WO2023168770A1 - 一种Rheb蛋白激活剂及其应用 - Google Patents

一种Rheb蛋白激活剂及其应用 Download PDF

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WO2023168770A1
WO2023168770A1 PCT/CN2022/083980 CN2022083980W WO2023168770A1 WO 2023168770 A1 WO2023168770 A1 WO 2023168770A1 CN 2022083980 W CN2022083980 W CN 2022083980W WO 2023168770 A1 WO2023168770 A1 WO 2023168770A1
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protein
rheb
atp6ap1
activator
polypeptide
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松阳洲
冯然
刘峰
吴苏
周志芬
李若菲
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中山大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to the field of biotechnology, and in particular to a Rheb protein activator and its application.
  • Rheb is a small GTPase of the Ras superfamily. It has two forms: Rheb-GDP and Rheb-GTP. It is an upstream positive regulator of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. factors, together with the mTORC1 signaling pathway, regulate cell growth, proliferation, differentiation and other processes.
  • mTORC1 mammalian target of rapamycin complex 1
  • GDP-GTP exchange requires the participation of specific factors, that is: Rheb-GTP is hydrolyzed to the GDP form under the catalysis of GAP, and Rheb-GDP can be reactivated by GEF (guanine nucleotide exchange factor) and converted into the GTP form.
  • GEF guanine nucleotide exchange factor
  • TSC2 is currently known to function as Rheb's GAP (G protein regulatory factor), but no known GEF of Rheb has been found.
  • GEF protein regulatory factor
  • Rheb's GEF which type of protein Rheb's GEF is and how its activation process is regulated are still scientific questions that need to be resolved. Since Rheb protein itself has weak GTPase activity, it exists in GTP-bound form in cells in most cases. This may be the reason why Rheb's GEF is not easy to detect. At the same time, because the binding of Rheb to its GEF is relatively instantaneous, traditional methods of detecting protein interactions are difficult to detect.
  • the object of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a Rheb protein activator and its application.
  • the present invention uses a new proximity labeling technology screening method to find the ATP6AP1 protein as Rheb's GEF (guanine nucleotide exchange factor), which can reactivate Rheb-GDP and convert it into GTP form, thereby exerting therapeutic effects.
  • Rheb's GEF guanine nucleotide exchange factor
  • the present invention provides a Rheb protein activator, and the Rheb protein activator is:
  • the Rheb protein activator is a protein with at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared to the ATP6AP1 protein.
  • the Rheb protein activator is ATP6AP1 protein or the polypeptide.
  • the second purpose of the present invention is to provide the use of ATP6AP1 protein or the above-mentioned polypeptide as a targeted Rheb protein activator.
  • the C-terminal polypeptide of the ATP6AP1 protein is used to activate the Rheb protein.
  • the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1.
  • the inventors screened the ATP6AP1 protein as a potential GEF for Rheb.
  • the C-terminal 30 amino acids of the ATP6AP1 protein (TYGLHMILSLKTMDRFDDHKGPTISLTQIV, SEQ ID NO: 1) can directly activate Rheb in vivo and in vitro as a direct target to activate Rheb. of activators used.
  • the present invention provides a method for screening Rheb protein activators, which includes the following steps:
  • the guanine nucleotide exchange factor screening conditions include the following conditions:
  • GEF renal nucleotide exchange factor
  • the present invention obtains Rheb protein activator-ATP6AP1 protein through novel proximity labeling technology screening.
  • the C-terminal 30 amino acids of ATP6AP1 protein can directly target and activate Rheb, which has great medicinal value, especially in the field of diabetes. It has better medicinal value. value.
  • the concentration of insulin is 0.1 nM-10 ⁇ M, and the concentration of biotin is 50 ⁇ M-500 ⁇ M.
  • concentrations of insulin and biotin are lower concentrations for cell lines under normal function, which are more consistent with physiological conditions.
  • the concentration of insulin is 0.9 ⁇ M and the concentration of biotin is 50 ⁇ M.
  • the stimulation time is 10-20 minutes.
  • GEF protein and Rheb The interaction between GEF protein and Rheb is relatively instantaneous, and stimulation time exceeding 20 minutes may cause some negative feedback phenomena to occur, making it impossible to accurately capture GEF protein.
