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Detection of Membrane Protein Interactions by Cell-based Tango Assays
采用基于细胞的Tango测定法检测膜蛋白相互作用   

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参见作者原研究论文

本实验方案简略版
The Journal of Biological Chemistry
Aug 2017

Abstract

The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells.

Keywords: Tango assay (Tango测定法), γ-Secretase (γ-分泌酶), Cleavage (裂解), Activity (活性), Interaction (相互作用), C99 (C99)

Background

The amyloid precursor protein (APP) has three dimerization domains in its N-terminal extracellular domain. In addition, APP can also form dimers through the membrane-bound C99 (C-terminal 99 amino acid fragment) region. Importantly, C99 dimerization has been linked to Aβ production in Alzheimer’s disease (AD) pathology. The Tango assay described here and schematically shown as cartoon in Figure 1 is a fast and sensitive method for investigating homodimerization of C99 and other membrane proteins (Yan et al., 2017).


Figure 1. Cartoon illustration of the Tango interaction assay. Upon membrane cleavage of the C99 hybrid protein by TEV protease, the rTA transactivator protein is released from the membrane into the cytoplasm. This allows rTA to enter the nucleus and bind the tetO DNA-binding site upstream of an integrated luciferase reporter gene to stimulate luciferase reporter gene activity as measured by luminescence. (Yan et al., 2017).

Here we use the Dual-luciferase reporter assay kit. The stably integrated luciferase-Firefly reads represent the γ-secretase cleavage activity, while the transfected Renilla luciferase reads serve as normalization standard.

Materials and Reagents

  1. Pipette tips (VWR)
  2. 24-well plate (Corning, Costar®, catalog number: 3524 )
  3. 96-well plate (Corning, catalog number: 3595 )
  4. Cell culture flask (Corning, catalog number: 430639 )
  5. 96-well OptiPlate (PerkinElmer, catalog number: 6005290 )
  6. Gel loading pipette tip
  7. PS1/PS2-deleted HTL cells (PS1/PS2 gene were deleted by CRISPR/Cas9 from HTL cells [Xu et al., 2016])
  8. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  9. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  10. Trypsin 0.25%-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  11. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  12. Dual-luciferase reporter assay kit (Promega, catalog number: E1960 )
  13. X-tremeGENE 9 Reagent (Roche Diagnostics, catalog number: 06365787001 )
  14. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-1 )
  15. Potassium phosphate monobasic (KH2PO4) (EMD Millipore, catalog number: PX1565-1 )
  16. Sodium chloride (NaCl) (EMD Millipore, catalog number: SX0420-5 )
  17. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-500 )
  18. 10x phosphate buffered saline (PBS buffer) (see Recipes)
  19. 1x passive lysis buffer (PLB) (see Recipes)
  20. LAR2 substrate (see Recipes)
  21. Stop & Glo® Reagent (see Recipes)

Equipment

  1. Micro pipette (Eppendorf)
  2. Vacuum pump
  3. 37 °C, 5% CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 Water-Jacketed)
  4. Cell culture microscope (Nikon Instruments, model: Eclipse TS100 )
  5. Shaker (ARMA LAB, model: Orbital Shaker 100 )
  6. Biosafety cabinet (The Baker, model: SterilGARD® e3 )
  7. EnVision Multilabel Plate Reader (PerkinElmer, model: EnVision Multilabel )

Software

  1. GraphPad Prism 5 (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Cell culture
    1. The PS1/PS2-deleted HTL cells are routinely grown in DMEM supplemented with 10% (v/v) FBS at 37 °C under a humidified 5% CO2 atmosphere.
    2. Treat the cells with 0.25% trypsin-EDTA at 37 °C for about 2 min. Dilute the cells to 0.4 x 106/ml with DMEM medium (supplemented with 10% [v/v] fetal bovine serum) and split at 50,000 per well into a 24-well plate one day prior to transfection.

