Nur77 methylation site mutation peptide based on structural modification design and application of Nur77 methylation site mutation peptide in promoting embryo implantation

Abstract

The invention discloses a Nur77 methylation site mutation peptide based on structural modification design and application of the Nur77 methylation site mutation peptide in promoting embryo implantation, according to the mutation peptide, arginine at the 346th site of Nur77 is mutated into lysine, and methylation modification of Nur77 is reserved while phosphorylation of Nur77 is inhibited, so that the transcriptional activity of Nur77 is enhanced. It is found that the mutant peptide can promote deciduation of endometrial interstitial cells, remarkably improve deciduation disorder of infertility mice in in-vivo experiments and improve the embryo planting rate of low-fertility mice. Therefore, the mutant peptide is expected to become a new medicine for treating the uterine-derived infertility and has important clinical application value. The invention also relates to a preparation method of the mutant peptide and application of the mutant peptide in a pharmaceutical composition and a treatment method.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically discloses a Nur77 methylation site mutant peptide designed based on structural modification and its application in promoting embryo implantation. Background Art

[0002] The endometrium is mainly composed of epithelial cells and stromal cells, which undergo cyclical proliferation and differentiation under the action of ovarian estrogen and progesterone. In fertile women, the endometrium has the ability to accept embryo implantation during the mid-luteal phase, and this period is called the "embryo implantation window" [1]. A characteristic change in the histology and transcriptomics of the endometrial implantation window is decidualization, which is characterized by the functional differentiation of endometrial stromal cells driven by multiple signaling pathways such as FOXO1, WNT4, Nur77, and AKT, which is a necessary condition for embryo implantation. Endometrial decidualization disorders are the main pathological basis of uterine infertility.

[0003] Nuclear receptor Nur77 (also known as TR3) is a product encoded by the immediate early gene NR4A1 and plays an important regulatory role in life activities such as cell proliferation, differentiation, apoptosis, autophagy, inflammation, and metabolism. Nur77 not only acts as a transcription factor to regulate the transcription and expression of downstream target genes, but also acts as a transcription-independent regulatory factor through protein interactions and changes in subcellular localization [2]. The transcriptional activity of Nur77 is regulated by phosphorylation. Studies have shown that phosphorylation modification of Nur77 at Ser351 mediated by AKT inhibits Nur77 from entering the cell nucleus and hinders its transcriptional activity [3].

[0004] In recent years, with the in-depth study of the function of Nur77 and its regulatory mechanism, people have begun to try to treat related diseases by regulating the activity of Nur77. However, most of the existing regulatory methods focus on directly regulating the transcriptional activity or protein expression level of Nur77. There is still a lack of effective intervention methods for the key regulatory link of Nur77 phosphorylation modification. In particular, specific inhibitors or regulators for phosphorylation modification of Nur77 Ser351 site are still blank in the market.

[0005] References

[0006] 1.Wang,H.,Zhang,S.,Lin,H.,Kong,S.,Wang,S.,Wang,H.,and Armant,DR(2013).Physiological and molecular determinants of embryoimplantation.Mol.Aspects Med.34,939–980.

[0007] 2. Zhou Bo, Chen Hangzi, Wu Qiao. Research progress on the multifunctionality of orphan nuclear receptor Nur77 and its related drug target action mechanism[J]. Journal of Xiamen University (Natural Science Edition), 2021, 60(02): 277-289.

[0008] 3.Pekarsky,Y.,Hallas,C.,Palamarchuk,A.,Koval,A.,Bullrich,F.,Hirata,Y.,Bichi,R.,Letofsky,J.,and Croce,CM(2001).Akt phosphorylates and regulates the orphan nuclear receptor Nur77.Proc.Natl.Acad.Sci.98,3690–3694. Summary of the invention

[0009] To solve the above problems, the present invention proposes a Nur77 methylation site mutant peptide designed based on structural modification and its application in promoting embryo implantation; the mutant peptide has a Ser351 site that can bind to AKT as a competitive substrate to reduce endogenous Nur77 phosphorylation, but its Arg346 site is mutated and cannot be methylated by PRMT5, so it does not affect the methylation modification of endogenous Nur77, and is named "Nur77-346 mutant peptide". Experiments have shown that the "Nur77-346 mutant peptide" has the effect of inhibiting Nur77 phosphorylation in vivo while retaining Nur77 methylation, and can improve the decidualization of infertile mice in vivo, and increase the embryo implantation rate of low-fertility mice, and is a potential new drug for the treatment of uterine infertility.

