WO2023108391A1 - 一种蛋白质液相分离的探测方法及其应用 - Google Patents

一种蛋白质液相分离的探测方法及其应用 Download PDF

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WO2023108391A1
WO2023108391A1 PCT/CN2021/137736 CN2021137736W WO2023108391A1 WO 2023108391 A1 WO2023108391 A1 WO 2023108391A1 CN 2021137736 W CN2021137736 W CN 2021137736W WO 2023108391 A1 WO2023108391 A1 WO 2023108391A1
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expression vector
protein
phase separation
liquid phase
fluorescence
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黄良宇
张淑雯
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深圳先进技术研究院
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • the invention belongs to the field of biotechnology, and in particular relates to a detection method for protein liquid phase separation and an application thereof.
  • Synapse is the structural and functional unit of interconnection and communication between all neurons. Its diameter is only 200-800 nanometers. Therefore, it is impossible to detect the liquid phase of proteins in synapses by observing the formation of submicron to micron-sized condensed phases. phenomenon of separation.
  • super-resolution fluorescence imaging has high hardware requirements (microscopes are expensive) and application limitations (usually for fixed cell samples).
  • PAM photoactivated localization microscopy
  • PROM stochastic optical reconstruction microscopy
  • the method has an upper limit on the imaging speed, and the strong laser light used will poison the cells, so it is not conducive to the detection of the liquid phase separation process on living neurons.
  • the purpose of the present invention is to realize the detection of the phase transition process of proteins in synapses under the condition of receiving various nerve stimuli on the sub-second time scale.
  • the present invention proposes a protein liquid phase Separation detection methods and their applications.
  • the process of protein phase transition (that is, liquid phase separation from scratch) will lead to the change of fluorescence resonance energy transfer efficiency, so the protein phase transition can be detected by measuring and calculating the change of energy transfer efficiency.
  • the first aspect of the present invention provides a detection method for protein liquid phase separation, comprising the following steps:
  • the target protein is a synapse protein
  • the primary cultured cells are primary cultured neuron cells.
  • the second expression vector contains 2-3 nucleotide sequences of energy acceptor fluorescent molecules in series.
  • an electric field is produced by an electric field stimulator to excite the primary cultured cells transferred into the first expression vector and the second expression vector;
  • the electric field has an intensity of 25-50 V/cm and a frequency of 20-50 Hz to stimulate nerve activity.
  • the fluorescence microscope has two or more excitation lasers or excitation filters.
  • fluorescence resonance energy transfer efficiency is calculated by sensitized emission, ratiometric method or fluorescence lifetime imaging (FLIM);
  • the fluorescence resonance energy transfer efficiency is calculated by a sensitized luminescence method.
  • the detection method is used to detect the process of liquid phase separation of proteins in living nerve synapses.
  • the second aspect of the present invention provides the application of the detection method in drug target screening.
  • the detection method of protein liquid phase separation proposed by the present invention is based on the principle of fluorescence resonance energy transfer.
  • the subcultured cells express energy donor fluorescent molecules and target proteins as well as energy acceptor fluorescent molecules and target proteins. After excitation, use a fluorescence microscope to collect fluorescence microscopic imaging data, and calculate the change in fluorescence resonance energy transfer efficiency to detect protein liquid phase. process of separation.
  • the detection method of the present invention realizes the detection of protein liquid phase separation in vitro or in living nerve cells (i.e.
  • neurons can be used to study the effects of protein phase transitions on neurophysiology and pathology, and to study the effects of protein phase transitions on neurophysiology and pathology, and to study the effects of protein phase transitions on neurophysiology and pathology in different genotypes or phenotypes.
  • the phase transition process of the target protein on a type of neuron so as to discover new disease mechanisms and drug targets.
  • the present invention realizes for the first time the detection of the liquid phase separation of proteins in the synapse of neurons on a sub-second time scale.
  • the invention detects the phase transition through the fluorescence resonance energy transfer phenomenon caused by the closer distance between proteins after liquid phase separation, so it is not limited by the size of the aggregate, and can be used to detect nerves with a diameter of only 200-800 nanometers Liquid phase separation in synapses.