  2. Transfection
    1. Prepare a master mix for one sample:
      To a sterile 1.5 ml Eppendorf tube, add:
      10 µl Opti-MEM medium
      0.195 µl X-tremeGENE 9 Transfection Reagent.
      Notes:
      1. Avoid touching the side of the tube while adding reagent.
      2. Make a master mix for larger sample numbers: for example, for 10 samples, multiply by 11 to prepare some extra mix. (10 x 11 = 110 µl medium; 0.195 x 11 = 2.145 µl X-tremeGENE 9 Transfection Reagent).
    2. Add 10 µl Opti-MEM medium per well in a 96-well plate and then add 65 ng total DNA into each well.
      Note: We usually dilute the DNA plasmids to a concentration of 100 ng/µl and store them at 4 °C.
    3. Add 10 µl master mix to DNA mix in each well of the 96-well plate and mix gently by pipetting.
    4. Incubate at room temperature for about 30 min.
    5. Carefully transfer 20 µl mix by pipetting into each well of cells cultured in a 24-well plate.
      Note: In this assay, 10 ng C99 (or its variants)-TEV site-rTA expression construct, 10 ng C99 (or its variants)-TEV protease construct and 5 ng of phRG-tk Renilla normalization luciferase expression vector were transfected together with 40 ng pBSK plasmid control into PS1/PS2-deleted HTL cells.
    6. Culture the cells in DMEM supplemented with 10% (v/v) fetal bovine serum at 37 °C under a humidified 5% CO2 atmosphere.

  3. Luciferase measurement
    1. One day post transfection, remove the medium from the cultured cells by vacuum pump attached by tubing to a liquid trap and a gel loading pipette tip, and gently apply a sufficient volume (i.e., 500 µl/well) of PBS (see Recipes) to rinse the bottom of each well.
    2. Dispense 100 µl of 1x PLB (see Recipes) into each well and gently shake the culture plate for 15 min at room temperature (e.g., 70 rpm).
      Note: We use Eppendorf® Research® Pro electronic single channel pipette (20-1,000 µl) to dispense the PLB buffer. However, any laboratory pipette that can be set to 100 µl can be used.
    3. Transfer 20 µl PLB lysate without cell debris to each well of a 96-well OptiPlate.
      Note: Generally, it is unnecessary to clear lysates of residual cell debris prior to performing the assay.
    4. Program the EnVision Multilabel Plate Reader to perform a 2-sec premeasurement delay, followed by a 10-sec measurement period using the Envision 96 Plate US Luminescence (700 nm emission filter) aperture for each reporter assay.
    5. Dispense 50 µl LAR2 substrate (see Recipes) into each well of the 96-well OptiPlate, mix by pipetting 2 or 3 times and measure Firefly luciferase activity using EnVision Multilabel Plate Reader. The Luminescence reads represent the Firefly luciferase activity.
    6. Dispense 50 µl Stop & Glo® Reagent (see Recipes) into the same plate, mix by pipetting 2 or 3 times and measure the Renilla luciferase activity by EnVision Multilabel Plate Reader. The Luminescence reads represent the Renilla luciferase activity.

Data analysis

Each data point represents the average from at least three independent experiments (Table 1), calculated activities are shown in Table 2. Data are presented as grouped bar graph type. The statistical analysis of data can be accomplished with GraphPad Prism using the two-tailed Student’s t-test versus control (Figure 2). A practical example of the applicability of this Tango interaction assay was recently shown in (Yan et al., 2017).

Table 1. Original Firefly and Renilla activity reads


Table 2. Relative activity and Normalized activity

Note: For activity normalization, each relative activity is divided by the average of the relative activity of WT (C99-T4L-rTA) and times 100.


Figure 2. Validation of C99 dimerization by Tango assay

Notes

The status of the cells is very important. When performing data analysis, pay attention to the Renilla luciferase signal (Table 3). It should be around 1,000,000 photo counts.

Table 3. Examples of bad cell status

These wells (three wells of Group ctrl1 and three wells of Group ctrl2 should be the negative control, the low Renilla activity, which to some degree reflects the cell status and transfection efficiency, makes the relative activity much higher than normal.

