Polypeptide for antagonizing Dkk3 protein function and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to a polypeptide for antagonizing a Dkk3 protein function and application of the polypeptide, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1 or has more than 90% of homology compared with the sequence shown as SEQ ID NO.1, and the polypeptide has an amino acid sequence with the Dkk3 protein antagonizing function. The polypeptide is the smallest function antagonism structural domain so far, not only can antagonize the atrophy-inducing ability of Dkk3 in vitro, but also successfully improves the skeletal muscle functions of skeletal muscle specific overexpression Dkk3 mice and senescence mice in a living experiment, and lays a foundation for delaying senescence and treating senescence-related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a polypeptide for antagonizing the function of Dkk3 protein and use thereof. BACKGROUND

[0002] Diseases characterized by reduced skeletal muscle mass (Mass) and weakened strength are clinically referred to as "sarcopenia", the concept of sarcopenia was first proposed by Rosenberg in 1988 to describe the age-related degeneration of skeletal muscle function. Loss of skeletal muscle mass and strength is one of the most common, harmful and insidious processes in the elderly, although there are great differences in the degree between different individuals, the impact of sarcopenia involves most of the elderly and is one of the factors leading to falls in the elderly (Cartee et al., 2016). And the reduction of muscle mass and strength is related to the occurrence of disability, and is a reliable predictor of death independent of other risk factors. Although sarcopenia has a great harm to population health, our understanding of this epidemic disease is still limited. Whether it is the pathogenesis of sarcopenia or the clinical treatment of this disease, most studies still remain at the level of description of the phenomenon, and cannot go deep into the stage of clinical treatment. How to improve the function of skeletal muscle, treat sarcopenia under the physiological condition of aging or disease, has important significance for healthy aging, delaying aging and treating metabolic diseases.

[0003] The Dkk (Dickkopf WNT signaling pathway inhibitor) protein family was discovered at the end of the twentieth century, and so far contains five members, Dkk1, Dkk2, Dkk3, Dkk4 and DkkL1, except DkkL1, the remaining members have N-terminal signal peptide (Signal peptide, SP) and two cysteine-rich domains (Cysteine rich domain, CRD) in structure. The relatively unique DkkL1 is similar to Dkk3 in sequence structure (Glinka et al., 1998; Grotewold et al., 1999; Krupnik et al., 1999; Monaghan et al., 1999). The members of this family are widely considered to be inhibitors of the Wnt signaling pathway, as secreted proteins, their receptors have been confirmed to be LRP5 / 6 or Kremen1 / 2 in experiments.

[0004] The functional study of Dkk family started from the identification of Wnt signaling pathway in embryonic development. Wnt has a wide influence on embryonic development. Dkk1 can strongly inhibit the Wnt signaling pathway by binding to the LRP5 / 6 receptor, and induce head development in zebrafish and African clawed toads. Similar phenomena have been found in Dkk2 and Dkk4.

[0005] Unlike family members, Dkk3 does not interact with the classic Wnt signaling pathway receptors LRP5 / 6 or Kremen1 / 2, and the mechanism and details related to its receptors have not been studied, which may indicate that Dkk3 has a significantly different function and mechanism of action from family members. Dkk3 has been proven to be a new type of skeletal muscle secretory factor (de Wilde et al., 2010; Yin et al., 2018), and as early as 2000, Dkk3 was found to be richly expressed in skeletal muscle and played a role in kidney and cardiovascular disease. Importantly, Dkk3 is also involved in the development of age-related muscle atrophy. In skeletal muscle, Dkk3 has been found to have different expression levels in different parts, specifically less expressed in muscles dominated by slow-oxidative muscle fibers and more expressed in muscles dominated by fast-fermentative muscle fibers (de Wilde et al., 2010). Clinical studies have revealed that after detecting the levels of Dkk3 in the plasma of healthy young people (n = 27, age less than 40) and healthy old people (n = 36, age greater than 60), the protein level of Dkk3 in the circulatory system of the aging population was significantly increased (Zenzmaier et al., 2008). Combining the above two findings, the applicant's previous results linked Dkk3 to aging-related sarcopenia. Dkk3 is up-regulated in skeletal muscle with sarcopenia and secretes a large amount of Dkk3 into the circulation. Further studies have shown that Dkk3 can induce the nuclear translocation of β-catenin in skeletal muscle cells and enhance its interaction with FoxO3, thereby activating the transcription and translation of E3 ubiquitin ligases Fbxo32 and Trim63, ultimately leading to muscle atrophy (Yin et al., 2018). In skeletal muscle, Dkk3 has been found to be up-regulated in expression and increased in content in serum during aging (Yin et al., 2018; Zenzmaier et al., 2008). Dkk3 is released from skeletal muscle and ultimately reaches and affects other tissues and organs, further linking it to sarcopenia, metabolic disease, skeletal disease, and aging. Obviously, Dkk3 is a new type of Myokine, which suggests that the function of Dkk3 is not limited to skeletal muscle, but also has an impact on other peripheral organs and tissues.

