discard the secondary antibody, then wash three times with PBST, 10 min each time.

[0101] 5. Enhanced chemiluminescence (ECL) color development

[0102] (1) Place the PVDF membrane flatly in the plastic film and then in the developer clip. Mix the ECL developer solution A and solution B in a volume ratio of 1:1.

[0103] (2) In a darkroom, add ECL droplets to the film surface, cover with plastic film, press the film onto the plastic film and expose it in the developing clip. After a period of time, take out the film, develop and fix it.

[0104] B. Co-Immunoprecipitation (Co-IP)

[0105] 1. Protein Sample Preparation

[0106] (1) Aspirate the cell culture medium, add 1 mL of pre-cooled 1× PBS, and wash twice.

[0107] (2) Add 1 mL of pre-cooled 1× PBS, scrape the cells with a cell scraper, and then transfer them to a 1.5 mL tube. Centrifuge at 2000 rpm at 4°C for 3 min, discard the supernatant, and collect the cells.

[0108] (3) Discard the supernatant and add 800 μL of 1% TNEN lysis buffer (with protease inhibitors added in advance) to each tube. Lyse on ice for 30 min and shake on a vortex shaker every 10 min.

[0109] (4) Centrifuge at 13,000 × g for 15 min at 4°C. Carefully aspirate the supernatant and transfer it to a new 1.5 mL EP tube.

[0110] 2. Immunoprecipitation

[0111] (1) Beads pretreatment: Recombinant Protein G beads were pre-washed 3 times with 1×PBS and centrifuged at 4°C, 3000 rpm for 2 min. Then, an equal amount of 1×PBS was mixed with the beads and set aside.

[0112] (2) Determine the protein concentration. Take 1 mg of the above protein lysate and add 0.5% TNEN lysate to 700 μL. Add 20 μL of the mixed Protein G beads and incubate at 4°C with rotation for 2 h. Take another 2% to 5% of the protein as input.

[0113] (3) Centrifuge at 3000 rpm for 3 min at 4°C. Pipette the supernatant into a new EP tube, add the antibody of the corresponding protein and 40 μL of the mixed Protein G beads. Incubate at 4°C with rotation overnight.

[0114] (4) Centrifuge at 3000 rpm for 3 min at 4°C, discard the supernatant, and wash three times with 1% TNEN lysis buffer, rotating and incubating for 10 min each time.

[0115] (5) Use a microinjector to discard the supernatant, add 2× loading buffer in an equal volume to the beads, mix well, and place in a sample cooker for 5 min for immunoblot analysis.

[0116] C. Tail vein injection

[0117] Dissolve the synthesized peptide in physiological saline and place the fully dissolved peptide on ice for later use. When injecting mice, ensure that the mouse is comfortably placed in the mouse tube and positioned with the tail vein syringe barrel, while placing the mouse tail in the groove of the squeeze plate. Adjust the light so that the blood vessels are clearly visible, and then start the injection from the bottom of the tail. Slowly push the peptide solution during the injection process and always observe the health of the mouse. After the injection, the needle stays to ensure the normal absorption of the drug. After the needle is removed, use a cotton swab to compress and stop bleeding for 1 minute. Inject once every other day for a total of 10 injections.

[0118] D. Volume multiphoton microscopy

[0119] Mice were anesthetized with isoflurane. After the mice entered deep anesthesia, part of the scalp was cut to expose the skull, and a 4 mm diameter cranial window was drilled on the dura mater with a skull drill. A glass coverslip was used to cover the surface of the cerebral cortex instead of the skull, and it was sealed with glue, and a custom-made fixture was fixed to the skull with dental cement. Multiphoton laser scanning microscope FVMPE-RS (Olympus) was used for imaging. Texas red-dextran was first injected intravenously to observe the blood vessels of the cortex, and then FITC-labeled peptides were injected. During imaging, a laser with a wavelength of 920 nm was used to excite Texas red-dextran and FITC, and the emitted light was detected using GaAsP PMTs with 575-645 and 495-540 bandpass filters, respectively.

[0120] E. Animal behavior testing

[0121] All mouse behavioral experiments were performed using TopScan Lite software (Clever Sys. Inc.) for data collection and analysis. Animal behavioral experiments were conducted between 9:00 a.m. and 18:00 p.m. every day, with the light intensity in the laboratory at 650 lux.

[0122] (1) Touch and stroke the mice once a day for 3 days before the start of the experiment. Gently grasp the mouse's tail and pick it up. Let the mouse stay in your hand for 30 seconds, then use a marker to draw a line on the mouse's tail. After marking the mouse, still hold it in your palm, grasp its tail, and then gently put it back into the cage.

