人细胞中HSV-1感染后RIPK3和MLKL的ZBP1依赖性磷酸化的免疫荧光荧光放大的酪胺信号扩增
Summary
免疫荧光染色期间的酪胺信号扩增能够在 HSV-1 感染后 ZBP1 诱导的坏死性凋亡期间灵敏地检测磷酸化的 RIPK3 和 MLKL。
Abstract
激酶受体相互作用丝氨酸/苏氨酸蛋白激酶3（RIPK3）及其底物混合谱系激酶结构域样（MLKL）是坏死性凋亡的关键调节因子，坏死性凋亡是一种具有重要抗病毒功能的细胞死亡的炎症形式。RIPK3的自磷酸化诱导坏死性凋亡MLKL的孔形成刽子蛋白的磷酸化和活化。细胞膜上磷酸化MLKL的运输和寡聚化导致细胞裂解，这是坏死性凋亡细胞死亡的特征。核酸传感器ZBP1在感染RNA和DNA病毒后通过与左旋Z型双链RNA（Z-RNA）结合来激活。ZBP1活化通过诱导受感染宿主细胞的调节细胞死亡（包括坏死性凋亡）来限制病毒感染。免疫荧光显微镜允许在每个细胞的基础上可视化ZBP1介导的坏死性凋亡下游的不同信号传导步骤。然而，使用目前市售的针对人RIPK3和MLKL的磷酸化特异性抗体的标准荧光显微镜的灵敏度排除了这些标志物的可重复成像。在这里，我们描述了感染单纯疱疹病毒1（HSV-1）的人HT-29细胞中丝氨酸（S）磷酸化RIPK3（S227）和MLKL（S358）的优化染色程序。在免疫荧光染色方案中加入酪胺信号放大（TSA）步骤可以特异性检测S227磷酸化的RIPK3。此外，TSA大大提高了S358磷酸化MLKL检测的灵敏度。总之，该方法能够在诱导ZBP1诱导的坏死性凋亡期间可视化这两个关键信号事件。
Introduction
受体相互作用的丝氨酸/苏氨酸蛋白激酶 3 （RIPK3） 和混合谱系激酶结构域样 （MLKL） 是坏死性凋亡细胞死亡的中枢调节因子1，2。坏死性凋亡是一种溶解性和炎症性调节性细胞死亡形式，涉及抗病毒免疫和自身炎症。病毒感染细胞的坏死性凋亡会立即关闭病毒复制。坏死性凋亡诱导后的细胞裂解也会释放与损伤相关的分子模式，从而刺激抗病毒免疫3，4。坏死性凋亡是由 RIP 同型相互作用基序 （RHIM） 介导的与三种上游活化分子之一的相互作用后激活 RIPK3 引发的：RIPK1（TNF 受体 1 [TNFR1] 接合时）、含 TIR 结构域的适配器诱导干扰素β（TRIF;在 Toll 样受体 3 和 4 接合时）或抗病毒核酸传感器 Z-DNA 结合蛋白 1 （ZBP1）1，2.坏死性凋亡信号传导通过一系列磷酸化事件进行，从RIPK3的自磷酸化开始。人RIPK3在其激酶结构域内的丝氨酸（S）227处的自磷酸化是通过与MLKL相互作用来使坏死性凋亡的先决条件，并且通常用作人RIPK3活化和坏死性凋亡细胞死亡的生化标志物1，5。一旦激活，RIPK3在苏氨酸（T）357和S3581处磷酸化MLKL的活化环。这会导致MLKL构象发生变化，导致N末端四螺旋束结构域暴露。然后MLKL寡聚并运输到细胞膜，在那里它通过在脂质双层中插入暴露的四个螺旋束形成孔，最终导致细胞死亡2，6。
ZBP1 是一种抗病毒核酸传感器，可识别左旋 Z 型核酸，包括 Z 构象 （Z-RNA） 中的双链 RNA。Z-RNA结合通过位于ZBP1的N末端的两个Zα结构域发生。在RNA和DNA病毒感染过程中积累的Z-RNA被认为直接参与ZBP17，8。活化的ZBP1通过其中枢RIM募集RIPK3并诱导调节细胞死亡，包括坏死性凋亡9，10。病毒已经采用了许多逃逸机制来抵消ZBP1诱导的宿主细胞坏死性凋亡11。例如，单纯疱疹病毒 1 （HSV-1） 核糖核苷酸还原酶亚基 1，称为 ICP6 并由 UL39 编码，在其 N 末端含有 RHIM，干扰人细胞中 ZBP1 介导的 RIPK3 活化12，13，14，15。ZBP1不仅限制病毒复制，而且小鼠研究表明，ZBP1激活引起炎症性疾病并刺激癌症免疫16，17，18，19，20，21。因此，检测ZBP1诱导的人类细胞坏死性凋亡期间发生的信号事件的协议对于评估ZBP1在这些过程中的作用很有价值。
酪胺信号放大（TSA），也称为催化报告沉积（CARD），已被开发用于提高基于抗体的免疫测定中的检测限和信噪比。在TSA期间，任何一抗都可用于检测目标抗原。辣根过氧化物酶（HRP）与二抗偶联，在过氧化氢存在下催化生物素化酪胺自由基的局部积聚。这些活化的生物素-酪胺自由基然后与近端酪氨酸残基反应形成共价键。潜在的酪胺-生物素底物包括抗原本身、一抗和二抗以及邻近的蛋白质。因此，虽然TSA显着提高了测定的灵敏度，但其一些空间分辨率会丢失。在最后一步中，使用荧光标记的链霉亲和素检测生物素分子。HRP反应在目标抗原上或附近沉积了许多酪酰胺-生物素分子。这大大增加了链霉亲和素-荧光染料结合位点的数量，从而大大增强了测定的灵敏度（图1）。或者，酪胺可以直接与荧光染料偶联，无需链霉亲和素偶联的荧光团。蛋白质免疫组织化学和DNA/RNA 原位 杂交是最早采用TSA提高信号强度的方法之一22，23。最近，TSA已与细胞内流式细胞术24 和质谱术25相结合。