[0006] In combination with previous studies, it can be known that skeletal muscle itself is an important metabolic organ and a secretory organ, and its function as a metabolic and aging regulator gradually emerges after long-term experimental exploration. How to maintain the normal function of skeletal muscle in the case of pathological and physiological aging and avoid muscle atrophy will become an important target for improving metabolic homeostasis and delaying aging. SUMMARY

[0007] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a polypeptide for antagonizing the function of Dkk3 protein and its use for solving the problems in the prior art.

[0008] To achieve the above-mentioned purpose and other related purposes, the present application provides a polypeptide for antagonizing the function of Dkk3 protein, the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1 or 2, or has more than 90% homology compared with the sequence shown in SEQ ID NO: 1 or 2, and has an amino acid sequence with the function of antagonizing Dkk3 protein.

[0009] The function of antagonizing Dkk3 protein is the function of antagonizing the full length of Dkk3 protein.

[0010] The full length of the amino acid sequence is shown in SEQ ID NO. 3 or 4, or has more than 90% homology compared with the sequence shown in SEQ ID NO. 3 or 4.

[0011] The present application also provides an isolated polynucleotide encoding the aforementioned polypeptide.

[0012] The present application also provides a nucleic acid construct comprising the isolated polynucleotide.

[0013] The present application also provides an engineered cell containing the nucleic acid construct or the isolated polynucleotide integrated into the genome of the engineered cell.

[0014] The present application also provides an adeno-associated virus vector system comprising the nucleic acid construct.

[0015] The adeno-associated virus vector expression system is selected from the group consisting of an E. coli expression system, a yeast expression system, an insect expression system, a mammalian expression system, and a plant expression system; preferably, the adeno-associated virus vector expression system is selected from any one of a plasmid transient transfection expression system, a baculovirus expression system, a stable cell line expression system, an adenovirus expression system, and a poxvirus expression system.

[0016] Further, the adeno-associated viral vector system further comprises a host cell. The host cell carries the adeno-associated viral vector. The host cell can be selected from various applicable host cells in the art, as long as it does not limit the inventive purpose of the present application. Specifically, the applicable cell can be a cell for producing adeno-associated virus, for example, can be 293T cell.

[0017] In the plasmid transient transfection expression system, the adeno-associated viral vector system further comprises an envelope plasmid and a helper plasmid.

[0018] The present application also provides an adeno-associated virus (AAV) which is packaged by the adeno-associated viral vector system.

[0019] The present application also provides the use of the polypeptide, the isolated polynucleotide, the nucleic acid construct, the engineered cell, the adeno-associated viral vector system or the adeno-associated virus in the preparation of a Dkk3 protein function antagonist.

[0020] The Dkk3 protein function antagonist is selected from one or more of the following: a sarcopenia prevention or treatment drug, a muscle atrophy prevention or treatment drug, a Duchenne muscular dystrophy prevention or treatment drug, an osteoporosis prevention or treatment drug, a bone fracture treatment drug, an arthritis treatment drug, a type 2 diabetes prevention or treatment drug, an obesity prevention or treatment drug, an insulin resistance treatment drug.

[0021] Preferably, the muscle in the sarcopenia or muscle atrophy is skeletal muscle.

[0022] As described above, the polypeptide for antagonizing the function of Dkk3 protein and its use of the present application have the following beneficial effects: based on the functional self-inhibition phenomenon of Dkk3, the smallest functional antagonizing domain of Dkk3 is found so far, the short peptide not only can antagonize the atrophy ability of Dkk3 in vitro, but also successfully improves the skeletal muscle function of skeletal muscle-specific overexpression Dkk3 mice and aging mice in vivo, which lays a foundation for delaying aging and treating aging-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Figure 1 shows the purified recombinant mouse Dkk3 protein, where (A) is a Coomassie blue staining image of the purified mouse Dkk3-Flag protein after SDS-PAGE, with a loading amount of 200 ng; (B) is a Western blot image of the purified mouse Dkk3-Flag protein, with a loading amount of 100 ng, using a Mouse anti Flag antibody; (C) is a Western blot image showing the change in the electrophoretic size of Dkk3 after mutation of each glycosylation site, with the numbers 96-106 / 121 / 204 indicating the mutated amino acid sites, and 204-1 and 204-2 being two independent clones of the site, and 96+204 and 121+204 indicating removal of the glycosylation sites at both sites; and (D) is a Western blot image showing the change in the electrophoretic size of Dkk3 after treatment with N-glycosidase

[0024] Figure 2 Figure 2 shows the purified recombinant human Dkk3 protein, where (A) is a Coomassie blue staining image of the purified human Dkk3-Flag protein after SDS-PAGE, with a loading amount of 100 ng; and (B) is a Western blot image of the purified human Dkk3-Flag protein, with a loading amount of 500 ng, using a Mouse anti Flag antibody.

[0025] Figure 3 Figure 3 shows that the recombinant Dkk3 protein can cause atrophy of myotube cells, where (A) is a bright field image of myotube cells after 4 days of induction of C2C12 cell differentiation, and then 1 nM of the recombinant Dkk3 protein was added for 24 h; (B) is a statistical analysis result of the diameter of the myotube cells in (A); (C) is a Western blot image showing the level of pAKT after 4 days of induction of C2C12 cell differentiation, and then 1 nM of the recombinant Dkk3 protein was added for 24 h; and (D) is a pAKT detection result after 4 days of induction of C2C12 cell differentiation, and then 1 nM of the recombinant Dkk3 protein was added for 24 h, and then insulin was added for 15 min.