Recipes

  1. 10x phosphate buffered saline (PBS buffer) (1 L)
    11.5 g Na2HPO4
    2 g KH2PO4
    80 g NaCl
    2 g KCl
    Dissolve in 1 L of sterile, deionized water
    The pH of 1x PBS should be 7.4
  2. 1x passive lysis buffer (PLB)
    Add 1 volume of 5x PLB from Dual-luciferase reporter assay kit to 4 volumes of distilled water and mix well
    The 1x PLB can be stored at 4 °C no more than one month
  3. LAR2 substrate (from Dual-luciferase reporter assay kit)
    Resuspend the lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer 2 (10 ml for one bottle) and store at -20 °C less than 1 month or -70 °C less than 1 year
  4. Stop & Glo® Reagent (from Dual-luciferase reporter assay kit)
    Freshly add 1 volume Stop & Glo® Substrate to 49 volumes Stop & Glo® Buffer and vortex 10 sec

Acknowledgments

This work was supported by the Van Andel Research Institute, the National Natural Science Foundation of China (31300607, 31300245 and 91217311), Ministry of Science and Technology grants 2012ZX09301001, 2012CB910403, and 2013CB910600, XDB08020303, 2013ZX09507001, Shanghai Science and Technology Committee (13ZR1447600), Shanghai Rising-Star Program (14QA1404300), and the National Institute of Health grants DK071662 (H.E.X.), GM102545 and GM104212 (K. M.). The authors declare no conflict of interest.

References

  1. Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R. and Lee, K. J. (2008). The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci U S A 105(1): 64-69.
  2. Xu, T. H., Yan, Y., Kang, Y., Jiang, Y., Melcher, K. and Xu, H. E. (2016). Alzheimer's disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio. Cell Discov 2: 16026.
  3. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem.

简介

Tango测定是蛋白质 - 蛋白质相互作用测定法,其中转录因子(rTA)通过含有TEV蛋白酶切割位点的接头与膜结合蛋白质融合,而可溶性相互作用配偶体与TEV蛋白酶融合( Barnea et al。,2008)。 两个相互作用伙伴之间的关联导致转录因子的高效切割,使其易位至核并激活荧光素酶报道基因作为相互作用的测量。 在该修改的测定中,我们将膜跨膜淀粉状蛋白前体蛋白(APP)C99区域的一个拷贝与TEV位点rTA(C99-TEV位点rTA)融合,并将另一个拷贝与TEV蛋白酶(C99-TEV)融合以分析膜内 活细胞中的C99-C99相互作用。
【背景】淀粉样蛋白前体蛋白(APP)在其N-末端胞外结构域中具有三个二聚化结构域。此外,APP还可以通过膜结合的C99(C-末端99个氨基酸片段)区域形成二聚体。重要的是,C99二聚化已经与阿尔茨海默氏病(AD)病理学中的Aβ产生相关联。这里所描述的并且在图1中示意性地显示为卡通的Tango测定是用于研究C99和其他膜蛋白的同源二聚化的快速且敏感的方法(Yan等人,2017)。

“”src
图1. Tango相互作用测定的卡通插图。TEV蛋白酶对C99杂交蛋白进行膜切割后,rTA反式激活蛋白从膜释放到细胞质中。这允许rTA进入细胞核并且结合整合的萤光素酶报道基因上游的tetO DNA结合位点以刺激荧光素酶报道基因活性,如通过发光测量的。 (Yan等人,2017)。

这里我们使用双荧光素酶报告基因检测试剂盒。稳定整合的萤光素酶萤火虫阅读代表γ-分泌酶切割活性,而转染的海肾荧光素酶阅读作为标准化标准。

关键字:Tango测定法, γ-分泌酶, 裂解, 活性, 相互作用, C99

材料和试剂

  1. 移液枪头(VWR)
  2. 24孔板(Corning,Costar ®,目录号:3524)
  3. 96孔板(康宁,目录号:3595)
  4. 细胞培养瓶(康宁,目录号:430639)
  5. 96孔OptiPlate(PerkinElmer,目录号:6005290)
  6. 凝胶加样枪头
  7. 来自HTL细胞的CRISPR / Cas9缺失了PS1 / PS2缺陷的HTL细胞(PS1 / PS2基因)[Xu等,2016] )
  8. Dulbecco改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,产品目录号:11965092)
  9. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  10. 胰蛋白酶0.25%-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  11. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:31985062)
  12. 双荧光素酶报告基因检测试剂盒(普洛麦格,目录号:E1960)
  13. X-tremeGENE 9试剂(Roche Diagnostics,目录号:06365787001)
  14. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S374-1)
  15. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:PX1565-1)
  16. 氯化钠(NaCl)(EMD Millipore,目录号:SX0420-5)
  17. 氯化钾(KCl)(Fisher Scientific,目录号:BP366-500)
  18. 10倍磷酸盐缓冲盐水(PBS缓冲液)(见食谱)
  19. 1x被动裂解缓冲液(PLB)(见食谱)
  20. LAR2底物(见食谱)
  21. 停止& Glo ®试剂(见食谱)