[0010] The technical solution adopted by the present invention is as follows:

[0011] A Nur77 methylation site mutant peptide designed based on structural modification, the amino acid sequence of the mutant peptide is shown in SEQ ID No.1.

[0012] Furthermore, in the above-mentioned Nur77 methylation site mutant peptide designed based on structural modification, the C-terminus of the mutant peptide is also coupled with a transmembrane peptide.

[0013] Furthermore, in the above-mentioned Nur77 methylation site mutant peptide designed based on structural modification, the amino acid sequence of the transmembrane peptide is shown in SEQ ID No.2.

[0014] The present invention also discloses a method for preparing the Nur77 methylation site mutant peptide, comprising the step of artificially synthesizing the mutant peptide in vitro by using a solid phase polypeptide synthesis method in a chemical synthesis method.

[0015] The present invention also discloses the use of the Nur77 methylation site mutant peptide in preparing a preparation for promoting embryo implantation.

[0016] Furthermore, in the above-mentioned use, the preparation is a medicine for treating uterine infertility.

[0017] The present invention also discloses a preparation for promoting embryo implantation, wherein the preparation comprises the above-mentioned Nur77 methylation site mutant peptide.

[0018] Furthermore, the above preparation also includes a pharmaceutically acceptable carrier or excipient.

[0019] Furthermore, in the above preparation, the preparation is an intrauterine infusion agent.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention, by deeply exploring the key regulatory role of Nur77 protein in the process of endometrial acceptance of embryo implantation, reveals for the first time the importance of symmetrical double methylation modification of Arg346 site of Nur77 protein in its biological function. This discovery has significant theoretical originality and provides a new perspective for understanding the molecular mechanism of endometrial decidualization and embryo implantation. Based on this important discovery, the present invention cleverly designed the "Nur77-346 mutant peptide" with Arg346 mutation, which achieves dual regulation of Nur77 function by accurately inhibiting the phosphorylation modification of endogenous Nur77 while retaining its methylation modification. The experimental results show that this mutant peptide can significantly promote the decidualization of endometrial stromal cells in vitro, providing strong support for the preparation of the endometrium; in in vivo experiments, it can effectively promote the decidualization process of infertile mice and significantly improve the embryo implantation rate of low fertility mice, fully demonstrating its great potential in the treatment of uterine infertility. In addition, the present invention also provides a detailed method for preparing the Nur77-346 mutant peptide, and explores its coupling technology with the membrane-penetrating peptide to enhance its cell permeability and further improve its application effect as a drug molecule. At the same time, taking into account the treatment needs of different patients, the present invention also proposes to prepare the Nur77-346 mutant peptide into various forms of preparations, such as intrauterine perfusion agents, etc., to provide more diversified options for clinical treatment. In summary, the present invention not only deepens our understanding of the function of Nur77 protein and its regulatory mechanism, but also opens up a new path for the treatment of uterine infertility, which has great scientific significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Nur77-346 mutant peptide sequence diagram;

[0023] Figure 2 The results of western blot experiments of human endometrial stromal cell lysates treated with Nur77-346 mutant peptide showed that Nur77-346 mutant peptide could effectively inhibit endogenous Nur77 Ser351 phosphorylation modification, but retained Nur77 Arg346 methylation modification (lane 1: human endometrial stromal cell lysate; lane 2: human endometrial stromal cell lysate undergoing decidualization; lane 2: human endometrial stromal cell lysate undergoing decidualization after treatment with Nur77-346 mutant peptide);

[0024] Figure 3Quantitative PCR analysis of IGFBP1 and PRL mRNA levels revealed the effect of Nur77-346 mutant peptide on promoting decidualization and differentiation of human endometrial stromal cells in vitro (first column: no decidualization; second column: hormone-induced decidualization; third column: hormone-induced decidualization after Nur77-346 mutant peptide was added to the culture medium);

[0025] Figure 4 Intrauterine perfusion of Nur77-346 mutant peptide to treat PRMT5 uterine-specific knockout infertile mice improved endometrial decidualization of the infertile mice (first picture: non-knockout control mice can undergo normal decidualization; second picture: PRMT5 uterine-specific knockout infertile mice cannot undergo decidualization; third picture: PRMT5 uterine-specific knockout mice can undergo decidualization after intrauterine perfusion of Nur77-346 mutant peptide; bar graph shows: uterine weight of PRMT5 uterine-specific knockout mice increased after intrauterine perfusion of Nur77-346 mutant peptide);