[0026] Figure 4 Figure 4 shows that the functional antagonistic segment of Dkk3 can be shortened, where (A) is a schematic diagram showing the position of each truncated body in the primary structure of Dkk3, with the yellow highlight indicating the position; (B) is a Western blot image showing the level of pAKT after 4 days of induction of C2C12 cell differentiation, and then each recombinant protein was added for 24 h.

[0027] Figure 5Figure 7 shows that 247a is a functional antagonistic segment of Dkk3. (A) Schematic diagram of the location of 247a in the primary structure of the protein; (B) photographs of myotube cells after inducing C2C12 cells to differentiate for 4 days, then adding Dkk3 1 nM, CRD2 10 nM and 247a 10 nM for 24 h treatment (scale bar, 100 μm); (C) statistical analysis results of the diameter of myotube cells in (B), at least five fields of view were taken for each treatment, and three fields of view were counted; (D) Western blot detection results of the pAKT level of cells after inducing C2C12 cells to differentiate for 4 days, then adding Dkk3 1 nM, CRD2 10 nM and 247a 10 nM for 24 h treatment.

[0028] Figure 6 Figure 8 shows that Myo-Dkk3 OE mice develop muscle hypoplasia at a young age. (A) Construction strategy of mice overexpressing Dkk3 in skeletal muscle; (B) results of in vivo fluorescence imaging of OE mice at 12 months of age, the red positive signal in the figure is the Dkk3-GFP protein; (C) detection results of Dkk3 expression in the tibialis anterior muscle of OE mice, n = 3, Mean ± SD; (D) statistical analysis of muscle fiber cross-sectional area of 6-month-old mice, n = 3, Mean ± SD; (F) test results of lower limb strength of 12-month-old mice, n = 5, Mean ± SD. In this figure, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.

[0029] Figure 7 Figure 9 shows that 247a can improve the muscle mass and strength of Myo-Dkk3 OE mice. (A) Flow chart of injecting AAV vectors overexpressing 247a short peptides into the TA of mice; (B) results of RT-qPCR detecting 247a expression level after AAV injection for 30 days, n = 3, Mean ± SD; (C) statistical results of lower limb strength test of mice after AAV injection for 30 days, n = 7, Mean ± SD; (D) dissection photograph of TA of mice after AAV injection for 30 days; (E) weighing results of skeletal muscle tissue of mice after AAV injection for 30 days, n = 4, Mean ± SD. In this figure, *P < 0.05, ***P < 0.001.

[0030] Figure 8247a can alleviate muscle atrophy in Myo-Dkk3 OE mice, wherein (A) immunofluorescence staining of gastrocnemius muscle of mice 30 days after AAV injection (scale bar, 100 μm); (C) measurement and statistical analysis of cross-sectional area of muscle fibers in (A) 30 days after AAV injection, all fields of each tissue section were taken, and all muscle fibers were counted, n = 5, Mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

[0031] Figure 9 247a can improve skeletal muscle function in aging mice, wherein (A) flow chart of injecting AAV vector overexpressing 247a short peptide into TA of mice; (B) statistical results of left and right lower limbs of mice after strength detection 30 days after AAV injection, n = 3, Mean ± SD; (C) immunofluorescence staining of gastrocnemius muscle of mice 30 days after AAV injection (scale bar, 100 μm); (D) measurement and statistical analysis of cross-sectional area of muscle fibers in (C) 30 days after AAV injection, all fields of each tissue section were taken, and all muscle fibers were counted, n = 5, Mean ± SD. In the figure, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. DETAILED DESCRIPTION

[0032] The present application provides a polypeptide 247a for antagonizing the function of Dkk3 protein, the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1 or 2, or an amino acid sequence having more than 90% homology compared with the sequence shown in SEQ ID NO: 1 or 2, and having the function of antagonizing Dkk3 protein.

[0033] SEQ ID NO. 1: RLLDLITWELEPDGALDRCPCASGLLCQPHSHSLVYVC

[0034] SEQ ID NO. 2: QLLDLITWELEPEGALDRCPCASGLLCQPHSHSLVYMC

[0035] In some embodiments of the present application, the function of Dkk3 protein includes any one or more of the following: causing muscle tube cell atrophy, inhibiting intracellular AKT phosphorylation, reducing the sensitivity of muscle tube cells to insulin, inducing nuclear transport of β-catenin in skeletal muscle cells, enhancing the interaction of β-catenin with FoxO3, and activating the transcription and translation of E3 ubiquitin ligase Fbxo32 and Trim63.

[0036] In some embodiments of the present application, the function of the Dkk3 protein is antagonized is the function of the full length of the Dkk3 protein.

[0037] The amino acid sequence of the full length of the Dkk3 protein is shown in SEQ ID NO. 3 or 4, or has more than 90% homology compared with the sequence shown in SEQ ID NO. 3 or 4.