  • the biotin ligase includes biotin ligase xxID, and the fusion expression vector of biotin ligase and Rheb is HAFlag-xxID-Rheb.
  • the biotin ligase mentioned in the present invention is not limited to the present invention, and can also be a biotin ligase commonly used in this field.
  • the cell line stably expressing Rheb protein is a HeLa-xxID-Rheb stably expressing cell line
  • the control cell line is a HeLa-xxID stably expressing cell line.
  • the host cells include HeLa cells, and may also be other cells.
  • the fourth object of the present invention is to provide a protein pharmaceutical composition comprising ATP6AP1 protein and a medically acceptable carrier.
  • the fifth object of the present invention is to provide the use of the above-mentioned Rheb protein activator or protein pharmaceutical composition in preparing drugs for preventing and/or treating diabetes.
  • the present invention has the following beneficial effects:
  • the present invention provides a Rheb protein activator and its application.
  • the present invention uses novel proximity labeling technology to screen and obtain the Rheb protein activator-ATP6AP1 protein as the GEF (guanine nucleotide exchange factor) of Rheb.
  • GEF guanine nucleotide exchange factor
  • the C-terminal 30 of the ATP6AP1 protein Each amino acid can directly target and activate Rheb, converting Rheb-GDP into GTP form, thereby exerting therapeutic effect.
  • the screened ATP6AP1 protein has great medicinal value, especially in the field of treating diabetes, and has better medicinal value. Compared with other small molecule activators, small peptide activators have higher specificity.
  • Figure 1 shows the construction process diagram and mass spectrometry detection chart of HeLa-xxID-Rheb stable expression cell line and HeLa-xxID stable expression cell line;
  • Figure 2 is a fold analysis chart of the Come/GO/Stay group in Example 1;
  • Figure 3 is a schematic diagram of the interaction between ATP6AP1 protein and Rheb in vivo and in vitro;
  • FIG. 4 is a schematic diagram of the mTORC1 downstream signaling pathway activated by overexpression of ATP6AP1 protein
  • Figure 5 is a schematic diagram of the activity of the mTORC1 downstream signaling pathway by knocking down or knocking out the ATP6AP1 protein;
  • Figure 6 shows the results of in vitro experiments proving that ATP6AP1 protein can directly promote Rheb to complete the GDP-GTP conversion process.
  • Example 1 A method for screening Rheb protein activators
  • a method for screening Rheb protein activators includes the following steps:
  • the enriched proteins in the HeLa-xxID-Rheb stable expression cell line were subtracted from the enriched proteins in the HeLa-xxID stable expression cell line to obtain the conditions with/without insulin stimulation.
  • Rheb neighboring proteins further analysis of this part of the enriched proteins (Rheb neighboring proteins) can be divided into Come/GO/Stay groups, that is, the protein group that appears after stimulation/the stable existence group before and after stimulation/the protein group that leaves after stimulation (such as As shown in Figure 2), the Come group includes ATP6AP1 protein, REEPS protein, and SC22B protein.
  • the GO group includes VAMP3 protein and SCD protein.
  • the Stay group includes RAB7A protein and TSC1 protein. Since GEF protein should appear after insulin stimulation, the Come group is The protein was validated as a GEF candidate protein.
  • GEF renal nucleotide exchange factor
  • the ATP6AP1 protein (C-terminal 30 amino acids, TYGLHMILSLKTMDRFDDHKGPTISLTQIV, SEQ ID NO: 1) can well meet the above conditions (the results are shown in Figure 3- Figure 6), proving that the ATP6AP1 protein is a potential GEF for Rheb.
  • Reference Figure 3 is a schematic diagram of the interaction between ATP6AP1 protein and Rheb in vivo and in vitro; through co-immunoprecipitation experiments, the interaction between ATP6AP1/C tail and Rheb was proven in vivo/in vitro, and in vitro experiments proved that there is a direct interaction between the two.
  • ATP6AP1-SFB/C tail-SFB and GST-Rheb were overexpressed in 293T cells. After Flag IP, the GST antibody was used to detect whether there was interaction with Rheb.