  • the measurement of fluorescence resonance energy transfer can be technically completed on a sub-second time scale, bypassing the bottleneck of super-resolution fluorescence imaging technology and avoiding the poisoning of cells by strong laser light.
  • Figure 1 is the detection principle and flow chart of protein liquid phase separation.
  • A. is connected to the target protein of fluorescent energy donor;
  • B. is connected to the target protein of fluorescent energy acceptor;
  • C. is connected to the target protein of multiple fluorescent energy acceptors in series;
  • D. has expressed protein A and B ( Or A and C) primary cultured neurons;
  • E. various types of fluorescence microscopes;
  • G neurons of different genetic types or phenotypes.
  • Fig. 2 is the experimental data for verifying the principle and feasibility of the present invention in the COS-7 cell line.
  • (Left image) General fluorescence imaging shows that the target protein forms a round condensed phase after liquid phase separation;
  • the data collected and calculated by the sensitized luminescence method show that, compared with the outside of the condensed phase (average value 0.11 ), there is a higher fluorescence resonance energy transfer efficiency (average value 0.22) in the condensed phase.
  • the present invention is based on fluorescence resonance energy transfer ( The principle of resonance energy transfer (FRET) detects the enhancement of fluorescence resonance energy transfer of proteins in the condensed phase.
  • FRET fluorescence resonance energy transfer
  • A is a target protein connected with a fluorescent molecule as an energy donor (donor)
  • B is a target protein connected with a fluorescent molecule as an energy acceptor (acceptor)
  • C is a target protein connected with multiple energy acceptor fluorescent molecules.
  • D is primary cultured neurons expressing proteins A and B (or A and C).
  • E is a confocal laser scanning microscope (or other types of fluorescence microscopes), with two or more excitation lasers (or excitation filters).
  • F is an electric field stimulator capable of exciting neurons by creating an electric field.
  • G are neurons of different genotypes or phenotypes.
  • the efficiency of FRET depends on the distance between the energy donor (A) and the acceptor molecule (B or C). Because the liquid phase separation of proteins is generated by the interaction between molecules, it is predicted that the distance between the molecules of proteins in the condensed phase will be brought closer, thereby improving the efficiency of fluorescence resonance energy transfer. This method can realize the detection of protein liquid phase separation in neurons of different genotypes or phenotypes.
  • a detection method for protein liquid phase separation comprising the steps of:
  • the second expression vector contains 2-3 nucleotide sequences of energy acceptor fluorescent molecules in series.
  • Fluorescence microscopy imaging data were collected using a confocal laser scanning microscope.
  • This example was verified in the COS-7 cell line expressing the target protein with fluorescent molecules.
  • the first expression vector is a pcDNA3.1+ expression vector with a fluorescent donor mEGFP connected to the N-terminal of miniShank3;
  • the second expression vector is a fluorescent acceptor mCherry connected to the N-terminal of miniShank3 pcDNA3.1+ expression vector.
  • Biyuntian’s Lipo6000 TM and 1.5 ⁇ g of two expression vectors were used to transfer into the COS-7 cell line, and the data were collected after 48 hours of culture.
  • the imaging of fluorescence resonance energy transfer efficiency is measured and calculated by sensitized luminescence method: the laser wavelengths for exciting the fluorescence donor and acceptor are 488 and 561nm, respectively, and the wavelength bands for fluorescence collection are 490-560nm (donor channel), respectively. 576-700nm (resonance energy transfer channel), and 576-700nm (acceptor channel).
  • the calculation of fluorescence resonance energy transfer efficiency is based on the formula 8 in the literature of Gordon GWet al.(1998) Biophys J.74(5):2702-13 and Xia Z.and Liu Y.(2001) Biophys J.81(4) : Formula 2 in the 2395-402 literature.