[0038] SEQ ID NO. 3:

[0039] MQRLGATLLCLLLAAAVPTAPAPAPTATSAPVKPGPALSYPQEEATLNEMFREVEELMEDTQHKLRSAVEEMEAEEAAAKASSEVNLANLPPSYHNETNTDTKVGNNTIHVHREIHKITNNQTGQMVFSETVITSVGDEEGRRSHECIIDEDCGPSMYCQFASFQYTCQPCRGQRMLCTRDSECCGDQLCVWGHCTKMATRGSNGTICDNQRDCQPGLCCAFQRGLLFPVCTPLPVEGELCHDPASRLLDLITWELEPDGALDRCPCASGLLCQPHSHSLVYVCKPTFVGSRDQDGEILLPREVPDEYEVGSFMEEVRQELEDLERSLTEEMALREPAAAAAALLGGEEI*

[0040] SEQ ID NO. 4

[0041] MQRLGGILLCTLLAAAVPTAPAPSPTVTWTPAEPGPALNYPQEEATLNEMFREVEELMEDTQHKLRSAVEEMEAEEAAAKTSSEVNLASLPPNYHNETSTETRVGNNTVHVHQEVHKITNNQSGQVVFSETVITSVGDEEGKRSHECIIDEDCGPTRYCQFSSFKYTCQPCRDQQMLCTRDSECCGDQLCAWGHCTQKATKGGNGTICDNQRDCQPGLCCAFQRGLLFPVCTPLPVEGELCHDPTSQLLDLITWELEPEGALDRCPCASGLLCQPHSHSLVYMCKPAFVGSHDHSEESQLPREAPDEYEDVGFIGEVRQELEDLERSLAQEMAFEGPAPVESLGGEEEI*

[0042] The present application also provides an isolated polynucleotide encoding the aforementioned polypeptide.

[0043] The polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded.

[0044] The polynucleotide encoding the polypeptide of the present application can be prepared by any suitable technique well known to those skilled in the art, for example, including but not limited to recombinant DNA technology, chemical synthesis and the like.

[0045] Based on the disclosure of the amino acid sequence of the polypeptide, the skilled person can obtain the nucleotide sequence of the isolated polynucleotide while keeping the encoded amino acid sequence unchanged due to the degeneracy of codons, which is a routine technique in the art.

[0046] The present application also provides a nucleic acid construct comprising the isolated polynucleotide.

[0047] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue, which can be various expression vectors including a vector backbone, i.e. an empty vector, and an expression framework.

[0048] The type of expression vector is not specifically limited. The expression vector refers to a nucleic acid molecule that allows the insertion of foreign nucleotides without destroying the ability of the vector to replicate and / or integrate in the host cell. The expression vector can include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. The expression vector can also include one or more selectable marker genes and other genetic factors. The expression vector is a vector that contains the necessary regulatory sequences to transcribe and translate an inserted gene or genes. The expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0049] The prokaryotic expression vector is selected from an E. coli expression vector, a B. subtilis expression vector or a Streptomyces expression vector. In a preferred embodiment, the prokaryotic expression vector is selected from an E. coli expression vector.

[0050] The eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector or a mammalian expression vector. The yeast expression vector is a Pichia expression vector. The mammalian expression vector is selected from a non-viral vector or a viral vector of any one of the following: a retroviral expression vector, a lentiviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector.

[0051] The host cell can be selected from a eukaryotic host cell or a prokaryotic host cell. The eukaryotic host cell can be selected from a fungus such as a yeast, an insect, an avian, a plant, a C. elegans or a nematode or a mammalian host cell. Examples of yeast host cells are S. cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as E. coli, Streptomyces, B. subtilis, Salmonella typhi or mycobacteria.

[0052] The present application also provides an engineered cell comprising the nucleic acid construct or the isolated polynucleotide integrated into the genome of the engineered cell.

[0053] The engineered cell can be obtained by transfecting the expression vector into a host cell according to methods well known in the art.

[0054] The present application also provides an adeno-associated viral vector system comprising the nucleic acid construct.

[0055] The adeno-associated viral vector expression system can be selected from any one of a E. coli expression system, a yeast expression system, an insect expression system, a mammalian expression system, a plant expression system; preferably, the adeno-associated viral vector expression system can be selected from any one of a plasmid transient transfection expression system, a baculovirus expression system, a stable cell line expression system, an adenovirus expression system, a poxvirus expression system.

[0056] Further, the adeno-associated viral vector system can further comprise a host cell. The host cell can carry the adeno-associated viral vector. The host cell can be selected from any suitable host cell available in the art as long as it does not limit the inventive purpose of the present application. Specifically, the suitable host cell can be a cell for producing adeno-associated virus, such as 293T cell.

[0057] In the plasmid transient transfection expression system, the adeno-associated viral vector system can comprise a packaging plasmid, a helper plasmid and a Helper plasmid.

[0058] The nucleic acid construct serves as a packaging plasmid, responsible for encoding the gene of interest as well as two inverted terminal repeats (ITRs). The helper plasmid carries the rep and cap genes necessary for AAV packaging, which encode the replication (Rep) and capsid (Cap) proteins of AAV. The Rep proteins are responsible for replicating the rAAV plasmid, while the Cap proteins are responsible for forming the viral capsid that can encapsidate the rAAV plasmid DNA. The helper plasmid serves to provide additional adenovirus proteins that are necessary for the packaging process but will not be included in the final rAAV particle, the functions of which include enhancing the replication, assembly, and cell release processes of the virus. The helper plasmid and the helper plasmid are routine choices in the art, and are not specifically limited in the present application.

