测量CD95死亡诱导信号传导复合物的组成和该复合物中Procaspase-8的处理
Summary
在这里，提出了一种实验工作流程，可以直接在死亡诱导信号复合物（DISC）上检测caspase-8处理并确定该复合物的组成。这种方法具有广泛的应用，从解开细胞死亡途径的分子机制到细胞凋亡网络的动态建模。
Abstract
外源性细胞凋亡由死亡受体（DR）的激活介导，例如CD95 / Fas / APO-1或肿瘤坏死因子相关的凋亡诱导配体（TRAIL）受体1 /受体2（TRAIL-R1 / R2）。这些受体及其同源配体的刺激导致死亡诱导信号复合物（DISC）的组装。DISC包括DR，具有死亡结构域的适应蛋白Fas相关蛋白（FADD），procaspases-8/-10和细胞FAD样白细胞介素（IL）-1β-转化酶抑制蛋白（c-FLIP）。DISC用作procaspase-8处理和激活的平台。后者 通过其 在DISC上组装的死亡效应域（DED）细丝中的二聚/寡聚化发生。
procaspase-8的激活之后是其处理，其分几个步骤进行。在这项工作中，描述了一个已建立的实验工作流程，该工作流程允许测量DISC形成和处理该复合物中的procaspase-8。该工作流程基于免疫沉淀技术，并辅以蛋白质印迹分析。该工作流程允许仔细监测PROCASPASE-8募集到DISC及其处理的不同步骤，并且与研究外源性凋亡的分子机制高度相关。
Introduction
研究最好的死亡受体（DRs）之一是CD95（Fas，APO-1）。外源性凋亡途径始于DR与其同源配体的相互作用，即CD95L与CD95相互作用或TRAIL与TRAIL-Rs结合。这导致在相应的DR处形成DISC，DISC由CD95，FADD，procaspase-8 / -10和c-FLIP蛋白1，2组成。此外，DISC是通过含死亡域（DD）的蛋白质（例如CD95和FADD）与含DED的蛋白质（例如FAD，procaspase-8 / -10和c-FLIP）之间的相互作用组装的（图1）。Procaspase-8通过其DED的结合经历寡聚化，导致DED细丝的形成，然后Procaspase-8的激活和处理。这触发了半胱天冬酶级联反应，导致细胞死亡（图1）3、4。因此，procaspase-8是由CD95或TRAIL-Rs介导的外源性凋亡途径的中枢引发子半胱天冬酶，在相应的大分子平台DISC上激活。
已知Procaspase-8的两种亚型，即procaspase-8a（p55）和-8b（p53），被招募到DISC5中。两种亚型均由两个DED组成。DED1和DED2位于procaspase-8a/b的N端部分，后跟催化p18和p10结构域。对procaspase-8 DEDs的详细冷冻电子显微镜（cryo-EM）分析揭示了Procaspase-8蛋白组装成称为DED细丝4，6的丝状结构。值得注意的是，线性procaspase-8链最初被认为参与二聚化，然后在DISC处进行procaspase-8激活。现在，已知这些链只是procaspase-8 DED细丝的子结构，后者由三个链组成三个螺旋3，4，6，7。
在DED灯丝处二聚化时，procaspase-8a / b的构象变化导致Procaspase-8活性中心的形成及其激活3，8。接下来是procaspase-8处理，其通过两种途径介导：第一种途径通过p43 / p41裂解产物的产生，第二种途径通过p30裂解产物的初始产生。p43 / p41途径由Asp374处procaspase-8a / b的裂解启动，导致p43 / p41和p12裂解产物（图2）。此外，这些片段在Asp384和Asp210/216处被自催化裂解，从而形成活性半胱天冬酶-8异四聚体p102/p1829，10，11。此外，研究表明，在p43 / p41处理途径的同时，procaspase-8a / b也在Asp216处被切割，这导致形成C端裂解产物p30，然后其蛋白水解至p10和p1810（图2）。
DED灯丝处的Procaspase-8a / b激活受到名为c-FLIPs12的蛋白质的严格调节。c-FLIP蛋白以三种亚型出现：c-FLIP Long（c-FLIPL），c-FLIP Short（c-FLIPS）和c-FLIP Raji（c-FLIP R）。所有三种亚型在其N端区域都包含两个DED。c-FLIPL还具有催化非活性的半胱天冬酶样结构域12、13。c-FLIP-c-FLIPS和c-FLIPR的短同种型都以抗凋亡的方式通过破坏DISC 6，14，15处的DED长丝形成而起作用。此外，c-FLIPL可以以浓度依赖性方式调节半胱天冬酶-8的活化。这可以导致促凋亡和抗凋亡作用16，17，18。通过形成催化活性的procaspase-8 / c-FLIPL异源二聚体，c-FLIPL导致procaspase-8活性中心的稳定及其活化。c-FLIPL的促凋亡或抗凋亡功能直接取决于其在DED细丝上的量以及随后组装的procaspase-8 / c-FLIPL异源二聚体19的量。DISC处的c-FLIPL浓度低或中等浓度导致DED长丝处产生足够量的procaspase-8 / c-FLIPL异源二聚体，这支持了Caspase-8的活化。相反，增加的c-FLIPL量直接导致其在DISC20处具有抗凋亡作用 。
综上所述，在DISC上procaspase-8a / b的激活和处理是一个高度监管的过程，涉及几个步骤。本文讨论了直接在DISC上测量procaspase-8处理以及分析该复合物的组成。这将使用CD95 DISC作为典型的DR复合物来呈现。
Protocol
Representative Results
Discussion
这种方法首先由Kischkel等人27 描述，并从那时起由几个小组成功开发。为了在该复合物中进行有效的DISC免疫沉淀和监测半胱天冬酶-8处理，必须考虑几个重要问题。