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Abstract

蛋白质液相分离的探测方法及其应用，方法包括构建表达能量供体荧光分子和目标蛋白的第一表达载体，表达能量受体荧光分子和目标蛋白的第二表达载体；将第一表达载体和第二表达载体转入原代培养的细胞中；激发转入第一表达载体和第二表达载体的原代培养的细胞，利用荧光显微镜采集荧光显微成像数据；计算荧光共振能量转移效率的改变，探测蛋白质液相分离的过程。实现在活体神经元突触内对蛋白质的液相分离进行亚秒时间尺度上的探测，绕过了超分辨率荧光成像技术的瓶颈，避免了强激光对细胞的毒害；探测蛋白质相变不受凝聚体大小的限制，用于探测在直径只有200-800纳米的神经突触内所发生的液相分离现象。

Description

一种蛋白质液相分离的探测方法及其应用 技术领域
本发明属于生物技术领域，具体涉及一种蛋白质液相分离的探测方法及其应用。
背景技术
研究指出有多种神经突触蛋白质在体外实验中能够进行液相分离，并且其中的一些蛋白质与神经退行性疾病有直接的关系，因此有一些研究人员提出了蛋白质相变失控而导致神经退行性疾病的一个理论。然而，只有直接观测蛋白质在神经元突触内进行液相分离并对其进行表征才能够证明此理论，并且有可能开拓新的研究方向。
现有的在体外实验中探测蛋白质液相分离的方法包括荧光显微镜技术以及光散射方法。其中，最为普遍的是通过调整各种物理化学条件(例如温度、蛋白质浓度、盐浓度等)，利用荧光显微镜观察亚微米至微米大小、呈液滴状的物体(即凝聚相)的形成，然后再利用光漂白荧光恢复技术(fluorescence recovery after photobleaching，FRAP)来测定蛋白质在凝聚相中的流动性，以断定蛋白质在相变后的物质形态。同时，凝聚相的形成也会增加可见光波长范围的光散射，因此可以利用该方法来探测蛋白质的液相分离。
神经突触是所有神经元之间相互连接和通讯的结构及功能单元，其直径只有200-800纳米，因此不能通过观测亚微米至微米大小的凝聚相形成来探测蛋白质在突触内进行液相分离的现象。目前，一些研究人员试图利用各种超分辨率荧光成像技术来观测由蛋白质所形成的纳米尺度的凝聚体。然而，超分辨率荧光成像具有较高的硬体要求(显微镜价格昂贵)以及应用局限(通常用于固定后的细胞样本)。由于光激活定位显微成像术(photoactivated localization microscopy，PALM)和随机光学重建显微法(stochastic optical reconstruction microscopy，STORM)都是基于重复激发、观测、漂白单个激活的荧光分子加上演算而得到定位的方法，在成像速度上具有上限，并且所利用的强激光会毒害细胞，所以并不利于在活的神经元上探测液相分离的过程。
发明内容
本发明旨在实现在亚秒时间尺度上探测在神经突触内蛋白质在接受各种神经刺激的情况下进行相变的过程，为了解决现有技术中的不足，本发明提出一种蛋白质液相分离的探测方法及其应用。蛋白质相变的过程(也就是液相分离从无到有)会导致荧光共振能量转移效率的改变，因此能够通过测量与计算该能量转移效率的改变来探测蛋白质相变。
本发明第一方面提供一种蛋白质液相分离的探测方法，包括如下步骤：
构建表达能量供体荧光分子和目标蛋白的第一表达载体，表达能量受体荧光分子和目标蛋白的第二表达载体；
将第一表达载体和第二表达载体转入原代培养的细胞中；
激发转入第一表达载体和第二表达载体的原代培养的细胞，利用荧光显微镜采集荧光显微成像数据；
计算荧光共振能量转移效率的改变，探测蛋白质液相分离的过程。
进一步地，所述目标蛋白为神经突触蛋白；
所述原代培养的细胞为原代培养的神经元细胞。
进一步地，所述第二表达载体包含2-3个串联的能量受体荧光分子核苷酸序列。
进一步地，通过电场激发器制造电场激发转入第一表达载体和第二表达载体的原代培养的细胞；