[0059] The present application also provides an adeno-associated virus (AAV) packaged by the adeno-associated virus vector system.

[0060] The process of virus packaging is routine in the art, and is not specifically limited in the present application.

[0061] The present application also provides use of the polypeptide, the isolated polynucleotide, the nucleic acid construct, the engineered cell, the adeno-associated virus vector system, or the adeno-associated virus in the preparation of a Dkk3 protein function antagonist.

[0062] The Dkk3 protein function antagonist is selected from one or more of the following: a sarcopenia prevention or treatment drug, a muscle atrophy prevention or treatment drug, a Duchenne muscular dystrophy prevention or treatment drug, an osteoporosis prevention or treatment drug, a bone fracture treatment drug, an arthritis treatment drug, a type 2 diabetes prevention or treatment drug, an obesity prevention or treatment drug, an insulin resistance treatment drug.

[0063] Preferably, the muscle in sarcopenia or muscle atrophy is skeletal muscle.

[0064] The present application also provides a method of treating a disease, the method comprising administering to a subject a therapeutically effective amount of the polypeptide or the adeno-associated virus.

[0065] A "subject" includes, but is not limited to, an animal, preferably a mammal; the mammal preferably is a rodent, an even-toed ungulate, an odd-toed ungulate, a lagomorph, a primate, and the like. The mammal includes, for example, a human, a non-human primate (e.g., a monkey), a mouse, a pig, a cow, a goat, a rabbit, a rat, a guinea pig, a hamster, a horse, a monkey, a sheep, or other non-human mammal; a non-mammal, including, for example, a non-mammalian vertebrate, such as a bird (e.g., a chicken or a duck) or a fish, and a non-mammalian invertebrate. The subject can be a human, for example, a patient suffering from sarcopenia or muscle atrophy.

[0066] "Treatment" or "therapy" of a condition includes preventing or diminishing a condition, reducing the speed of occurrence or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0067] A "therapeutically effective amount" or "effective dose" in the present application means a dose or concentration of a drug effective to treat Dkk3 protein-induced disease. For example, for the use of the polypeptide disclosed in the present application, a therapeutically effective amount is a dose or concentration at which the polypeptide can reverse or alleviate any one or more of the following: Dkk3 protein-induced myotube cell atrophy, Dkk3 protein-induced decrease in intracellular AKT phosphorylation, Dkk3 protein-induced decrease in cell sensitivity to insulin, Dkk3 protein-induced nuclear transport of β-catenin in skeletal muscle cells, and interaction of β-catenin with FoxO3 or transcriptional translation of E3 ubiquitin ligase Fbxo32 and Trim63.

[0068] Specifically, when administered to a subject, the dosage varies depending on the age and body weight of the patient, the nature and severity of the disease, and the route of administration, and the total amount of administration can not exceed a certain range, which can be determined based on the results of animal experiments and various conditions.

[0069] In some embodiments, the methods described herein can further comprise administration in combination with other compounds or other treatment regimens of the prior art.

[0070] The disease is selected from sarcopenia, muscle atrophy, Duchenne muscular dystrophy, osteoporosis, bone fracture, arthritis, type 2 diabetes, or obesity.

[0071] Other advantages and benefits of the present application will become apparent to those skilled in the art, upon consideration of the following detailed description of embodiments of the application. The present application can be practiced with other specific embodiments, and modifications and changes can be made thereto without departing from the spirit and scope of the present application. It is to be understood that the following detailed description of the application is given primarily by way of example and that various modifications and changes can be made thereto by those skilled in the art.

[0072] Before further description of the present application, it is to be understood that the application is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting; in this specification and in the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0073] When the embodiments give numerical ranges, it should be understood that unless the application indicates otherwise, every numerical range's two endpoints, and any number falling between the two endpoints, can be selected. Unless otherwise defined, all technical and scientific terms used in the application have the same meaning as commonly understood by one of ordinary skill in the art in the field of the application. In addition to specific methods, devices, materials, and the like recited in the embodiments, any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the application

[0074] Example 1 In vitro expression, purification and activity verification of Dkk3 recombinant protein

[0075] 1.1 In vitro expression and purification of murine Dkk3 recombinant protein

[0076] Dkk3 is a secreted glycoprotein, with glycosylation modification at multiple sites, and also contains other modifications such as phosphorylation. In order to preserve the post-translational modifications of Dkk3 as much as possible, maintain its complete physiological activity, and also in order to more directly and intuitively study its effects produced by autocrine or exocrine, the present application directly selected the following eukaryotic system to express and purify Dkk3, and used a Flag tag to label the recombinant protein, and the specific expression and purification method is as follows:

[0077] 293F suspension cells were cultured to an appropriate density at 37°C, 7% CO2, and 130 rpm, then adenovirus expressing Dkk3 was added for infection, and after infection, the culture solution containing secreted recombinant protein was obtained by continuing to culture; the obtained culture solution was added with affinity beads with Flag tag peptide segment antibody to adsorb and precipitate the recombinant protein, then a high-concentration short peptide that can compete with the antibody was used to elute the recombinant protein, and the purified recombinant protein was obtained by dialysis.