  • the in vitro experiment was to perform a GST pull-down experiment on the in vitro purified GST-Rheb protein and Ctail short peptide, which proved that there is a direct interaction between GST-Rheb and Ctail.
  • Figure 4 is a schematic diagram of overexpressing ATP6AP1 protein activating the mTORC1 downstream signaling pathway; by overexpressing ATP6AP1-SFB and C tail-SFB respectively in HeLa cells to detect whether the mTORC1 signaling pathway is activated, it can be seen that compared with the control group GFP, Overexpression of ATP6AP1 and C tail can significantly activate the mTORC1 downstream signaling pathway.
  • Figure 5 is a schematic diagram of the activity of the mTORC1 downstream signaling pathway when knocking down or knocking out the ATP6AP1 protein.
  • the impact on the mTORC1 downstream signaling pathway was detected, and it was found that the mTORC1 downstream signaling pathway was inhibited.
  • Figure 6 shows the results of in vitro experiments proving that ATP6AP1 protein can directly promote Rheb to complete the GDP-GTP conversion process.
  • the present invention uses a new proximity labeling technology to screen and obtain the Rheb protein activator-ATP6AP1 protein as the GEF (guanine nucleotide exchange factor) of Rheb.
  • the C-terminal 30 amino acids of the ATP6AP1 protein can directly target and activate Rheb, causing Rheb-GDP conversion. In the form of GTP, it can exert therapeutic effects.
  • the selected ATP6AP1 protein has greater medicinal value, especially in the field of diabetes.

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Abstract

一种Rheb蛋白激活剂及其应用。利用新型邻近标记技术筛选得到Rheb蛋白激活剂-ATP6AP1蛋白作为Rheb的GEF(鸟嘌呤核苷酸交换因子)，ATP6AP1蛋白的C末端30个氨基酸可以直接靶向激活Rheb，使得Rheb-GDP转变为GTP形式，进而发挥疗效，筛选出的ATP6AP1蛋白具有较大的药用价值，尤其在治疗糖尿病领域具有更佳的药用价值。

Description

一种Rheb蛋白激活剂及其应用 技术领域
本发明涉及生物技术领域，尤其是涉及一种Rheb蛋白激活剂及其应用。
背景技术