[0123] (2) On the day of the experiment, the mice were transferred to the preparation room and allowed to adapt for 60 minutes. Before the experiment, the boxes and mazes were cleaned with 70% alcohol. After each experiment, the boxes and mazes were cleaned with 70% alcohol to remove the feces and urine excreted by the mice during the experiment and to eliminate the interference of the residual smell of the mice on the experiment.

[0124] 1. Y maze test: The Y maze is a "Y"-shaped maze consisting of three arms of 30 cm (L) × 6 cm (W) × 15 cm (H). The test mouse is placed in the center of the Y maze and allowed to freely shuttle in each arm for 5 min. The order in which each mouse enters each arm and the number of times it enters the arm are recorded. Continuous shuttles between three different arms are recorded as a correct autonomous alternation behavior, and the percentage of autonomous alternation is the ratio of the number of correct shuttles to the total number of shuttles.

[0125] 2. Morris water maze test: The Morris water maze test is conducted in a large circular pool (120 cm in diameter). An appropriate amount of water is injected and the water temperature is maintained at 22°C. Titanium dioxide is added to the water to make it opaque to facilitate hiding the platform. The platform is hidden 1 cm below the water surface. The water maze wall is pasted with graphics of different shapes in four different directions to help mice form spatial memory. The test specifically includes:

[0126] a. Training phase: Place the mouse in the water maze from different starting positions facing the pool wall, and record the time it takes to reach and climb onto the platform (i.e., escape latency). If the mouse fails to find the platform within 60 seconds, guide it to the platform and allow it to stay on the platform for 10 seconds. Choose two different directions for training every day, and train for 6 consecutive days.

[0127] b. Test phase: On the 7th day, the platform under the water surface was removed, and the mice were placed in the water maze from the position farthest from the platform, facing the pool wall, and allowed to swim freely for 60 seconds. The swimming speed, moving distance, time spent in each quadrant, and number of times the mice crossed the platform were recorded and analyzed.

[0128] 3. Fear conditioning test, including:

[0129] a. Training phase: On the first day, mice were placed in a test box (square, with an electric grid at the bottom to provide foot shocks for mice) and adapted to the environment for 2 minutes. Then, 80 dB noise stimulation was given for 30 seconds, and 0.7 mA electric shock stimulation was given within the last 2 seconds of the noise stimulation. This process was repeated three times, with an interval of 30 seconds each time. After the last electric shock stimulation, wait for 1 minute and put the mice back into the cage.

[0130] b. Contextual memory test: On the second day, place the mice in the same test box for 5 min, and record the percentage of freezing time.

[0131] c. Cue memory test: On the third day, the mice were placed in a test box that was completely different from the previous training environment (cylindrical, with walls and bottoms of different colors and materials). After the mice adapted for 3 minutes, they were then exposed to 80 dB noise stimulation for 3 minutes. The percentage of time the mice showed freezing behavior under sound stimulation and without sound stimulation was recorded.

[0132] 4. mNSS (modified Neurologic severity score) comprehensive score: The neurological function damage scale (mNSS) was used to comprehensively evaluate the neurological function of each group of rats after brain ischemia. The main observations were the movement (muscle state, abnormal actions), sensation (vision, touch, proprioception), balance, nerve reflexes and overall performance of the rats, and then the severity of nervous system damage was systematically evaluated. The total score of the scale is 18 points, and the higher the score, the more severe the neurological damage: 1-6 points represent mild damage, 7-12 points represent moderate damage, and 13 points or more represent severe damage. The details are as follows:

[0133]

[0134]

[0135] 5. Rotarod test: This test is used to evaluate the motor coordination and balance impairment of mice after ischemic stroke modeling. The mice were trained continuously for 3 days before modeling, 3 times a day. The mice were placed on the rotarod to adapt for 1 minute, and then the rotarod was started, rotating at a constant speed of 20r / min, and training was carried out for 5 minutes. If the animal falls, the rotarod is put back until the exercise time reaches 5 minutes. On the third day of training, a baseline test was performed after 2 training sessions. That is, the rotarod accelerates from 0 until it rotates at a constant speed of 40r / min, and the time when the mouse falls is recorded. The test of mice after modeling is the same as the baseline test. Clean the instrument with alcohol and wipe it dry after training or testing.

[0136] 6. Adhesive removal test: This test is used to evaluate the loss of self-sensation in mice after ischemic stroke modeling. The mice should be familiar with the test environment before the experiment. The mice were trained 3 days before modeling. During the experiment, a small round sticky stick was pasted on the palm of the mouse, and the sticking method and strength were kept consistent as much as possible. Then the mouse was placed in a transparent sterilized box to move freely. The mouse would touch and try to remove the sticky stick with its teeth, and the time the mouse touched the sticky stick and tried to remove it was recorded.