首先，在免疫沉淀期间必须遵循所有洗涤步骤。特别重要的是琼脂糖珠的最终洗涤步骤和蜂蜡珠的干燥。必须正确完成此操作以增加免疫沉淀的信噪比，从而允许在DISC上检测caspase-8募集和处理。?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢威廉·桑德基金会（2017.008.02），动态系统中心（CDS），由欧盟计划ERDF（欧洲区域发展基金）和DFG（LA 2386）资助，以支持我们的工作。我们感谢Karina Guttek支持我们的实验。我们感谢Dirk Reinhold教授（OvGU，Magdeburg）为我们提供了原代T细胞。
Materials
| 12.5% SDS gel | self made | for two separating gels: 3.28 mL distilled H2O 2.5 mL Tris; pH 8.8; 1.5 M 4.06 mL acrylamide 100 µL 10% SDS 100 µL 10% APS 7.5 µL TEMED for two collecting gels: 3.1 mL distilled H2O 1.25 mL Tris; pH 6.8; 1.5 M 0.5 mL acrylamide 50 µL 10% SDS 25 µL 10% APS 7.5 µL TEMED |
|
| 14.5 cm cell dishes | Greiner | 639160 | |
| acrylamide | Carl Roth | A124.1 | |
| anti-actin Ab | Sigma Aldrich | A2103 | dilution: 1:4000 in PBST + 1:100 NaN3 |
| anti-APO-1 Ab | provided in these experiments by Prof. P. Krammer or can be purchased by Enzo | ALX-805-038-C100 | used only for immunoprecipitation |
| anti-caspase-10 Ab | Biozol | MBL-M059-3 | dilution: 1:1000 in PBST + 1:100 NaN3 |
| anti-caspase-3 Ab | cell signaling | 9662 S | dilution: 1:2000 in PBST + 1:100 NaN3 |
| anti-caspase-8 Ab C15 | provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-804-242-C100 | dilution: 1:20 in PBST + 1:100 NaN3 |
| anti-CD95 Ab | Santa Cruz | sc-715 | dilution: 1:2000 in PBST + 1:100 NaN3 |
| anti-c-FLIP NF6 Ab | provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-804-961-0100 | dilution: 1:10 in PBST + 1:100 NaN3 |
| anti-FADD 1C4 Ab | provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ADI-AAM-212-E | dilution: 1:10 in PBST + 1:100 NaN3 |
| anti-PARP Ab | cell signaling | 9542 | dilution: 1:1000 in PBST + 1:100 NaN3 |
| APS | Carl Roth | 9592.3 | |
| β-mercaptoethanol | Carl Roth | 4227.2 | |
| Bradford solution Protein Assay Dye Reagent Concentrate 450ml |
Bio Rad | 500-0006 | used according to manufacturer's instructions |
| CD95L | provided in these experiments by Prof. P. Krammer or can be purchased by ENZO | ALX-522-020-C005 | |
| chemoluminescence detector Chem Doc XRS+ |
Bio Rad | ||
| cOmplete Protese Inhibitor Cocktail (PIC) | Sigma Aldrich | 11 836 145 001 | prepared according to manufacturer's instructions |
| DPBS (10x) w/o Ca, Mg | PAN Biotech | P04-53500 | dilution 1:10 with H2O, storage in the fridge |
| eletrophoresis buffer | self made | 10x electrophoresis buffer: 60.6 g Tris 288 g glycine 20 g SDS ad 2 L H2O 1:10 dilution before usage |
|