[0026] Figure 5 Intrauterine infusion of Nur77-346 mutant peptide significantly increased the pregnancy rate of subfertile mice (first picture: approximately 7 embryos were implanted in the subfertile mouse model; second picture: the number of embryo implantations in the subfertile mouse model after intrauterine infusion of Nur77-346 mutant peptide was 13; first bar graph: comparison of the number of embryo implantations in the subfertile mouse model after intrauterine infusion of Nur77-346 mutant peptide and the control group; second bar graph: comparison of the pregnancy rate in the subfertile mouse model after intrauterine infusion of Nur77-346 mutant peptide and the control group, pregnancy rate = number of embryo implantations / number of ovarian ovulations*100%). DETAILED DESCRIPTION

[0027] The technical scheme in the embodiment of the present invention is clearly and completely described below, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The reagent or instrument used in the embodiment of the present invention do not indicate the manufacturer, and are all conventional reagent products that can be obtained by commercial purchase.

[0028] The present invention comprises the following main experimental steps:

[0029] 1. Preparation of small peptides:

[0030] Solid phase peptide synthesis in chemical synthesis was used to artificially synthesize in vitro a segment of amino acids 338 to 355 in which the arginine at position 346 of Nur77 was replaced by lysine (i.e., Nur77-346 mutant peptide). In order to promote the effective entry of the small peptide into cells to function, we coupled an amino acid sequence with cell membrane penetration function (sequence: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg). The resulting Nur77-346 mutant peptide sequence with therapeutic effects is shown in the following figure. Figure 1 shown.

[0031] 2. Effect of Nur77-346 mutant peptide on promoting decidualization of endometrial stromal cells in vitro

[0032] After exogenous addition of Nur77-346 mutant peptide, the mRNA expressions of IGFBP1 and PRL, marker genes of endometrial stromal cells after induction of decidualization in vitro, were detected to observe the effect of adding Nur77-346 mutant peptide in culture medium on the decidualization and differentiation of endometrial stromal cells.

[0033] 3. Effect of Nur77-346 mutant peptide on promoting decidualization in uterine infertile mice

[0034] A uterine-derived infertility mouse model was constructed by uterine-specific knockout of PRMT5, and decidualization of mice was induced by exogenous oil. The effect of intrauterine perfusion of Nur77-346 mutant peptide on uterine decidualization in the uterine-derived infertility mouse model was observed.

[0035] 4. Effect of mutant peptides on promoting embryo implantation

[0036] A subfertility mouse model was constructed by ovarian removal and exogenous hormone supplementation, and the effect of intrauterine perfusion of Nur77-346 mutant peptide on promoting embryo implantation and enhancing fertility was observed.

[0037] Example 1

[0038] 1. Preparation of small peptides:

[0039] A segment of amino acids 338 to 355 of Nur77 (i.e., a short peptide of Nur77) was artificially synthesized in vitro by solid phase peptide synthesis in chemical synthesis, wherein the arginine at position 346 of Nur77 was changed to lysine, and the specific sequence was SEQ ID No.1 = Arg-Thr-Asp-Ser-Leu-Lys-Gly-Arg-Lys-Gly-Arg-Leu-Pro-Ser-Lys-Pro-Lys-Gln. In order to promote the short peptide to enter the cell and exert its biological effect, a segment of amino acids with cell membrane penetration function (sequence SEQ ID No.2 = Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg) was fused to the carboxyl end (C-terminus) of the short peptide of Nur77, and the resulting mutant peptide sequence of Nur77 was as follows: Figure 1 and SEQ ID No. 3=Arg-Thr-Asp-Ser-Leu-Lys-Gly-Arg-Lys-Gly-Arg-Leu-Pro-Ser-Lys-Pro-Lys–Gln-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.

[0040] Example 2

[0041] 2. In vitro application of small peptides:

[0042] Primary human endometrial stromal cells were isolated and cultured in DMEM / F12 medium containing 2.5% carbon-adsorbed serum, and antibiotics were routinely added. 0.5mM 8-Br-cAMP (commercial number: Sigma, #B7880) and 1μM medroxyprogesterone acetate (commercial number: MedChemExpress, #HY-B0469) were added for 3 days to induce decidualization and differentiation of endometrial stromal cells. 20ug of Nur77-346 mutant peptide was added to the culture medium before inducing differentiation. The results showed that the addition of Nur77-346 mutant peptide to the culture medium can effectively inhibit the endogenous Nur77 Ser351 phosphorylation modification in stromal cells, but retain the Nur77 Arg346 methylation modification ( Figure 2 ), and functionally can promote cell decidualization and differentiation ( Figure 3 ).