[0137] 7. TTC (2,3,5-triphenyltetrazolium chloride) staining: After the mouse is anesthetized and killed, the whole brain of the mouse is quickly and carefully removed, rinsed in PBS and mounted on a brain mold, quickly frozen in a -20°C refrigerator for 20 minutes, and then taken out and cut into brain slices of about 2 mm each. The cut brain slices are quickly placed in a 0.5% TTC solution, heated in a 37°C water bath in the dark for 30 minutes, and turned over three times during the process to ensure uniform staining. After staining, the brain slices are placed in 4% paraformaldehyde in the dark for 24 hours, and the images are analyzed using Image J software after being photographed and saved. The cerebral infarction area rate = ischemic side infarction area / total brain tissue area × 100%.

[0138] 2. Materials and Reagents

[0139]

[0140] Example 1: Determination of the ATG16L1 core sequence with strong binding ability to TMEM59

[0141] Studies have reported that the intracellular segment (CTF) of TMEM59 interacts with the WD repeat region of ATG16L1. In order to further determine the regions of mutual binding, the plasmids of WD1-WD7 of human ATG16L1 modified with GFP were constructed in this example (the amino acid sequence information is shown in Table 2). These expression plasmids were co-transfected with the myc-modified human TMEM59-CTF expression plasmid into HEK293T cells, and the interaction between them was analyzed by Co-IP experiments. The results showed that the WD7 region of ATG16L1 (SEQID NO.05) had the strongest interaction with TMEM59 ( Figure 1 A, B). Further, this example divides the WD7 region into three fragments: D1 (aa575-590), D2 (aa582-598) and D3 (aa591-607, SEQ ID NO.07) ( Figure 1 C) to determine the shortest sequence that binds to TMEM59. The results showed that D3 has the strongest binding ability to TMEM59 ( Figure 1 CE). In order to determine whether D3 can bind to mouse TMEM59, in this example, plasmids expressing D3 and mouse TMEM59 were co-transfected into HEK293T cells. Co-IP experimental analysis found that the D3 sequence can bind to mouse TMEM59 through oxygen ( Figure 1 F).

[0142] The above results indicate that the 591-607 sequence region of ATG16L1 is the key region for interaction with TMEM59.

[0143] Table 2: Truncations of human ATG16L1

[0144]

[0145]

[0146] Example 2: Detection of the efficiency of ATG16L1-based polypeptide penetrating the blood-brain barrier

[0147] In this example, the D3-containing peptide (CTM) of human ATG16L1 was designed and artificially synthesized using the principle of LYTACs. WD7-D3 (HVVSVDKGCKAVLWAQY; SEQ ID NO.07) was connected to the lysosomal targeting motif (KFERQKILDQRFFE; SEQ ID NO.10) and TAT (YGRKKRRQRRR; SEQ ID NO.11) through a linker (GSGS; SEQ ID NO.12), and modified with FITC; a peptide without the lysosomal targeting motif was used as a control (Scr)( Figure 2 A). The above polypeptides were synthesized by Sangon Biotech Co., Ltd. In order to detect whether the synthesized polypeptides can penetrate the blood-brain barrier, this example uses in vivo multiphoton microscopy to detect the efficiency of the above two polypeptides in penetrating the blood-brain barrier. The imaging results show that both Scr and CTM polypeptides can penetrate the blood-brain barrier and enter the brain parenchyma ( Figure 2 B, C).

[0148] Example 3: Evaluation of the therapeutic efficacy of ATG16L1-based polypeptides in Alzheimer's disease (AD) model mice

[0149] 5xFAD mice are widely used as a typical AD model. Previous studies have shown that the expression of TMEM59 in 5xFAD mice is abnormally increased, and targeted degradation of TMEM59 may become a drug for AD treatment. Therefore, this example further evaluates the therapeutic effect of ATG16L1-based polypeptides on AD model mice.

[0150] In this example, synthetic Scr and CTM polypeptides were injected into 6-month-old 5xFAD mice or littermate control WT mice via tail vein injection, once every other day. After 10 injections, the mice were subjected to behavioral testing and biochemical analysis.

[0151] Through immunoblotting analysis, this example found that abnormally elevated TMEM59 in the hippocampus and cortex tissues of 5xFAD mice returned to normal levels after CTM polypeptide treatment ( Figure 3 AC). The results of mouse behavioral tests showed that in the Y-maze test, the working memory ability of 5xFAD mice given CTM peptide was significantly improved, but the motor ability was not affected ( Figure 3D, E); In the Morris water maze test, CTM peptide can significantly improve the learning and memory ability of 5xFAD mice compared with Scr peptide ( Figure 3 F, G); In the conditioned fear behavior test, the contextual memory and cue memory abilities of 5xFAD mice given CTM peptide were significantly restored ( Figure 3 H, I). The above results confirm that ATG16L1-based peptides can significantly improve the disease-related phenotypes of AD model mice by targeting and degrading TMEM59.

