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γ-Secretase Epsilon-cleavage Assay
γ-分泌酶ε-裂解分析   

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Abstract

γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO operator element, in which C99 C terminally fused to a reversed tetracyclin-inducible activator (rTA) transcriptional activator is expressed. Endogenous or transfected γ-secretase cleaves a C terminally fused rTA transcriptional activator from C99, allowing rTA to move to the nucleus to activate a luciferase reporter gene as a measurement for γ-secretase cleavage activity.

Keywords: γ-Secretase(γ-分泌酶), Cleavage(裂解), Activity(活性), Epsilon-cleavage assay(ε-裂解分析), C99(C99)

Background

Alzheimer’s disease (AD) is the most prevalent chronic neurodegenerative disease. AD is closely associated with the formation of amyloid plaques. These plaques mainly consist of aggregated amyloid β, which is generated by the cleavage of the C terminal 99 amino acids fragment (C99) of the amyloid precursor protein by γ-secretase. In spite of extensive efforts, it remains unknown how γ-secretase recognizes its substrates. The conventional Tango assay was designed to monitor the activation of GPCRs (Barnea et al., 2008) by engineering a TEV cleavage site and a transcription activator to the cytoplasmic C terminus to of GPCRs and a TEV protease linked to human β-arrestin2. Here we used endogenous or transfected γ-secretase to cleave the transmembrane portion of its substrates, i.e., the C terminus of C99 is fused with rTA to establish the γ-secretase Epsilon-cleavage assay (Figure 1). The assay was first established to investigate the initial cleavage of C99 by γ-secretase (Xu et al., 2016).


Figure 1. The principle of the γ-secretase epsilon-cleavage assay. Upon cleavage of the C99 (or other γ-secretase substrates) hybrid protein by γ-secretase, the rTA is released from the membrane and enters nucleus to bind tetO DNA-binding site to stimulate luciferase reporter gene activity as measurement for total cleavage, both by endogenous and by transfected γ-secretase variants. (Xu et al., 2016)

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 a 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. Loading pipette tips
  5. Cell culture flask (Corning, catalog number: 430639 )
  6. 96-well OptiPlate (PerkinElmer, catalog number: 6005290 )
  7. HTL cells (a gift from G Barnea and R Axel, Brown University and Columbia University, resp.)
  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. X-tremeGENE 9 Reagent (Roche Diagnostics, catalog number: 06365787001 )
  13. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S374-1 )
  14. Potassium phosphate monobasic (KH2PO4) (EMD Millipore, catalog number: PX1565-1 )
  15. Sodium chloride (NaCl) (EMD Millipore, catalog number: SX0420-5 )
  16. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-500 )
  17. Dual-luciferase reporter assay kit (Promega, catalog number: E1960 )
  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. Eppendorf® Research® Pro electronic single channel pipette (20-1,000 µl)
  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. Grow HTL cells in DMEM supplemented with 10% (v/v) FBS at 37 °C under a humidified 5% CO2 atmosphere to about 80% confluence.
    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 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 number of samples: 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 plasmid to the concentration of 100 ng/µl and store them at 4 °C.
    3. Add 10 µl Master Mix to DNA mix per well 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 24-well plate.
      Note: In this particular case, for coexpression with γ-secretase, 20 ng substrate-encoding DNA, 5 ng phRG-tk Renilla normalization standard and 10 ng of each of the γ-secretase subunit expression plasmids were transfected. For the substrate-only control, 20 ng substrate, 5 ng phRG-tk Renilla and 40 ng pBSK mock plasmid were transfected. As a positive control, 10 ng Rho(4M)-TEV-site-rTA, 10 ng Arr(3A)-TEV, 5 ng phRG-tk Renilla and 40 ng pBSK mock plasmid were co-transfected (Kang et al., 2015).
    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. After one day growth, remove the medium from the cultured cells by vacuum pump using loading pipette tips, 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 pipettes which can reach 100 µl should be fine.
    3. Transfer 20 µl PLB lysate without cell debris into 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 96 Plate US Luminescence aperture (700 nm emission filter) for each reporter assay.
    5. Dispense 50 µl LAR2 substrate (see Recipes), 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 freshly prepared Stop & Glo® Reagent (see Recipes) in 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.

    7. Note: Normalize the relative activity using WT activity as 100.

Data analysis

Each experiment is performed at least in triplicate (Table1), 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 current γ-secretase Epsilon-cleavage assay was recently shown in (Yan et al., 2017a and 2017b).

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. Relative reporter gene activity using C99-T4L-rTA. Error bars = SEM, n = 3, P-values (two-tailed Student’s t-test): *P < 0.05; **P < 0.01; ***P < 0.001).