设备

  1. 微量移液器(Eppendorf)
  2. 真空泵
  3. 37℃,5%CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Series 3110水套)
  4. 细胞培养显微镜(尼康仪器,型号:Eclipse TS100)
  5. 摇床(ARMA LAB,型号:轨道摇床100)
  6. 生物安全柜(贝克,型号:SterilGARD e3)
  7. EnVision Multilabel Plate阅读器(PerkinElmer,型号:EnVision Multilabel)

软件

  1. GraphPad Prism 5( https://www.graphpad.com/scientific-software/prism/

程序

  1. 细胞培养
    1. 在37℃,潮湿的5%CO 2气氛下,通常使用PS1 / PS2去除的HTL细胞在补充有10%(v / v)FBS的DMEM中生长。
    2. 用0.25%胰蛋白酶-EDTA在37℃处理细胞约2分钟。用DMEM培养基(补充有10%[v / v]胎牛血清)将细胞稀释至0.4×10 6 / ml,并在每孔50,000个孔中分裂到24孔板中,转。

  2. 转染
    1. 准备一个样本的主混合:
      向无菌1.5 ml Eppendorf管中加入:
      10μlOpti-MEM培养基
      0.195μlX-tremeGENE 9转染试剂。
      注意:
      1. 避免在添加试剂时触摸试管的一侧。
      2. 为更大的样本数量做一个主混合:例如,对于10个样本,乘以11来准备一些额外的混合。 (10×11 =110μl培养基; 0.195×11 =2.145μlX-tremeGENE 9转染试剂)。
    2. 每孔加入10μlOpti-MEM培养基到96孔板中,然后每孔加入65 ng总DNA。
      注:我们通常将DNA质粒稀释至100 ng /μl的浓度,并将其保存在4°C。
    3. 在96孔板的每个孔中加入10μl主混合物到DNA混合物中,并通过移液轻轻混合。

    4. 在室温下孵育约30分钟
    5. 小心转移20μL混合物移液到24孔板培养细胞的每个井中。
      注意:在该测定中,10ng C99(或其变体)-TEV位点-rTA表达构建体,10ng C99(或其变体)-TEV蛋白酶构建体和5ng phRG-tk Renilla归一化萤光素酶表达载体分别是与40ng pBSK质粒对照一起转染到PS1 / PS2缺失的HTL细胞中。
    6. 将培养细胞在补充有10%(v / v)胎牛血清的DMEM中在37℃,5%CO 2的潮湿空气中培养。

  3. 萤光素酶测量
    1. 转染后一天,通过真空泵将培养细胞中的培养基从管道上连接到液体收集器和凝胶装载移液器尖端,并轻轻地施加足够的体积(500μl/孔)的PBS(见食谱)冲洗每口井的底部。
    2. 将100μl的1x PLB(参见食谱)分配到每个孔中,并轻轻地将培养板在室温下摇动15分钟(,例如<70rpm)。
      注意:我们使用Eppendorf研究 Pro电子单通道移液器(20-1,000μl)分配PLB缓冲液。但是,任何可以设置为100μl的实验室移液器都可以使用。
    3. 将20μlPLB裂解液转移至96孔OptiPlate的每个孔中,不含细胞碎片。
      注意:一般来说,在进行测定之前不需要清除残余细胞碎片的裂解物。
    4. 对EnVision Multilabel Plate Reader进行编程,以执行2秒的预先测量延迟,然后使用Envision 96 Plate US发光(700 nm发射滤波器)孔径进行10秒的测量,以进行每个报告分析。
    5. 向96孔OptiPlate的每个孔中分配50μlLAR2底物(参见配方),通过移液2次或3次混合并使用EnVision Multilabel Plate Reader测量萤火虫萤光素酶活性。发光读取代表萤火虫萤光素酶活性。
    6. 分配50μl停止&amp; Glo试剂(参见配方)放入同一平板,通过移液2次或3次混合并通过EnVision Multilabel Plate Reader测量海肾荧光素酶活性。发光读取代表海肾荧光素酶活性。
    7. “”src