[0152] Example 4: Evaluation of the therapeutic efficacy of ATG16L1-based polypeptides in ischemic stroke model mice

[0153] The mouse middle cerebral artery occlusion (MCAO) model is a classic model of ischemic stroke. This example found that the level of TMEM59 protein in the brain tissue of MCAO mice was significantly reduced compared with the control group ( Figure 4 A, B). In order to explore whether targeted degradation of TMEM59 can be used to treat ischemic stroke, this example further evaluated the therapeutic effect of ATG16L1-based polypeptides on MCAO model mice.

[0154] In this example, after MCAO modeling, the synthetic Scr and CTM polypeptides were then injected into the MCAO mice via tail vein injection, once every 12 hours. After two injections, the mice were subjected to behavioral testing and biochemical analysis.

[0155] Through immunoblotting analysis, this example found that CTM polypeptide treatment can further reduce the expression level of TMEM59 in MCAO model mice ( Figure 4 C, D). The infarct volume after ischemic stroke in mice was evaluated by TTC staining. The results showed that CTM peptide treatment could reduce the area of ​​cerebral infarction in MCAO mice compared with the control group mice ( Figure 4 E, F). Behavioral results showed that CTM peptide treatment could improve the mNSS score of MCAO mice ( Figure 4 G), prolonged the time that MCAO mice stayed on the rotarod ( Figure 4 H), and reduced the time required for MCAO mice to remove sticky stickers ( Figure 4 L). The above results confirm that ATG16L1-based peptides can significantly improve the neurological function and motor impairment of MCAO model mice by targeting and degrading TMEM59.

[0156] The above description is only the preferred embodiment, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for targeted degradation of TMEM59 polypeptide, characterized in that: It consists of 17-33 consecutive amino acids of ATG16L1 protein and includes HVVSVDKGXXAVLWAQX (SEQ ID NO.09), wherein X is C, K, S, R, Y or P.

2. A targeted degradation of TMEM59 polypeptide according to claim 1, characterized in that: The amino acid sequence thereof comprises HVVSVDKGCKAVLWAQY (SEQ ID NO. 07) or HVVSVDKGSRAVLWAQP (SEQ ID NO. 08).

3. A conjugate, characterized in that: It has the targeted degradation TMEM59 polypeptide according to claim 1 or 2 and a modified portion, wherein the modified portion is selected from another polypeptide, a detectable marker or any combination thereof, and the other polypeptide is selected from CPP, a targeting portion and / or a protein tag.

4. A fusion protein, characterized in that: It has the targeted degradation TMEM59 polypeptide according to claim 1 or 2 and another polypeptide, and the other polypeptide is selected from CPP, a targeting part, a protein tag or any combination thereof.

5. An isolated nucleic acid molecule, characterized in that: It has the nucleotide sequence of the targeted degradation TMEM59 polypeptide according to claim 1 or 2, or the fusion protein according to claim 4.

6. A carrier, characterized in that: It has the isolated nucleic acid molecule according to claim 5.

7. A host cell, characterized in that: It has the isolated nucleic acid molecule of claim 5 or the vector of claim 7.

8. The method for preparing the targeted degradation TMEM59 polypeptide according to claim 1 or 2 or the fusion protein according to claim 4, characterized in that: include: The host cell of claim 7 is cultured under suitable conditions, and the TMEM59 polypeptide or the fusion protein targeted for degradation is recovered from the culture of the host cell.

9. A pharmaceutical composition, characterized in that: It comprises the targeted degradation TMEM59 polypeptide of claim 1 or 2, the conjugate of claim 3, the fusion protein of claim 4, the isolated nucleic acid molecule of claim 5, the vector of claim 6 or the host cell of claim 7, and a pharmaceutically acceptable carrier and / or excipient.

10. Use of the targeted degradation TMEM59 polypeptide of claim 1 or 2, the conjugate of claim 3, the fusion protein of claim 4, the isolated nucleic acid molecule of claim 5, the vector of claim 6 or the host cell of claim 7 in the preparation of a pharmaceutical composition for treating a disease associated with excessive expression of TMEM59 or the expression of TMEM59.

11. A method for reducing the expression of TMEM59 in vitro for non-diagnostic therapeutic purposes, characterized in that: The targeted degradation TMEM59 polypeptide of claim 1 or 2, the conjugate of claim 3 or the fusion protein of claim 4 is contacted with a cell in need thereof.

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