在这里，我们提出了一种方案，用于检测丝氨酸227磷酸化人RIPK3（p-RIPK3 [S227]）和磷酸化人MLKL（p-MLKL [S358]）在HSV-1感染激活ZBP1时使用免疫荧光显微镜。我们使用坏死性凋亡敏感的HT-29人结直肠腺癌细胞系，该细胞系被转导以稳定表达人ZBP1。这些细胞感染了表达突变ICP6蛋白（HSV-1 ICP6mutRHIM）的HSV-1菌株，其中病毒RHIM（VQCG）内的四个核心氨基酸被丙氨酸（AAAA）取代，从而使ICP6无法阻断ZBP1介导的坏死性凋亡13，14，15。为了克服目前市售的针对p-RIPK3和p-MLKL的抗体在免疫染色26中的低信噪比问题，我们执行了酪胺信号放大（TSA）步骤（图1），该步骤可对人p-RIPK3（S227）进行稳健检测，并将人p-MLKL（S358）的检测灵敏度提高一个数量级。
Protocol
Representative Results
Discussion
该免疫荧光染色方案描述了使用酪胺信号扩增（TSA）来提高难以检测的人类坏死性凋亡信号通路信号事件的灵敏度，包括RIPK3和MLKL26的磷酸化。TSA步骤的加入显著提高了p-RIPK3（S227）和p-MLKL（S358）的检测阈值，并提高了p-MLKL（S358）应变的灵敏度。TSA显示模拟处理的样品中已经存在p-RIPK3（S227）信号。在人细胞中，RIPK3在S227处的自磷酸化是通过与MLKL稳定相互作用来激活坏死性凋亡?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们要感谢VIB生物成像核心的培训，支持和进入仪器园区。J.N.得到了法兰德斯研究基金会（FWO）的博士奖学金支持。J.M.小组的研究得到了奥德修斯II资助（G0H8618N），EOS INFLADIS（40007512），法兰德斯研究基金会（FWO）的初级研究资助（G031022N），CRIG青年研究者概念验证资助和根特大学的支持。P.V.组的研究得到了EOS MODEL-IDI（30826052），EOS INFLADIS（40007512），FWO高级研究资助（G.0C76.18N，G.0B71.18N，G.0B96.20N，G.0A9322N），Methusalem（BOF16 / MET_V / 007），iBOF20 / IBF / 039 ATLANTIS，抗癌基金会（F / 2016 / 865，F / 2020 / 1505），CRIG和GIGG联盟以及VIB的支持。
Materials
| Antibodies | |||
| Anti-rabbit HRP | Agilent Technologies Belgium | K4002 | Envision+ System-HRP Labelled Polymer anti-rabbit |
| Goat anti-mouse DyLight 633 | Thermofisher | 35513 | Secundary antibody |
| HSV-1 ICP0 | Santa Cruz | sc-53070 | Mouse anti-ICP0(HSV-1) antibody |
| IAV-PR8 mouse serum | In house production | xx | Mouse anti-IAV-PR8 polyclonal antibody |
| pMLKL | Abcam | ab187091 | Rabbit anti-MLKL-phospho S358 antibody |
| pRIPK3 | Abcam | ab209384 | Rabbit anti-RIPK3-phospho S227 antibody |
| Fluorophores | |||
| DAPI | Thermofisher | D21490 | To visualise the nucleus of the cells |
| Streptavidin coupled to Alexa Fluor 568 | Thermofisher | S11226 | To visulalise biotin molecules |
| Compounds | |||
| 30% H2O2 | Sigma | H1009 | Oxidising substrate, necessary for HRP activity |
| 4% PFA | SANBIO | AR1068 | To fix/crosslink the cells |
| Biotinyl-tyramide | R&D Systems | 6241 | To amplify signal, HRP substrate |
| BV-6 | Selleckchem | S7597 | BV6 IAP Inhibitor |
| For cell culture: to detach the cells | |||
| 8.0 g/L NaCl | |||
| 0.4 g/L disodium salt of EDTA | |||
| EDTA 0.04% | In house formulation | 1.1 g/L Na2HPO4 | |
