Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.

Necroptosis is a type of programmed cell death with great significance in many pathological processes. Tumour necrosis factor-α(TNF), a proinflammatory cytokine, is a prototypic trigger of necroptosis. It is known that mitochondrial reactive oxygen species (ROS) promote necroptosis, and that kinase activity of receptor interacting protein 1 (RIP1) is required for TNF-induced necroptosis. However, how ROS function and what RIP1 phosphorylates to promote necroptosis are largely unknown. Here we show that three crucial cysteines in RIP1 are required for sensing ROS, and ROS subsequently activates RIP1 autophosphorylation on serine residue 161 (S161). The major function of RIP1 kinase activity in TNF-induced necroptosis is to autophosphorylate S161. This specific phosphorylation then enables RIP1 to recruit RIP3 and form a functional necrosome, a central controller of necroptosis. Since ROS induction is known to require necrosomal RIP3, ROS therefore function in a positive feedback circuit that ensures effective induction of necroptosis.

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.

Reactive oxygen species (ROS) production in phagocytes is a major defense mechanism against pathogens. However, the cellular self-protective mechanism against such potential damage from oxidative stress remains unclear. Here we show that the kinases Mst1 and Mst2 (Mst1/2) sense ROS and maintain cellular redox balance by modulating the stability of antioxidant transcription factor Nrf2. Site-specific ROS release recruits Mst1/2 from the cytosol to the phagosomal or mitochondrial membrane, with ROS subsequently activating Mst1/2 to phosphorylate kelch like ECH associated protein 1 (Keap1) and prevent Keap1 polymerization, thereby blocking Nrf2 ubiquitination and degradation to protect cells against oxidative damage. Treatment with the antioxidant N-acetylcysteine disrupts ROS-induced interaction of Mst1/2 with phagosomes or mitochondria, and thereby diminishes the Mst-Nrf2 signal. Consistently, loss of Mst1/2 results in increased oxidative injury, phagocyte ageing and death. Thus, our results identify the Mst-Nrf2 axis as an important ROS-sensing and antioxidant mechanism during an antimicrobial response.

Oligomerization of the mixed-lineage kinase domain-like protein (MLKL) is essential for its cation channel function in necroptosis. Here we show that the MLKL channel is an octamer comprising two previously identified tetramers most likely in their side-by-side position. Intermolecule disulfide bonds are present in the tetramer but are not required for octamer assembly and necroptosis. MLKL forms oligomers in the necrosome and is then released from the necrosome before or during its membrane translocation. We identified two MLKL mutants that could not oligomerize into octamers, although they formed a tetramer, and also, one MLKL mutant could spontaneously form a disulfide bond-linked octamer. Subsequent analysis revealed that the tetramers fail to translocate to the plasma membrane and that the MLKL octamer formation depends on ␣-helices 4 and 5. While MLKL could be detected from outside the cells, its N-and C-terminal ends could not be detected, indicating that the MLKL octamer spans across the plasma membrane, leaving its N and C termini inside the cell. These data allowed us to propose a 180°symmetry model of the MLKL octamer and conclude that the fully assembled MLKL octamers, but not the previously described tetramers, act as effectors of necroptosis.

Due to the fact that Chinese tourists are becoming the main players in the international tourism market, with large growth potential, this research empirically identifies the psychological factors that affect Chinese outbound tourists’ memorable tourism experiences (MTEs), and investigates the relationships among MTEs, the “fun” emotion, and their recommendation and revisit intentions based on the stimulus–organism–response (S-O-R) model. In addition, the moderating role of perceived risk between the causal relationships among the “fun” emotion and recommendation and revisit intentions is identified. A total of 560 survey participants who had experienced overseas tourism in the last two years were selected as the target population, and the data from 413 valid surveys were empirically analyzed via structural equation modeling (SEM). Chinese outbound tourists’ MTEs, such as hedonism, novelty, meaningfulness, and social interaction, are found to positively influence their experience of the “fun” emotion, such as emotional spark and flow. Additionally, it is found that when Chinese outbound tourists have achieved a high level of the “fun” emotion, their intentions to recommend and revisit their destination will be stronger. In addition, perceived risk is found to have a negative moderating role in the causal relationships among emotional spark and recommendation and revisit intentions, and also has negative moderating effects on the causal relationship between flow and recommendation intentions. This study provides important practical insights for how international tourist destination marketers and managers provide Chinese tourists with MTEs, and how to elicit the “fun” emotion and loyalty behaviors from Chinese tourists via the investigation of the mechanism of MTEs, fun, and recommendation and revisit intentions. It is also found that it is beneficial for international tourist destination managers to lower the level of tourists’ perceived risk to improve tourists’ recommendation and revisit intentions. These findings can aid in the development of more effective strategies to increase the market share and the international competitiveness of destinations.

Highlights d TRPV cation channels are required for glucose starvationinduced AMPK activation d FBP-unoccupied aldolase inhibits Ca 2+ release of TRPVs at the ER-lysosome contact d The decrease of the local Ca 2+ allow TRPV proteins to interact with v-ATPase