数据分析

每个数据点表示至少三次独立实验的平均值(表1),计算出的活动如表2所示。数据以分组条形图类型呈现。数据的统计分析可以用GraphPad Prism使用双尾Student's测试与对照(图2)完成。
这个Tango相互作用测定的适用性的一个实际例子最近被显示在(Yan等人,2017年)。

表1.原始的萤火虫和海肾活动读


表2.相对活动和正常化活动

注意:对于活动标准化,每个相对活动除以WT(C99-T4L-rTA)的相对活动的平均值和100倍。


图2.通过Tango测定验证C99二聚化

笔记

细胞的状态是非常重要的。进行数据分析时,注意海肾荧光素酶信号(表3)。它应该是大约100万张照片。

表3.不良细胞状态的例子

这些孔(ctrl1组3孔和ctrl2组3孔应为阴性对照,低Renilla活性,这在一定程度上反映了细胞状态和转染效率,使得相对活性远高于正常水平。

食谱

  1. 10倍磷酸盐缓冲盐水(PBS缓冲液)(1升)
    11.5克Na 2 HPO 4 4 2克KH 2 PO 4 4克/克 80克NaCl
    2克KCl
    溶于1升无菌去离子水中 1x PBS的pH应该是7.4
  2. 1x被动裂解缓冲液(PLB)
    从双荧光素酶报告基因检测试剂盒中加入1倍体积的5x PLB到4倍体积的蒸馏水中并充分混合

    1x PLB可以储存在4°C以下,不超过一个月
  3. LAR2底物(来自双荧光素酶报告基因检测试剂盒)
    在萤光素酶测定缓冲液2(每瓶10ml)中重悬冻干的荧光素酶测定底物,-20°C保存少于1个月或-70°C少于1年。
  4. 停止&amp; Glo试剂(来自双荧光素酶报告基因检测试剂盒)
    新增1个音量停止&amp; Glo ® Substrate to 49 volume Stop&amp; Glo ®缓冲液和涡旋10秒

致谢

国家自然科学基金面上项目(31300607,31300245和91217311),科技部2012ZX09301001,2012CB910403,2013CB910600,XDB08020303,2013ZX09507001,上海市科委(13ZR1447600),国家自然科学基金),上海新星计划(14QA1404300),国家卫生研究院赠款DK071662(HEX),GM102545和GM104212(KM)。作者宣称没有利益冲突。

参考

  1. Barnea,G.,Strapps,W.,Herrada,G.,Berman,Y.,Ong,J.,Kloss,B.,Axel,R.and Lee,K.J。(2008)。 信号级联的遗传设计来记录受体激活 Proc Natl Acad Sci USA 105(1):64-69。
  2. Xu,T.H.,Yan,Y.,Kang,Y.,Jiang,Y.,Melcher,K。和Xu,H.E。(2016)。 阿尔茨海默氏病相关突变增加淀粉样蛋白前体蛋白对γ-分泌酶切割的抗性,而Aβ42/Aβ40比率。 Cell Discov 2:16026.
  3. Yan,Y.,Xu,T.H.,Harikumar,K.G.,Miller,L.J.,Melcher,K.and Xu,H.E。(2017)。 淀粉样蛋白前体蛋白的跨膜结构域的二聚化由γ-分泌酶切割位点周围的残基决定。 J Biol Chem 。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yan, Y., Xu, T., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). Detection of Membrane Protein Interactions by Cell-based Tango Assays. Bio-protocol 7(22): e2903. DOI: 10.21769/BioProtoc.2903.
  2. Yan, Y., Xu, T. H., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017a). Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the gamma-secretase cleavage sites. J Biol Chem, 292: 15826-15837.
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