Generation and Use of Chimeric RIP Kinase Molecules to Study Necroptosis.
Generation and Use of Chimeric RIP Kinase Molecules to Study Necroptosis.
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DOI：
10.1007/978-1-4939-8754-2_7
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发表时间：
2018
影响因子：
--
通讯作者：
D. Rodriguez;D. Green
中科院分区：
文献类型：
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作者：
D. Rodriguez;D. Green
坏死性凋亡是一种调节性坏死，由多种信号触发，这些信号汇聚以激活受体相互作用蛋白激酶-3（RIPK 3），从而促进混合谱系激酶样（MLKL）蛋白的直接磷酸化和激活。活性MLKL通过破坏质膜的完整性来执行坏死性凋亡。可以诱导坏死性凋亡的刺激包括死亡受体（TNFR家族的子集）、toll样受体（特别是TLR 3和TLR 4）、干扰素和细胞内病毒传感器DAI/ZBP 1等的连接。为了更详细地研究该过程，具有直接激活RIPK 3的方法是有用的。在这里，我们提供了通过药物诱导的RIPK 3的强制二聚化来人工诱导坏死性细胞死亡的方案和程序。我们还提供有关特定激酶抑制剂的信息，监测RIPK 3和MLKL激活的程序，以及细胞死亡的实时定量。
Necroptosis, a form of regulated necrosis, is triggered by a variety of signals that converge to activate receptor interacting protein kinase-3 (RIPK3), consequently promoting the direct phosphorylation and activation of the mixed lineage kinase like (MLKL) protein. Active MLKL executes necroptosis by disrupting the integrity of the plasma membrane. Stimuli that can induce necroptosis include ligation of death receptors (a subset of the TNFR family), toll-like receptors (in particular, TLR3 and TLR4), interferons, and the intracellular viral sensor, DAI/ZBP1, among others. To study the process in more detail, it is useful to have a means to directly activate RIPK3. Here we provide protocols and procedures to artificially induce necroptotic cell death by drug-induced forced dimerization of RIPK3. We also provide information on specific kinase inhibitors, procedures to monitor RIPK3 and MLKL activation, and real-time quantification of cell death.