| glycine | Carl Roth | 3908.3 | |
| Goat Anti-Mouse IgG1 HRP | SouthernBiotech | 1070-05 | dilution 1:10.000 in PBST + 5% milk |
| Goat Anti-Mouse IgG2b | SouthernBiotech | 1090-05 | dilution 1:10.000 in PBST + 5% milk |
| Goat Anti-Rabbit IgG-HRP | SouthernBiotech | 4030-05 | dilution 1:10.000 in PBST + 5% milk |
| Interleukin-2 Human(hIL-2) | Merckgroup/ Roche | 11011456001 | for activation of T cells |
| KCl | Carl Roth | 6781.2 | |
| KH2PO4 | Carl Roth | 3904.1 | |
| loading buffer 4x Laemmli Sample Buffer,10 mL |
Bio Rad | 161-0747 | prepared according to manufacturer's instructions |
| Luminata Forte Western HRP substrate | Millipore | WBLUFO500 | |
| lysis buffer | self made | 13.3 mL Tris-HCl; pH 7.4; 1.5 M 27.5 mL NaCl; 5 M 10 mL EDTA; 2 mM 100 mL Triton X-100 add 960 mL H2O |
|
| medium for adhaerent cells DMEM F12 (1:1) w stable Glutamine, 2,438 g/L | PAN Biotech | P04-41154 | adding 10% FCS, 1% Penicillin-Streptomycin and 0.0001% Puromycin to the medium |
| medium for primary T cells | gibco by Life Technologie | 21875034 | adding 10% FCS and 1% Penicillin-Streptomycin to the medium |
| milk powder | Carl Roth | T145.4 | |
| Na2HPO4 | Carl Roth | P030.3 | |
| NaCl | Carl Roth | 3957.2 | |
| PBST | self made | 20x PBST: 230 g NaCl 8 g KCl 56.8 g Na2HPO4 8 g KH2PO4 20 mL Tween-20 ad 2 L H2O dilution 1:20 before usage |
|
| PBST + 5% milk | self made | 50 g milk powder + 1 L PBST | |
| PHA | Thermo Fisher Scientific | R30852801 | for actavation of T ells |
| Power Pac HC | Bio Rad | ||
| Precision Plus Protein Standard All Blue | Bio Rad | 161-0373 | use between 3-5 µL |
| Protein A Sepharose CL-4B beads | Novodirect/ Th.Geyer | GE 17-0780-01 | affinity resin beads prepared according to manufacturer's instructions |
| scraper | VWR | 734-2602 | |
| SDS | Carl Roth | 4360.2 | |
| shaker | Heidolph | ||
| sodium azide | Carl Roth | K305.1 | |
| TEMED | Carl Roth | 2367.3 | |
| Trans Blot Turbo mini-size transfer stacks | Bio Rad | 170-4270 | used according to manufacturer's instructions |
| TransBlot Turbo 5x Transfer Buffer | Bio Rad | 10026938 | prepared according to manufacturer's instructions |
| TransBlot Turbo Mini-size nictrocellulose membrane | Bio Rad | 170-4270 | used according to manufacturer's instructions |
| Trans-Blot-Turbo | Bio Rad | ||
| Tris | Chem Solute | 8,08,51,000 | |
| Triton X-100 | Carl Roth | 3051.4 | |
| Tween-20 | Pan Reac Appli Chem | A4974,1000 |
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