Characterization of the ripoptosome and its components: implications for anti-inflammatory and cancer therapy

Abstract

Most intracellular signaling cascades rely on the formation of multiprotein signaling complexes assembled in large protein signaling platforms. Especially in cell death signaling, there is a large variety of these complexes, including the apoptosome, the necrosome, or the death-inducing signaling complex (DISC), to name only a few. During the last years, a number of cellular conditions were identified that lead to the formation of another signaling platform, the so-called ripoptosome. Diverse stimuli such as genotoxic stress, death receptor or Toll-like-receptor (TLR) ligation, or degradation of cellular inhibitor of apoptosis proteins (cIAPs) are able to induce ripoptosome formation. The ripoptosome is tightly regulated by cIAPs that control intracellular RIP1 assembly and the association with other cell death-regulating proteins, most likely by ubiquitin linkage. The suppression of cIAP activity results in accumulation of RIP1 platforms that ultimately triggers necroptosis by activation of RIP3-MLKL-dependent necrosis signaling pathways. The ripoptosome is a 2-MDa protein complex, which consists of the core components caspase-8, FADD, different cFLIP isoforms, and RIP1. It represents one of the rheostats in cell death signaling, as it can activate apoptotic and necroptotic cell death responses. The specific formation and activation of the ripoptosome in cancer but not in primary cells suggests that this complex is a potential novel target for cancer or anti-inflammatory therapy, as suggested by the potential proinflammatory effects of necroptosis. Therefore, the better understanding and characterization of this signaling platform is of enormous importance for the development of novel cancer therapeutics. In this chapter, we describe several methods for purification and investigation of the ripoptosome in human cells. We also describe methods for monitoring apoptotic as well as necroptotic cell death.

Keywords: Apoptosis; IAP antagonist; Immunoprecipitation; Necroptosis; RIP1; Ripoptosome; cFLIP isoforms.