Rheb是Ras超家族的小GTP酶，有Rheb-GDP和Rheb-GTP两种形式，是哺乳动物雷帕霉素靶蛋白复合体1(mammalian target of rapamycin complex 1，mTORC1)信号通路的上游正调控因子，与mTORC1信号通路共同调控着细胞生长、增殖和分化等过程。
GDP-GTP交换需要特定因子的参与，即：在GAP催化下Rheb-GTP水解为GDP形式，Rheb-GDP可以被GEF(鸟嘌呤核苷酸交换因子)重新激活，转变为GTP形式。目前已知TSC2作为Rheb的GAP(G蛋白调控因子)发挥功能，但目前并未发现已知Rheb的GEF。同时，目前也并未发现体内外可以直接靶向激活Rheb的小分子药物或短肽药物。
Rheb的GEF是哪一类蛋白、其激活过程受什么调控，目前仍是有待解决的科学问题。由于Rheb蛋白本身存在较弱的GTPase活性，因此在细胞中多数情况以GTP结合形式存在，这可能是Rheb的GEF不容易被发现的原因。同时，由于Rheb与其GEF的结合较为瞬时，因此，传统的检测蛋白互作的手段难以发现。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种Rheb蛋白激活剂及其应用。本发明利用新型邻近标记技术筛选方法找到了ATP6AP1蛋白作为Rheb的GEF(鸟嘌呤核苷酸交换因子)，可以使得Rheb-GDP重新被激活，转变为GTP形式，进而发挥疗效。
为实现上述目的，本发明采取的技术方案为：
第一目的，本发明提供了一种Rheb蛋白激活剂，所述Rheb蛋白激活剂为:
1)多肽，所述多肽具有与SEQ ID NO:1所示的氨基酸序列至少9/10、14/15或29/30的序列一致性；或
2)C端为所述多肽的蛋白质。
进一步地，所述Rheb蛋白激活剂为相比ATP6AP1蛋白具有至少95％、96％、97％、98％或99％氨基酸序列一致性的蛋白质。
更进一步地，所述Rheb蛋白激活剂为ATP6AP1蛋白或所述多肽。
第二目的，本发明提供了ATP6AP1蛋白或上述多肽作为靶向Rheb蛋白激活剂的应用。
作为本发明所述应用的优选实施方式，采用ATP6AP1蛋白的C末端多肽激活Rheb蛋白，所述多肽的氨基酸序列如SEQ ID NO:1所示。
本发明人经过大量的实验，筛选得到ATP6AP1蛋白可以作为Rheb的潜在GEF，ATP6AP1蛋白的C末端30个氨基酸(TYGLHMILSLKTMDRFDDHKGPTISLTQIV，SEQ ID NO:1)可以直接在体内外激活Rheb，作为直接靶向激活Rheb的激活剂使用。
第三目的，本发明提供了一种筛选Rheb蛋白激活剂的方法，包括以下步骤：
1)构建生物素连接酶与Rheb融合表达载体，将载体转入宿主细胞内，药物筛选稳定表达Rheb蛋白的细胞株，同时构建对照细胞株；
2)向稳定表达Rheb蛋白的细胞株中加入胰岛素和生物素进行刺激，向对照细胞株中加入生物素刺激，然后裂解样品、提取蛋白、富集蛋白，再进行质谱检测，分析蛋白；以及
3)分别将稳定表达Rheb蛋白的细胞株中的富集蛋白和对照细胞株中的富集蛋白之间重叠的蛋白扣除(扣除表示为将两组数据中重叠的蛋白去掉，以达到去除对照组干扰的目的)，获得有/无进行胰岛素刺激条件下的Rheb邻近蛋白，分析Rheb邻近蛋白，经鸟嘌呤核苷酸交换因子筛选条件，得ATP6AP1蛋白。
更优选地，所述鸟嘌呤核苷酸交换因子筛选条件包括以下条件：
(1)GEF(鸟嘌呤核苷酸交换因子)与Rheb存在体内外相互作用；
(2)过表达GEF后可以激活mTORC1下游信号通路；
(3)敲低或敲除GEF后，mTORC1下游信号通路活性受到大幅度抑制；
(4)体外实验证明GEF可以直接促进Rheb完成GDP-GTP转换过程。
本发明通过新型邻近标记技术筛选得到Rheb蛋白激活剂-ATP6AP1蛋白， ATP6AP1蛋白的C末端30个氨基酸可以直接靶向激活Rheb，具有较大的药用价值，尤其在糖尿病领域具有更佳的药用价值。
作为本发明所述方法的优选实施方式，所述胰岛素的浓度为0.1nM-10μM，所述生物素的浓度为50μM-500μM。
胰岛素和生物素采用上述浓度为细胞株在正常发挥功能下的较低浓度，更为符合生理状态。
更优选地，所述胰岛素的浓度为0.9μM，所述生物素的浓度为50μM。
作为本发明所述方法的优选实施方式，所述刺激的时间为10-20min。