| 0.2 g/L NaH2PO4 | |||
| 0.2 g/L KCl | |||
| 0.2 g/L Glucose | |||
| Fetal Bovine serum | TICO | FBS EU XXX | For cell culture, maintaining cell culture; lot number: 90439 |
| GSK'840 | Aobious | AOB0917 | RIPK3 kinase inhibitor |
| L-Glutamine | Sigma-Aldrich | G7513 | For cell culture, maintaining cell culture |
| MAXblock | Active Motif | 15252 | Blocking solution |
| PBS | Gibco | 10444402 | |
| Sodium pyruvate | Sigma-Aldrich | S8636 | For cell culture, maintaining cell culture |
| TNF-α | In house production | – | Signaling molecule, able to trigger cell death in combination with BV6 and zVAD |
| Triton X-100 | Sigma Aldrich | T8787-50ML | To permeabilise the cells |
| Trypan blue | Merck | 11732 | For cell counting, used as live/dead marker at 0,1% |
| Trypsine | Sigma-Aldrich | T4424 | For cell culture: to detach the cells |
| zVAD | Bachem | BACE4026865.0005 | Z-Val-Ala-DL-Asp-fluoromethylketone |
| Material | |||
| µ-Slide 8 well high glass bottom | iBidi | 80807 | To culture the cells |
| Cotton Preping Balls-size medium | Electron Microscopy Sciences | 71001-10 | To clean the objectives |
| Immersol 518 F / 30 °C | ZEISS | 444970-9000-000 | To visualise the sample at high magnifications |
| Lens Cleaner | ZEISS | 000000-0105-200 | To clean the objectives |
| LSM880 Fast Airyscan confocal microscope | To visualise the sample | ||
| Software | |||
| Excel | Office | xx | To process the data |
| Prism 9 | Graphpad | xx | To analyse the data- statistical testing and graph generation |
| Volocity 6.3 | Volocity | xx | To perform quantifications |
| Zen black | ZEISS | xx | To aquire and process images |
| Zen blue | ZEISS | xx | To visualise images |
| Viruses | |||
| HSV-1 (mutRHIM) F strain | produced by Dr. Jiahuai Han | in house replication | HSV-1 as a trigger for necroptosis; RHIM core domain of UL39/ICP6 is mutated (VQCG>AAAA) |
| HSV-1 (WT) F strain | Produced by Dr. Jiahuai Han | in house replication | HSV-1 (WT) as a negative control for necroptosis induction (ICP6 inhibition) |
| IAV PR8 | in house stock | in house replication | IAV as a trigger for necroptosis |
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