Nuclear RIPK3 and MLKL contribute to cytosolic necrosome formation and necroptosis

Necroptotic signaling converges in the assembly of a cytosolic signaling platform, the necrosome, with the activation of its downstream effector, MLKL. RIPK1 and RIPK3, key components of the necrosome, act as signaling intermediates for the activation of MLKL. We report that RIPK3 and MLKL continuously shuttle between the nucleus and the cytoplasm, whereas RIPK1 is constitutively present in both compartments. During TNF-induced necroptosis, nuclear RIPK1 becomes ubiquitinated, after which nuclear MLKL becomes phosphorylated and oligomerized. Pharmacological inhibition of the nuclear export machinery leads to retention of RIPK3 and MLKL in the nucleus, prevents the nucleation of cytosolic RIPK3/MLKL oligomerization, and reduces cell death. Our results suggest that passage of necroptotic signaling components through the nucleus is a mechanism for regulating cytosolic necrosome formation and consequently necroptotic cell death

Delivery of mixed-lineage kinase domain-like protein by vapor nanobubble photoporation induces necroptotic-like cell death in tumor cells

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis

In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptorinteracting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types

Serine 25 phosphorylation inhibits RIPK1 kinase-dependent cell death in models of infection and inflammation

RIPK1 regulates cell death and inflammation through kinase-dependent and -independent mechanisms. As a scaffold, RIPK1 inhibits caspase-8-dependent apoptosis and RIPK3/MLKL-dependent necroptosis. As a kinase, RIPK1 paradoxically induces these cell death modalities. The molecular switch between RIPK1 pro-survival and pro-death functions remains poorly understood. We identify phosphorylation of RIPK1 on Ser25 by IKKs as a key mechanism directly inhibiting RIPK1 kinase activity and preventing TNF-mediated RIPK1-dependent cell death. Mimicking Ser25 phosphorylation (S > D mutation) protects cells and mice from the cytotoxic effect of TNF in conditions of IKK inhibition. In line with their roles in IKK activation, TNF-induced Ser25 phosphorylation of RIPK1 is defective in TAK1- or SHARPIN-deficient cells and restoring phosphorylation protects these cells from TNF-induced death. Importantly, mimicking Ser25 phosphorylation compromises the in vivo cell death-dependent immune control of Yersinia infection, a physiological model of TAK1/IKK inhibition, and rescues the cell death-induced multi-organ inflammatory phenotype of the SHARPIN-deficient mice

Epithelial HMGB1 delays skin wound healing and drives tumor initiation by priming neutrophils for NET formation

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation

cIAP1/2 Are Direct E3 Ligases Conjugating Diverse Types of Ubiquitin Chains to Receptor Interacting Proteins Kinases 1 to 4 (RIP1–4)

The RIP kinases have emerged as essential mediators of cellular stress that integrate both extracellular stimuli emanating from various cell-surface receptors and signals coming from intracellular pattern recognition receptors. The molecular mechanisms regulating the ability of the RIP proteins to transduce the stress signals remain poorly understood, but seem to rely only partially on their kinase activities. Recent studies on RIP1 and RIP2 have highlighted the importance of ubiquitination as a key process regulating their capacity to activate downstream signaling pathways. In this study, we found that XIAP, cIAP1 and cIAP2 not only directly bind to RIP1 and RIP2 but also to RIP3 and RIP4. We show that cIAP1 and cIAP2 are direct E3 ubiquitin ligases for all four RIP proteins and that cIAP1 is capable of conjugating the RIPs with diverse types of ubiquitin chains, including linear chains. Consistently, we show that repressing cIAP1/2 levels affects the activation of NF-κB that is dependent on RIP1, -2, -3 and -4. Finally, we identified Lys51 and Lys145 of RIP4 as two critical residues for cIAP1-mediated ubiquitination and NF-κB activation

MLKL reveals its friendly face : a role in nerve regeneration

In this issue of Molecular Cell, Ying et al. (2018) report on a novel function of MLKL in sciatic nerve regeneration after injury through myelin sheath destabilization. This function of MLKL is independent of necroptosis execution and requires phosphorylation at MLKL-S441

MLKL in cancer : more than a necroptosis regulator

Mixed lineage kinase domain-like protein (MLKL) emerged as executioner of necroptosis, a RIPK3-dependent form of regulated necrosis. Cell death evasion is one of the hallmarks of cancer. Besides apoptosis, some cancers suppress necroptosis-associated mechanisms by for example epigenetic silencing of RIPK3 expression. Conversely, necroptosis-elicited inflammation by cancer cells can fuel tumor growth. Recently, necroptosis-independent functions of MLKL were unraveled in receptor internalization, ligand-receptor degradation, endosomal trafficking, extracellular vesicle formation, autophagy, nuclear functions, axon repair, neutrophil extracellular trap (NET) formation, and inflammasome regulation. Little is known about the precise role of MLKL in cancer and whether some of these functions are involved in cancer development and metastasis. Here, we discuss current knowledge and controversies on MLKL, its structure, necroptosis-independent functions, expression, mutations, and its potential role as a pro- or anti-cancerous factor. Analysis of MLKL expression patterns reveals that MLKL is upregulated by type I/II interferon, conditions of inflammation, and tissue injury. Overall, MLKL may affect cancer development and metastasis through necroptosis-dependent and -independent functions

Punching holes in cellular membranes : biology and evolution of gasdermins

The gasdermin (GSDM) family has evolved as six gene clusters (GSDMA-E and Pejvakin, PJVK), and GSDM proteins are characterized by a unique N-terminal domain (N-GSDM). With the exception of PJVK, the N-GSDM domain is capable of executing plasma membrane permeabilization. Depending on the cell death modality, several protease- and kinase-dependent mechanisms directly regulate the activity of GSDME and GSDMD, the two most widely expressed and best-studied GSDMs. We provide an overview of all GSDMs in terms of biological function, tissue expression, activation, regulation, and structure. In-depth phylogenetic analysis reveals that GSDM genes show many gene duplications and deletions, suggesting that strong evolutionary forces and a unique position of the PJVK gene are associated with the occurrence of complex inner-ear development in vertebrates