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


Three wells of Group ctrl1 and three wells of Group ctrl2 should be the negative controls, 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)
    Add 1 volume Stop & Glo® Substrate to 49 volumes Stop & Glo® Buffer and vortex for 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. F. (2008). The genetic design of signaling cascades to record receptor activation. Proc the Natl Acad Sci U S A 105(1): 64-69
  2. Kang, Y., Zhou, X. E., Gao, X., He, Y., Liu, W., Ishchenko, A., Barty, A., White, T. A., Yefanov, O., Han, G. W., Xu, Q., de Waal, P. W., Ke, J., Tan, M. H., Zhang, C., Moeller, A., West, G. M., Pascal, B. D., Van Eps, N., Caro, L. N., Vishnivetskiy, S. A., Lee, R. J., Suino-Powell, K. M., Gu, X., Pal, K., Ma, J., Zhi, X., Boutet, S., Williams, G. J., Messerschmidt, M., Gati, C., Zatsepin, N. A., Wang, D., James, D., Basu, S., Roy-Chowdhury, S., Conrad, C. E., Coe, J., Liu, H., Lisova, S., Kupitz, C., Grotjohann, I., Fromme, R., Jiang, Y., Tan, M., Yang, H., Li, J., Wang, M., Zheng, Z., Li, D., Howe, N., Zhao, Y., Standfuss, J., Diederichs, K., Dong, Y., Potter, C. S., Carragher, B., Caffrey, M., Jiang, H., Chapman, H. N., Spence, J. C., Fromme, P., Weierstall, U., Ernst, O. P., Katritch, V., Gurevich, V. V., Griffin, P. R., Hubbell, W. L., Stevens, R. C., Cherezov, V., Melcher, K. and Xu, H. E. (2015). Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523(7562): 561-567.
  3. 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.
  4. 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.
  5. Yan, Y., Xu, T. H., Melcher, K. and Xu, H. E. (2017b). Defining the minimum substrate and charge recognition model of gamma-secretase. Acta Pharmacol Sin.

简介

γ-分泌酶ε-切割测定来源于基于细胞的Tango测定法(Kang等人,2015),并且是确定γ-分泌酶对C99的初始切割的快速且灵敏的方法。 在该协议中,我们使用HTL细胞,其是在细菌tetO操纵元件的控制下具有稳定整合的萤光素酶报道基因的HEK293细胞,其中C99C末端融合至反向四环素诱导型活化剂(rTA)转录激活剂。 内源性或转染的γ-分泌酶从C99切割C末端融合的rTA转录激活物,允许rTA移动到核以激活萤光素酶报道基因作为γ-分泌酶切割活性的测量。

【背景】阿尔茨海默病(AD)是最普遍的慢性神经退行性疾病。 AD与淀粉样斑块的形成密切相关。这些斑块主要由聚集的β淀粉样蛋白组成,β淀粉样蛋白是由γ-分泌酶裂解淀粉样蛋白前体蛋白的C端99个氨基酸片段(C99)产生的。尽管进行了广泛的努力,γ-分泌酶如何识别其底物尚不清楚。通过将TEV切割位点和转录激活子工程化至GPCR的胞质C末端并连接TEV蛋白酶,设计常规Tango测定以监测GPCR的活化(Barnea等人,2008)到人β-arrestin2。在这里,我们使用内源性或转染的γ-分泌酶切割其底物的跨膜部分,即C99的C末端与rTA融合以建立γ-分泌酶Epsilon切割测定(图1) 。首先建立该测定法以研究γ-分泌酶对C99的初始裂解(Xu等人,2016)。

“”src
图1.γ-分泌酶ε-裂解测定的原理当用γ-分泌酶裂解C99(或其他γ-分泌酶底物)杂合蛋白质时,rTA从膜上释放,进入细胞核以结合tetO DNA结合位点以刺激萤光素酶报道基因活性,作为内切和由转染的γ-分泌酶变体测量总切割。 (Xu等人,2016年)

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:γ-分泌酶, 裂解, 活性, ε-裂解分析, 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. 加载移液器吸头
  5. 细胞培养瓶(康宁,目录号:430639)
  6. 96孔OptiPlate(PerkinElmer,目录号:6005290)
  7. HTL细胞(来自G Barnea和R Axel,布朗大学和哥伦比亚大学的一份礼物)
  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. X-tremeGENE 9试剂(Roche Diagnostics,目录号:06365787001)
  13. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S374-1)
  14. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:PX1565-1)
  15. 氯化钠(NaCl)(EMD Millipore,目录号:SX0420-5)
  16. 氯化钾(KCl)(Fisher Scientific,目录号:BP366-500)
  17. 双荧光素酶报告基因检测试剂盒(普洛麦格,目录号:E1960)
  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&amp; Glo ®试剂(见食谱)