优选地，所述电场的强度为25-50伏/厘米，频率为20-50赫兹，刺激神經活動。
进一步地，所述荧光显微镜具有两个或以上的激发激光器或激发滤光片。
进一步地，所述荧光共振能量转移效率通过敏化发光法(sensitized emission)、比例法(ratiometric method)或荧光寿命法(fluorescence lifetime imaging，FLIM)计算；
优选地，所述荧光共振能量转移效率通过敏化发光法计算。
进一步地，所述探测方法用于探测蛋白质在活体神经突触内液相分离的过程。
本发明第二方面提供所述探测方法在药物靶点筛选中的应用。
本发明的有益效果为：
1、本发明提出的蛋白质液相分离的探测方法基于荧光共振能量转移的原理，根据蛋白质液相分离后蛋白质在凝聚相中分子之间的距离拉近，荧光共振能量转移的效率提升，通过在原代培养的细胞中表达能量供体荧光分子和目标蛋白以及能量受体荧光分子和目标蛋白，激发后利用荧光显微镜采集荧光显微成像数据，计算荧光共振能量转移效率的改变，从而探测蛋白质液相分离的过程。本发明探测方法实现了在体外或活体神经细胞(即神经元)内对蛋白质液相分离的探测，可以用于研究蛋白质相变对神经生理以及病理的作用，用于研究在不同遗传型或表型的神经元上目标蛋白质的相变过程，从而发现新的疾病机理与药物靶点。
在一个优选的方案中，通过串联多个能量受体荧光分子，进一步提升荧光共振能量转移的效率，增强探测蛋白质在神经元突触内液相分离现象的精准度。
2、本发明第一次实现在神经元突触内对蛋白质的液相分离进行亚秒时间尺度上的探测。 本发明通过液相分离后蛋白质之间的距离拉近而造成的荧光共振能量转移现象来探测其相变，因此不会受到凝聚体大小的限制，可用于探测在直径只有200-800纳米的神经突触内所发生的液相分离现象。其次，测量荧光共振能量转移在技术上能够在亚秒时间尺度上完成，绕过了超分辨率荧光成像技术的瓶颈，并且避免了强激光对细胞的毒害。
附图说明
图1为蛋白质液相分离的探测原理和流程图。其中，A.接上荧光能量供体的目标蛋白；B.接上荧光能量受体的目标蛋白；C.接上串联多个荧光能量受体的目标蛋白；D.已表达蛋白A与B(或者A与C)的原代培养神经元；E.各种类型的荧光显微镜；F.激发神经元的电场激发器；G.不同遗传型或表型的神经元。
图2为在COS-7细胞系中验证本发明原理和可行性的实验数据。(左图)一般的荧光成像显示目标蛋白液相分离后形成了呈圆形的凝聚相；(右图)通过敏化发光法采集数据及计算的成像显示，相对于凝聚相外(平均值0.11)，凝聚相内有较高的荧光共振能量转移效率(平均值0.22)。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
本发明是基于荧光共振能量转移(
Figure PCTCN2021137736-appb-000001
resonance energy transfer，FRET)的原理，探测蛋白质在凝聚相中荧光共振能量转移的增强。蛋白质液相分离的探测原理和流程图如图1所示。A是接上作为能量供体(donor)荧光分子的目标蛋白，B是接上作为能量受体(acceptor)荧光分子的目标蛋白，C是接上多个能量受体荧光分子的目标蛋白。D是已表达蛋白A与B(或者A与C)的原代培养神经元。E是共聚焦激光扫描显微镜(或者其他类型的荧光显微镜)，具有两个或以上的激发激光器(或者激发滤光片)。F是能够通过制造电场激发神经元的电场激发器。G是不同遗传型或表型的神经元。
荧光共振能量转移的效率取决于能量供体(A)与受体分子(B或C)之间的距离。因为蛋白质的液相分离是通过分子之间的相互作用所产生的，因此预测蛋白质在凝聚相中分子之间的距离拉近，从而提升了荧光共振能量转移的效率。该方法可以实现对不同遗传型或表型 的神经元内蛋白质液相分离的探测。
在一个优选的方案中，通过串联多个能量受体荧光分子，将进一步提升荧光共振能量转移的效率，增强探测蛋白质在神经元突触内液相分离现象的精准度。
