Abstract
Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, cIAPs, A20, or CYLD. Finally, we show that Triad3a-dependent necrosomal degradation limits necroptosis and production of inflammatory cytokines. These results reveal a new mechanism of shutting off necrosome signaling and may pave the way to new strategies for therapeutic manipulation of inflammatory responses.
Highlights
Macrophage-like cells are present throughout the body and play a major role in initiating inflammatory responses to control pathogens[1,2,3,4]
Necrosome activation leads to the degradation of various interacting proteins Combined TLR4 stimulation and caspase inhibition in macrophages causes the phosphorylation of RipK1 and RipK3, which can be observed as a slightly slower migrating protein band in western blot
Consistent with this, RipK1 and RipK3 were resolved as a single band upon dephosphorylation by calf intestinal phosphatase (CIP) (Fig. 1a)
Summary
Macrophage-like cells are present throughout the body and play a major role in initiating inflammatory responses to control pathogens[1,2,3,4]. TLR signaling induces MyD88dependent and TRIF-dependent signaling, which leads to the production of cytokines and chemokines[5,6,7,8], and recruitment of myeloid cells[9]. Apoptosis promotes cell death during embryogenesis and in the elimination of self-reactive cells[10,11]. Inflammatory cell death results in cell-rupture and release of intracellular contents including cytokines and numerous danger-associated molecular patterns (DAMPs) to the. Activated RipK3 phosphorylates mixed lineage kinase domain-like protein (MLKL), resulting in trimerization of MLKL and its relocation to the cell membrane, causing membrane rupture and necroptosis[29]
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