[0043] Example 3

[0044] 3. In vivo application of small peptides:

[0045] 1) Nur77-346 mutant peptide improves endometrial decidualization

[0046] According to our previous experiments, genetically engineered mice Prmt5f / f Mice (strain No. T007047, commissioned by Nanjing Jicui Pharmaceutical Co., Ltd.) were mated with genetically engineered mice Amhr2-Cre mice (commercial strain: MMRRC, 014245-UNC) to obtain PRMT5 uterine-specific knockout mice, which cannot induce decidualization normally and are suitable for the treatment of decidualization disorder mice. 6-8 week-old PRMT5 uterine-specific knockout mice underwent bilateral ovariectomy via the dorsal approach, rested for 2 weeks, and subcutaneously administered estrogen (100 ng / time / day) for 2 days, rested for 2 days, and subcutaneously administered estrogen (10 ng / time / day) and progesterone (1 mg / time / day) for 3 days. 8 hours after the last administration, 20 ul was injected into the right uterus via the dorsal approach to induce decidualization. After the second injection of estrogen and the third injection of estrogen + progesterone, 10ul of Nur77-346 mutant peptide containing 50ug was infused into the vaginal uterine cavity, and the control group was infused with 10ul of normal saline into the vaginal uterine cavity at the corresponding time point. After 3 days, the uterine decidualization was examined, including morphological analysis and uterine weight weighing. The results showed that vaginal uterine infusion of Nur77-346 mutant peptide can significantly improve decidualization disorders ( Figure 4 ).

[0047] 2) Nur77-346 mutant peptide improves embryo implantation

[0048] 8-10 week old ICR female mice were caged with male mice. The female mice with vaginal plugs at 8 am the next day were recorded as pregnant day 1 (D1). Bilateral ovariectomy was performed via the dorsal approach at 8 am on D4. Progesterone (2 mg / time / day) was administered subcutaneously for 3 consecutive days from D5 to D7 to maintain embryo dormancy. After progesterone administration on D7, 3.75 ng of estradiol was given to activate dormant embryos and initiate embryo implantation. This model has a significantly reduced number of embryos implanted compared with normal mice, making it a suitable therapeutic model for subfertile mice. After subcutaneous administration on D3 and D7, 10 ul of Nur77-346 mutant peptide containing 50 ug was infused into the vaginal uterine cavity, and the control group was infused with 10 ul of normal saline at the corresponding time points. Embryo implantation was examined on D8, and Chicago blue dye was injected through the tail vein to examine embryo implantation in both uteri. The results showed that vaginal uterine infusion of Nur77-346 mutant peptide could significantly increase embryo implantation rate and improve mouse pregnancy rate ( Figure 5 ).

[0049] As can be seen from the above examples, the Nur77-346 mutant peptide disclosed in the present invention has shown significant beneficial effects in both in vitro and in vivo applications. In vitro, the peptide can significantly promote the decidualization and differentiation of primary human endometrial stromal cells, laying an important foundation for understanding its biological functions and subsequent application research. In vivo, whether it is the decidualization disorder model of PRMT5 uterine-specific knockout mice or the low fertility mouse model, vaginal intrauterine perfusion of Nur77-346 mutant peptide can significantly improve decidualization disorders and significantly increase embryo implantation rate and mouse pregnancy rate. These findings not only verify the effectiveness and application potential of Nur77-346 mutant peptide, but also provide new ideas and methods for the treatment of related diseases such as infertility, and have broad application prospects.

[0050] The above embodiments show and describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A Nur77 methylation site mutant peptide designed based on structural modification, characterized in that: The amino acid sequence of the mutant peptide is shown in SEQ ID No.

1.

2. The Nur77 methylation site mutant peptide according to claim 1, characterized in that: The C-terminus of the mutant peptide is also coupled with a cell-penetrating peptide.

3. The Nur77 methylation site mutant peptide according to claim 2, characterized in that: The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID No.

2.

4. A method for preparing the Nur77 methylation site mutant peptide according to any one of claims 1 to 3, characterized in that: The method comprises the step of artificially synthesizing the mutant peptide in vitro by using a solid phase polypeptide synthesis method in a chemical synthesis method.

5. Use of the Nur77 methylation site mutant peptide according to any one of claims 1 to 3 in the preparation of a preparation for promoting embryo implantation.

6. The use according to claim 5, characterized in that The preparation is a medicine used for treating uterine-derived infertility.

7. A preparation for promoting embryo implantation, characterized in that: The preparation comprises the Nur77 methylation site mutant peptide as described in any one of claims 1-3.

8. The preparation according to claim 7, characterized in that The preparation also includes a pharmaceutically acceptable carrier or excipient.

9. The preparation according to claim 7, characterized in that The preparation is an intrauterine infusion agent.

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