设备

  1. 微量移液器(Eppendorf)
  2. Eppendorf®Research™Pro电子单道移液器(20-1,000μl)
  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. 在补充有10%(v / v)FBS的DMEM中,在37℃,潮湿的5%CO 2气氛下至约80%汇合培养HTL细胞。
    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1天前以每孔50,000个分裂到24孔板中转染。

  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. 做大量样品的主混合物:例如,对于10个样品,乘以11来准备一些额外的混合物。 (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。
      注意:我们通常将DNA质粒稀释到100 ng /μl的浓度,并将它们保存在4°C。

    3. 每孔加入10μlMaster Mix至DNA混合物,轻轻混匀

    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:在这个特例中,为了与γ-分泌酶共表达,转染20ng底物编码DNA,5ng phRG-tk Renilla标准化标准品和10ng每种γ-分泌酶亚基表达质粒。对于仅底物对照,转染20ng底物,5ng phRG-tk海肾和40ng pBSK模拟质粒。作为阳性对照,将10ng Rho(4M)-TEV-site-rTA,10ng Arr(3A)-TEV,5ng phRG-tk Renilla和40ng pBSK模拟质粒共转染(Kang等,2015 )。
    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. 培养一天后,使用加样枪头,通过真空泵从培养细胞中取出培养基,轻轻地加入足够量的PBS(500μl/孔)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,任何可以达到100μl的实验室移液器都应该没问题。
    3. 将20μl无细胞碎片的PLB裂解液转移到96孔OptiPlate的每个孔中。
      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96板美国发光孔径(700 nm发射滤光片)进行10秒的测量,用于每个记者测定。
    5. 分配50μLLAR2底物(见食谱),通过移液2次或3次混合,并使用EnVision Multilabel Plate Reader来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新鲜制备的Stop&amp; Glo试剂(参见配方)放在同一平板上,用移液器混匀2或3次,用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
      注意:将WT活动的相对活动标准化为100。

数据分析

每个实验至少进行三次(表1),计算的活动如表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。最近在(Yan等人,2017a和2017b)中显示了当前γ-分泌酶Epsilon-裂解测定的实际例子。

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.使用C99-T4L-rTA的相对报道基因活性误差棒= SEM,n = 3,P-value(双尾学生 / em> -test):* P &lt; 0.05; ** P 0.01; *** P &lt; 0.001)。

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.不良细胞状态示例


ctrl1组的3个孔和ctrl2组的3个孔应为阴性对照,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)
    添加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.F。(2008)。 信号级联的遗传设计来记录受体激活。 Proc the Natl美国科学院院士105(1):64-69
  2. Kang,Y.,Zhou,XE,Gao,X.,He,Y.,Liu,W.,Ishchenko,A.,Barty,A.,White,TA,Yefanov,O.,Han,GW,Xu,Q ,Wa Wa,PW,Ke,J.,Tan,MH,Zhang,C.,Moeller,A.,West,GM,Pascal,BD,Van Eps,N.,Caro,LN,Vishnivetskiy,SA,Lee, RJ,Suino-Powell,KM,Gu,X.,Pal,K.,Ma,J.,Zhi,X.,Boutet,S.,Williams,GJ,Messerschmidt,M.,Gati,C.,Zatsepin,NA ,Wang,D.,James,D.,Basu,S.,Roy-Chowdhury,S.,Conrad,CE,Coe,J.,Liu,H.,Lisova,S.,Kupitz,C.,Grotjohann,I Fromme,R.,Jiang,Y.,Tan,M.,Yang,H.,Li,J.,Wang,M.,Zheng,Z.,Li,D.,Howe,N.,Zhao,Y 。,Standfuss,J.,Diederichs,K.,Dong,Y.,Potter,CS,Carragher,B.,Caffrey,M.,Jiang,H.,Chapman,HN,Spence,JC,Fromme,P.,Weierstall (Ernst,OP,Katritch,V.,Gurevich,VV,Griffin,PR,Hubbell,WL,Stevens,RC,Cherezov,V.,Melcher,K.和Xu,HE(2015))。 用飞秒X射线激光结合视紫红质的视紫红质晶体结构 自然 523(7562):561-567。
  3. 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.
  4. Yan,Y.,Xu,T.H.,Harikumar,K.G.,Miller,L.J.,Melcher,K。和Xu,H.E。(2017a)。 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 。
  5. Yan,Y.,Xu,T. H.,Melcher,K.和Xu,H. E.(2017b)。 定义γ-分泌酶的最小底物和电荷识别模型 学术报告Pharmacol Sin 。
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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. Xu, T., Yan, Y., Harikumar, K. G., Miller, L. J., Melcher, K. and Xu, H. E. (2017). γ-Secretase Epsilon-cleavage Assay. Bio-protocol 7(22): e2900. DOI: 10.21769/BioProtoc.2900.
  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.
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