GEF蛋白与Rheb的相互作用是较为瞬时的，刺激时间超过20min可能会导致一些负反馈现象的发生，不能准确捕捉到GEF蛋白。
作为本发明所述方法的优选实施方式，所述生物素连接酶包括生物素连接酶xxID，生物素连接酶与Rheb融合表达载体为HAFlag-xxID-Rheb。本发明中提到的生物素连接酶不仅限于本发明，还可以为本领域中常规使用的生物素连接酶。
优选地，所述稳定表达Rheb蛋白的细胞株为HeLa-xxID-Rheb稳定表达细胞株，所述对照细胞株为HeLa-xxID稳定表达细胞株。
更优选地，所述宿主细胞包括HeLa细胞，还可以为其他细胞。
第四目的，本发明提供了一种蛋白质药物组合物，包含ATP6AP1蛋白以及医学上可接受的载体。
第五目的，本发明提供了上述Rheb蛋白激活剂或蛋白质药物组合物在制备预防和/或治疗糖尿病药物中的用途。
与现有技术相比，本发明具有以下有益效果：
本发明提供了一种Rheb蛋白激活剂及其应用，本发明利用新型邻近标记技术筛选得到Rheb蛋白激活剂-ATP6AP1蛋白作为Rheb的GEF(鸟嘌呤核苷酸交换因子)，ATP6AP1蛋白的C末端30个氨基酸可以直接靶向激活Rheb，使得Rheb-GDP转变为GTP形式，进而发挥疗效，筛选出的ATP6AP1蛋白具有较大的药用价值，尤其在治疗糖尿病领域具有更佳的药用价值，同时相比于其他小分子激活剂，小肽激活剂的特异性较高。
附图说明
图1为HeLa-xxID-Rheb稳定表达细胞株和HeLa-xxID稳定表达细胞株构建过程图及质谱检测图；
图2为实施例1中Come/GO/Stay组的差异倍数分析图；
图3为ATP6AP1蛋白与Rheb存在体内外相互作用示意图；
图4为过表达ATP6AP1蛋白激活mTORC1下游信号通路示意图；
图5为敲低或敲除ATP6AP1蛋白，mTORC1下游信号通路活性示意图；
图6为体外实验证明ATP6AP1蛋白可以直接促进Rheb完成GDP-GTP转换过程的结果图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
在以下实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、一种筛选Rheb蛋白激活剂的方法
一种筛选Rheb蛋白激活剂的方法，包括以下步骤：
1)构建生物素连接酶(xxID)与Rheb融合表达载体(HAFlag-xxID-Rheb)，将载体转入HeLa细胞内，药物筛选HeLa-xxID-Rheb稳定表达细胞株，同时构建对照细胞系HeLa-xxID稳定表达细胞株，利用含10％透析血清培养基培养；实验前16小时，使用无血清培养基培养，规避胰岛素的干扰。
2)然后向HeLa-xxID-Rheb稳定表达细胞株中加入胰岛素(0.9μM)和生物素(50μM)进行刺激，刺激15min，向HeLa-xxID稳定表达细胞株中加入生物素(50μM)刺激，不加入胰岛素，刺激15min，对样品进行裂解，提取蛋白后，利用链霉亲和素下拉，富集生物素化的蛋白，对链霉亲和磁珠进行还原烷基化，胰酶酶解，脱盐，最后将样品真空干燥后干粉保存，进行质谱检测(如图1所示)，分析蛋白。
3)在有/无进行胰岛素刺激条件下，分别将HeLa-xxID-Rheb稳定表达细胞株中的富集蛋白扣除HeLa-xxID稳定表达细胞株中的富集蛋白，获得有/无进行胰岛素刺激条件下的Rheb邻近蛋白，将这部分富集蛋白(Rheb邻近蛋白)进一步分析，可分为Come/GO/Stay组，即刺激后出现蛋白组/刺激前后稳定存在 组/刺激后离开蛋白组(如图2所示)，Come组包括ATP6AP1蛋白、REEPS蛋白、SC22B蛋白，GO组包括VAMP3蛋白、SCD蛋白，Stay组包括RAB7A蛋白、TSC1蛋白，由于GEF蛋白应该在胰岛素刺激后出现，因此将Come组蛋白作为GEF候选蛋白进行验证。
4)对质谱表单的候选蛋白进行验证，Rheb的GEF候选蛋白需满足以下条件：
(1)GEF(鸟嘌呤核苷酸交换因子)与Rheb存在体内外相互作用；
(2)过表达GEF后可以激活mTORC1下游信号通路；
(3)敲低或敲除GEF后，mTORC1下游信号通路活性受到大幅度抑制；
(4)体外实验证明GEF可以直接促进Rheb完成GDP-GTP转换过程。