[0078] The above method was used to first purify the murine Dkk3 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO. 4, and the amino acid sequence of the Flag tag is shown in SEQ ID NO. 5: DYKDDDDK.

[0079] After obtaining the recombinant protein product, it was verified by Coomassie brilliant blue staining and Western blot, and a single band of the target product was obtained near 70 kDa Figure 1A-B), which is quite different from the theoretical size of Dkk3, 38 kDa, due to the post-translational modifications of Dkk3. When the glycosylation sites of Dkk3 were mutated or the Dkk3 was treated with glycosidase, the size of Dkk3 protein in the electrophoretic results changed Figure 1 C-D). This result further indicates that the complex post-translational modification of Dkk3 has a significant impact on its properties, and its function should be studied while maintaining its intact modification state.

[0080] 1.2 In vitro expression and purification of human Dkk3 recombinant protein

[0081] To further verify the role of Dkk3 in human diseases, the present application also expresses and purifies human Dkk3 recombinant protein. The same process as the mouse protein expression and purification is used to obtain human recombinant Dkk3 protein, and the amino acid sequence is shown in SEQ ID NO. 3:

[0082] The target product band between 50-70 kDa is obtained by Coomassie blue staining and Western blot verification Figure 2 A-B).

[0083] 1.3 Activity verification of recombinant Dkk3 protein

[0084] To identify the activity of the purified recombinant Dkk3 protein, according to the previous results, the protein was used to induce atrophy of differentiated C2C12 myotube cells, and the diameter of the myotube cells was used as an indicator to detect the activity of the protein. The results show that the recombinant protein successfully induced the atrophy of myotube cells, reducing the diameter of myotube cells Figure 3 A-B). On the other hand, our previous research results also found that Dkk3 can significantly inhibit the phosphorylation of AKT in vivo, therefore, the present application further detects the biological activity of the recombinant Dkk3 protein by taking the phosphorylation level of AKT as another indicator. The results show that after treatment with 1 nM of Dkk3, the pAKT (Phospho-AKT) level in C2C12 myotube cells is significantly reduced Figure 3 C), indicating that the purified recombinant protein has biological activity.

[0085] In addition, by treating with gradient concentration of insulin, it is also found that after treatment with Dkk3 recombinant protein, the sensitivity of myotube cells to insulin is significantly reduced, and higher concentration of insulin is required to make the phosphorylation level of AKT reach the peak Figure 3D), which is an important result, suggesting that Dkk3 is most likely related to the glucose metabolic capacity of skeletal muscle, and the gradually high expression of Dkk3 in the aging process will destroy the insulin signaling pathway of skeletal muscle, hinder the absorption and utilization of peripheral glucose by skeletal muscle, and lead to the occurrence of insulin resistance and even T2D.

[0086] Example 2 Truncation screening of Dkk3 functional antagonism segment

[0087] 2.1 Preliminary screening of Dkk3 functional antagonism segment

[0088] Our previous research results found that CRD2-C is the functional antagonism segment of Dkk3, based on this, the present application further truncates and reduces this segment. First, separate CRD2 and C to form two basic truncations; and then divide the three parts according to the front, middle and rear sections on the basis of the two truncations, and finally obtain eight truncations as shown in Figure 4 A, recombine and express the truncations to obtain recombinant proteins, and preliminarily verify their effects in in vitro experiments taking pAKT in myotube cells as the detection index.

[0089] The experimental results are shown in Figure 4 B, the results show that the truncations numbered 5 and 7 can significantly antagonize the function of full-length Dkk3 protein in in vitro experiments, and restore the reduced pAKT level. Accordingly, one of the two truncated polypeptides is subjected to more detailed functional detection to confirm that it indeed has the function of antagonizing full-length Dkk3.

[0090] 2.2 247a is a functional antagonism segment of Dkk3

[0091] The aforementioned experiment screened out a truncated peptide that can restore the pAKT level in myotube cells, which is the latter half of the CRD2 peptide (i.e. the truncated peptide numbered 7, hereinafter referred to as "247a"), with a length of 38 amino acids (the amino acid sequence is shown as SEQ ID NO. 2). It is finally found that the 247a peptide segment can antagonize full-length Dkk3. Similar to CRD2, 10nM 247a synthetic peptide alone does not change the morphology of myotube cells and does not affect the insulin signaling pathway thereof, which can be explained by the fact that it has no cytotoxicity. After mixing 1nM Dkk3 (to make myotube cells atrophy) and 10nM 247a synthetic peptide and treating C2C12 myotube cells for 48h, the phenomenon similar to CRD2 antagonizing full-length Dkk3 was observed in myotube cells, i.e. the atrophy phenotype of myotube cells was alleviated, and the intracellular pAKT level was also increased Figure 5 A-D).

[0092] This indicates that 247a short peptide is the smallest Dkk3 protein functional antagonistic segment discovered so far, only having 38 amino acids. This discovery can help us further study the mechanism of Dkk3 triggering a series of physiological phenomena, and also help develop methods to treat muscle atrophy and related diseases caused by elevated Dkk3 expression.