蛋白质液相分离的探测方法，包括如下步骤：
(1)构建表达能量供体荧光分子和目标蛋白的第一表达载体，表达能量受体荧光分子和目标蛋白的第二表达载体，将第一表达载体和第二表达载体转入原代培养的神经元细胞中。在一个优选的实施方案中，第二表达载体包含2-3个串联的能量受体荧光分子核苷酸序列。
(2)通过电场激发器制造电场激发神经元。
(3)利用共聚焦激光扫描显微镜采集荧光显微成像数据。
(4)通过敏化发光法计算荧光共振能量转移效率的改变，从而完成整个探测目标蛋白质在神经元内的相变过程。
实施例2
本实施例在表达带有荧光分子的目标蛋白的COS-7细胞系得到了验证。
在本实施例当中，第一表达载体为在miniShank3的N端接上了荧光供体mEGFP的pcDNA3.1+表达载体；第二表达载体为在miniShank3的N端接上了一个荧光受体mCherry的pcDNA3.1+表达载体。采用了碧云天的Lipo6000 TM以及加入两个表达载体个别1.5μg的量转入COS-7细胞系里，培养48小时后采集数据，利用了蔡司的Plan-Apochromat 63x/1.4 Oil DIC M27物镜，像素为40nm x 40nm。荧光共振能量转移效率的成像是通过敏化发光法测量与计算的：激发荧光供体与受体的激光波长分别为488与561nm，而荧光收集的波段分别为490-560nm(供体频道)，576-700nm(共振能量转移频道)，以及576-700nm(受体频道)。荧光共振能量转移效率的计算是根据Gordon G.W.et al.(1998)Biophys J.74(5):2702-13文献里的公式8以及Xia Z.and Liu Y.(2001)Biophys J.81(4):2395-402文献里的公式2。
如图2所示，神经突触蛋白miniShank3在COS-7细胞内过表达时会发生液相分离现象(左图)，并且在凝聚相中的荧光共振能量转移效率(平均值0.22)与凝聚相外的(平均值0.11)有明显的差异(右图)，由此证明了本发明的原理，通过测量荧光共振能量转移效率的变化来探测目标蛋白相变的过程。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (8)

  1. 一种蛋白质液相分离的探测方法，其特征在于，包括如下步骤：
    构建表达能量供体荧光分子和目标蛋白的第一表达载体，表达能量受体荧光分子和目标蛋白的第二表达载体；
    将第一表达载体和第二表达载体转入原代培养的细胞中；
    激发转入第一表达载体和第二表达载体的原代培养的细胞，利用荧光显微镜采集荧光显微成像数据；
    计算荧光共振能量转移效率的改变，探测蛋白质液相分离的过程。
  2. 根据权利要求1所述的探测方法，其特征在于，所述目标蛋白为神经突触蛋白；
    所述原代培养的细胞为原代培养的神经元细胞。
  3. 根据权利要求1所述的探测方法，其特征在于，所述第二表达载体包含2-3个串联的能量受体荧光分子核苷酸序列。
  4. 根据权利要求1所述的探测方法，其特征在于，通过电场激发器制造电场激发转入第一表达载体和第二表达载体的原代培养的细胞；
    优选地，所述电场的强度为25-50伏/厘米，频率为20-50赫兹。
  5. 根据权利要求1所述的探测方法，其特征在于，所述荧光显微镜具有两个或以上的激发激光器或激发滤光片。
  6. 根据权利要求1所述的探测方法，其特征在于，所述荧光共振能量转移效率通过敏化发光法、比例法或荧光寿命法计算；
    优选地，所述荧光共振能量转移效率通过敏化发光法计算。
  7. 根据权利要求1所述的探测方法，其特征在于，所述探测方法用于探测蛋白质在活体神经突触内液相分离的过程。
  8. 权利要求1-7任一项所述探测方法在药物靶点筛选中的应用。
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