通过筛选，发现ATP6AP1蛋白(C末端30个氨基酸，TYGLHMILSLKTMDRFDDHKGPTISLTQIV，SEQ ID NO:1)可以很好的满足上述条件(结果示意图如图3-图6所示)，证明ATP6AP1蛋白是Rheb的潜在GEF。
参考图3为ATP6AP1蛋白与Rheb存在体内外相互作用示意图；通过免疫共沉淀实验，分别在体内/体外证明ATP6AP1/C tail与Rheb的存在相互作用，体外实验证明二者存在直接相互作用。体内实验是将ATP6AP1-SFB/C tail-SFB和GST-Rheb在293T细胞中过表达，通过Flag IP后，利用GST抗体检测是否与Rheb存在相互作用。体外实验是将体外纯化的GST-Rheb蛋白与C tail短肽进行GST下拉实验，证明GST-Rheb与C tail存在直接相互作用。
图4为过表达ATP6AP1蛋白激活mTORC1下游信号通路示意图；通过在HeLa细胞中，分别过表达ATP6AP1-SFB与C tail-SFB，检测mTORC1信号通路是否被激活，可以看到相比于对照组GFP，过表达ATP6AP1和C tail均能明显激活mTORC1下游信号通路。
图5为敲低或敲除ATP6AP1蛋白，mTORC1下游信号通路活性示意图；在HeLa细胞中，敲低ATP6AP1后，检测对mTORC1下游信号通路的影响，发现会抑制mTORC1下游信号通路。
图6为体外实验证明ATP6AP1蛋白可以直接促进Rheb完成GDP-GTP转换过程的结果图。
本发明利用新型邻近标记技术筛选得到Rheb蛋白激活剂-ATP6AP1蛋白作为Rheb的GEF(鸟嘌呤核苷酸交换因子)，ATP6AP1蛋白的C末端30个氨基 酸可以直接靶向激活Rheb，使得Rheb-GDP转变为GTP形式，进而发挥疗效，筛选出的ATP6AP1蛋白具有较大的药用价值，尤其在糖尿病领域具有更佳的药用价值。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (11)

  1. 一种Rheb蛋白激活剂，其特征在于，所述Rheb蛋白激活剂为:
    1)多肽，所述多肽具有与SEQ ID NO:1所示的氨基酸序列至少9/10、14/15或29/30的序列一致性；或
    2)C端为所述多肽的蛋白质。
  2. 如权利要求1所述的Rheb蛋白激活剂，其特征在于，所述Rheb蛋白激活剂为相比ATP6AP1蛋白具有至少95％、96％、97％、98％或99％氨基酸序列一致性的蛋白质。
  3. 如权利要求1所述的Rheb蛋白激活剂，其特征在于，所述Rheb蛋白激活剂为ATP6AP1蛋白或所述多肽。
  4. ATP6AP1蛋白或权利要求1中的多肽作为靶向Rheb蛋白激活剂的应用。
  5. 如权利要求4所述的应用，其特征在于，采用ATP6AP1蛋白的C末端多肽激活Rheb蛋白，所述多肽的序列如SEQ ID NO:1所示。
  6. 一种筛选Rheb蛋白激活剂的方法，其特征在于，包括以下步骤：
    1)构建生物素连接酶与Rheb融合表达载体，将载体转入宿主细胞内，药物筛选稳定表达Rheb蛋白的细胞株，同时构建对照细胞株；
    2)向稳定表达Rheb蛋白的细胞株中加入胰岛素和生物素进行刺激，向对照细胞株中加入生物素刺激，然后裂解样品、提取蛋白、富集蛋白，再进行质谱检测，分析蛋白；以及
    3)分别将稳定表达Rheb蛋白的细胞株中的富集蛋白和对照细胞株中的富集蛋白之间重叠的蛋白扣除，获得有/无进行胰岛素刺激条件下的Rheb邻近蛋白，分析Rheb邻近蛋白，经鸟嘌呤核苷酸交换因子筛选条件，得ATP6AP1蛋白。
  7. 如权利要求6所述的方法，其特征在于，所述胰岛素的浓度为0.1nM-10μM，所述生物素的浓度为50μM-500μM。
  8. 如权利要求6所述的方法，其特征在于，所述刺激的时间为10-20min。
  9. 如权利要求6所述的方法，其特征在于，所述生物素连接酶包括生物素连接酶xxID，所述生物素连接酶与Rheb融合表达载体为HAFlag-xxID-Rheb。
  10. 一种蛋白质药物组合物，其特征在于，包含ATP6AP1蛋白以及医学上可接受的载体。
  11. 如权利要求1所述的Rheb蛋白激活剂或权利要求10所述的蛋白质药物组合物在制备预防和/或治疗糖尿病药物中的用途。
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