Thapa Roshan J, Nogusa Shoko, Balachandran Siddharth
Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA, USA.
Methods Mol Biol. 2018;1857:93-99. doi: 10.1007/978-1-4939-8754-2_9.
In multicellular organisms, regulated cell death plays a vital role in development, adult tissue homeostasis, and clearance of damaged or infected cells. Necroptosis is one such form of regulated cell death, characterized by its reliance on receptor-interacting protein kinase 3 (RIPK3). Once activated, RIPK3 nucleates a protein complex, termed the "necrosome," which includes the adaptors RIPK1 and FADD, and the effector protein MLKL. From the necrosome, RIPK3 phosphorylates MLKL to drive necroptosis, and can also induce RIPK1/FADD-mediated apoptosis, via caspase-8. Assembly of the necrosome thus serves as a useful readout of RIPK3 activation. In this chapter, we describe molecular methods for examining necrosome activation in response to the cytokines TNF-α, IFN-β, and IFN-γ, and upon infection with influenza A virus (IAV).
在多细胞生物体中，程序性细胞死亡在发育、成体组织稳态以及受损或感染细胞的清除过程中发挥着至关重要的作用。坏死性凋亡就是程序性细胞死亡的一种形式，其特征在于依赖受体相互作用蛋白激酶3（RIPK3）。一旦被激活，RIPK3会形成一种名为“坏死小体”的蛋白质复合物，该复合物包括衔接蛋白RIPK1和FADD以及效应蛋白MLKL。在坏死小体中，RIPK3会磷酸化MLKL以驱动坏死性凋亡，并且还可通过半胱天冬酶-8诱导RIPK1/FADD介导的凋亡。因此，坏死小体的组装可作为RIPK3激活的有效指标。在本章中，我们描述了用于检测坏死小体激活的分子方法，这些激活是对细胞因子肿瘤坏死因子-α（TNF-α）、干扰素-β（IFN-β）和干扰素-γ（IFN-γ）的反应，以及甲型流感病毒（IAV）感染后的反应。