[0093] Example 3 247a short peptide can alleviate muscle atrophy in vivo in mice

[0094] 3.1 Construction of muscle atrophy model mice

[0095] To verify whether 247a short peptide can play an antagonistic role in vivo, first of all, a transgenic mouse that can specifically overexpress Dkk3 in skeletal muscle was constructed using conventional techniques in the field. By conventional homologous recombination, an LSL-CAG-SP-Flag-GFP-DKK3 wpre-polyA expression frame was inserted into the Rosa26 gene site, and then the mouse was mated with Ckm Cre mice that can constitutively express Cre enzyme in mature skeletal muscle cells, thereby obtaining a transgenic mouse that can constitutively overexpress Dkk3 in skeletal muscle (Transgenic mice with skeletal muscle-specific overexpression of Dkk3, Myo-Dkk3 OE mice, referred to herein as OE mice) Figure 6 A). Using in vivo imaging of mice ( Figure 6 B) and RT-qPCR ( Figure 6 C) methods, the overexpression of Dkk3 in mouse skeletal muscle was verified.

[0096] Consistent with our previous observations, when Dkk3 expression in skeletal muscle increases, the skeletal muscle shows obvious symptoms of muscle atrophy, and the muscle fibers become thinner and the muscle strength is more weakened ( Figure 6 D-F). This means that skeletal muscle overexpression of Dkk3 can simulate the premature aging of skeletal muscle and is a reliable model of muscle atrophy.

[0097] 3.2 247a can alleviate the symptoms of muscle atrophy in Myo-Dkk3 OE mice

[0098] According to the following steps, an AAV virus (Adeno-associated virus, AAV) that can overexpress and secrete the SP-247a-HA peptide segment in skeletal muscle was constructed.

[0099] 1) Construct the expression plasmid pAAV-mCherryKASH-SP-247A-HA-P2A-mCherry based on pAAV-mCherryKASH (Addgene 139654), the nucleotide sequence of SP-247A-HA-P2A is shown in SEQ ID NO. 6:

[0100] atgcagcggctcgggggtattttgctgtgtacactgctggcggcggcggtccccactgctcctgctcagctgctggatctcatcacctgggaactggagcctgaaggagctttggaccgatgcccctgcgccagtggcctcctatgccagccacacagccacagtctggtgtacatgtgctacccatacgatgttccagattacgctgccactaacttctccctgttgaaacaagcaggggatgtcgaagagaatcccgggcca (SEQ ID NO. 6)

[0101] 2) Prepare pHelper (Addgene 112867, pAdDeltaF6), AAV9 (Addgene 112865, pAAV2 / 9n) plasmids.

[0102] 3) Seed 10 6 Number of 293T cells in a 10 cm diameter culture dish and culture with DMEM medium containing 10% fetal bovine serum. Place the culture dish in a cell incubator at 37°C for incubation so that transfection can be performed the next day.

[0103] 4) 4h before transfection, replace the culture medium of 293T with fresh culture medium to maintain stable culture conditions.

[0104] 5) Use Lipo293 TM Transfection reagent to transfect three plasmids (6 μg of expression plasmid and AAV9, 9 μg of pHelper) into cells.

[0105] 6) 12h later, observe whether the cells emit red fluorescence. If it is a positive signal, replace the fresh serum-free medium.

[0106] 7) 48h after changing the medium, remove the culture medium, scrape all the cells, and resuspend in 0.5ml PBS after PBS washing.

[0107] 8) Resuspend the cells on ice and use an ultrasonic disrupter to break the cells.

[0108] 9) Centrifuge at 13000 rpm for 1 minute at 4°C, discard the pellet and transfer the supernatant to a new tube.

[0109] 8) Use Benzonase nuclease to remove genomic DNA in the supernatant.

[0110] 9) Use Millex Syringe Filter, PES, Non-sterile (0.45 μm) to remove impurities.

[0111] 10) Use HITRAP SP HP cation exchange column to further remove impurities and obtain AAV.

[0112] In the experiment of using 12-month-old OE mice as experimental subjects, empty control AAV was injected into the left tibialis anterior muscle on the ventral side of each mouse, and SP-247a-HA overexpression AAV was injected into the right side. After waiting for 30 days Figure 7 A-B), the muscle strength of the lower limbs of the mice was detected, and the results showed that the right lower limb strength injected with SP-247a-HA overexpression AAV was significantly improved, and the muscle response from contraction to relaxation was also more rapid and sensitive Figure 7 C).

[0113] Then the tibialis anterior muscle and gastrocnemius muscle of the left and right sides of the mice were peeled off separately, and when comparing the right side of each mouse to the left side, it was found that the right muscle of almost all mice was heavier than the left side Figure 7 D-E). This result shows that although there are individual differences between mice, the improvement of 247a short peptide on the skeletal muscle function of OE mice is significantly effective in terms of individuals.

[0114] Then the gastrocnemius muscle of the aforementioned AAV-injected mice was compared, and it was found that the cross-sectional area distribution of the muscle fibers was significantly larger than that of the control group, which shows that the 247a short peptide secreted by the tibialis anterior muscle can act on other surrounding skeletal muscle tissues Figure 8 A-B).

[0115] 3.3 247a can improve the skeletal muscle mass of aging mice

[0116] An experiment was conducted on 24-month-old aging mice using 247a peptide segment. Similarly, empty control AAV was injected into the left tibialis anterior muscle on the ventral side of each mouse, and SP-247A-HA overexpression AAV was injected into the right side. After waiting for a month Figure 9A), followed by detecting the muscle strength of both lower limbs of the mice, and the results are similar to the overexpression model. The right lower limb strength is improved by injecting SP-247a-HA overexpression AAV, and the improvement effect of 247a is more intuitive when comparing the treatment effect of each mouse alone Figure 9 B).

[0117] After comparing the cross-sectional area of muscle fibers of the gastrocnemius muscle, it is found that after comparing the treatment effect of each old mouse alone, it can be seen that the right side of 247a overexpression has more thick muscle fiber count in general Figure 9 C-D).

[0118] Based on the experimental results of this part, the present application successfully screens out a Dkk3 functional antagonism segment with a length of only 38 amino acids. This segment can not only alleviate the symptoms of muscle atrophy in the Myo-Dkk3 OE muscle atrophy model, but also improve skeletal muscle function in the naturally occurring muscle atrophy model of aging. These experimental results further indicate that Dkk3 is a potential muscle atrophy diagnosis and treatment target, and its functional antagonism segment 247a is a promising muscle atrophy treatment molecule.

[0119] The present study has found that an increased level of muscle-derived Dkk3 can promote bone loss to a greater extent and accelerate the aging process. After reducing the level of muscle-derived Dkk3, the state of the skeletal system can be protected during aging of the mouse, so that the joint function can be maintained better.

[0120] In summary, the present application proves that inhibiting the function of Dkk3 helps to alleviate muscle atrophy and improve the metabolic capacity and bone mass of the body. After reversing these functional degenerations, the progression of the aging phenotype can be significantly delayed, and the quality of life of old mice can be improved. Further, the present application uses the functional antagonism of Dkk3 to find a short peptide that can antagonize the function of the full-length protein of Dkk3, which will help to develop Dkk3 inhibition therapy for treating muscle atrophy and other related diseases in the future.

[0121] The above examples are intended to illustrate the embodiments disclosed in the present application and should not be construed as limiting the present application. In addition, various modifications listed herein and changes in the method of the application are obvious to those skilled in the art without departing from the scope and spirit of the present application. Although the present application has been specifically described in conjunction with various preferred embodiments thereof, it should be understood that the present application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the application that are obvious to those skilled in the art should be included within the scope of the present application.

Claims

1. A polypeptide for antagonizing the function of Dkk3 protein, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1 or 2, or has more than 90% homology to the sequence shown in SEQ ID NO: 1 or 2, and has an amino acid sequence that antagonizes the function of Dkk3 protein.

2. The polypeptide of claim 1, wherein, The function of the Dkk3 protein includes any one or more of the following: atrophying myotube cells, inhibiting intracellular AKT phosphorylation, reducing the sensitivity of myotube cells to insulin, inducing the nuclear transport of β-catenin in skeletal muscle cells, enhancing the interaction of β-catenin with FoxO3, and activating the transcription and translation of E3 ubiquitin ligases Fbxo32 and Trim63; preferably, the function of the Dkk3 protein is the function of the full-length Dkk3 protein; more preferably, the amino acid sequence of the full-length Dkk3 protein is shown in SEQ ID NO. 3 or 4, or has more than 90% homology to the sequence shown in SEQ ID NO: 3 or 4.

3. An isolated polynucleotide, comprising, The polynucleotide encodes the polypeptide of claim 1 or 2.

4. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the isolated polynucleotide of claim 3.

5. An engineered cell, characterized by, The engineered cell contains the nucleic acid construct of claim 4 or the genome of the engineered cell has integrated therein the exogenous isolated polynucleotide of claim 3.

6. An adeno-associated viral vector system, characterized in that, The adeno-associated viral vector system comprises the nucleic acid construct of claim 4.

7. An adeno-associated virus, characterized in that, The adeno-associated virus is packaged from the adeno-associated viral vector system of claim 6.

8. Use of the polypeptide of claim 1 or 2, the isolated polynucleotide of claim 3, the nucleic acid construct of claim 4, the engineered cell of claim 5, the adeno-associated viral vector system of claim 6, or the adeno-associated virus of claim 7 in the preparation of an antagonist of the function of Dkk3 protein.

9. Use according to claim 8, characterized in that, The antagonist of the function of Dkk3 protein is selected from one or more of the following: a drug for preventing or treating sarcopenia, a drug for preventing or treating muscle atrophy, a drug for preventing or treating Duchenne muscular dystrophy, a drug for preventing or treating osteoporosis, a drug for treating bone fracture, a drug for treating arthritis, a drug for preventing or treating type 2 diabetes, a drug for preventing or treating obesity, and a drug for treating insulin resistance.

10. Use according to claim 9, characterized in that, The muscle in the sarcopenia or muscle atrophy is skeletal muscle.

Images