Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in ...disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.
•Protein synthesis is one of the most energy consuming processes in the cell.•Nutrient availability modulates growth signaling and protein synthesis rates.•Autophagy is a survival mechanism providing ...metabolic substrates during stress.•Nutrient sensing, protein synthesis and autophagy are coordinated via mTORC1.
Translation is a pivotal step in the regulation of gene expression as well as one of the most energy consuming processes in the cell. Dysregulation of translation caused by the aberrant function of upstream signaling pathways and/or perturbations in the expression or function of components of the translation machinery is frequent in cancer. In this review, we discuss emerging findings that highlight hitherto unappreciated aspects of signaling to the translation apparatus with the particular focus on emerging connections between protein synthesis, autophagy and energy homeostasis in cancer.
Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to ...the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2–associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak.
Cells are constantly subjected to a vast range of potentially lethal insults, which may activate specific molecular pathways that have evolved to kill the cell. Cell death pathways are defined partly ...by their morphology, and more specifically by the molecules that regulate and enact them. As these pathways become more thoroughly characterized, interesting molecular links between them have emerged, some still controversial and others hinting at the physiological and pathophysiological roles these death pathways play. We describe specific molecular programs controlling cell death, with a focus on some of the distinct features of the pathways and the molecular links between them.
Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor ...prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.
The eukaryotic translation initiation factor 4AI (eIF4AI) is the prototypical DEAD-box RNA helicase. It has a “dumbbell” structure consisting of two domains connected by a flexible linker. Previous ...studies demonstrated that eIF4AI, in conjunction with eIF4H, bind to loop structures and repetitively unwind RNA hairpins. Here, we probe the conformational dynamics of eIF4AI in real time using single-molecule FRET. We demonstrate that eIF4AI/eIF4H complex can repetitively unwind RNA hairpins by transitioning between an eIF4AI “open” and a “closed” conformation using the energy derived from ATP hydrolysis. Our experiments directly track the conformational changes in the catalytic cycle of eIF4AI and eIF4H, and this correlates precisely with the kinetics of RNA unwinding. Furthermore, we show that the small-molecule eIF4A inhibitor hippuristanol locks eIF4AI in the closed conformation, thus efficiently inhibiting RNA unwinding. These results indicate that the large conformational changes undertaken by eIF4A during the helicase catalytic cycle are rate limiting.
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•The conformational dynamics of eIF4AI are directly probed by single-molecule FRET•EIF4AI undergoes large conformational transitions, while unwinding RNA hairpins•EIF4H binds to the N-terminal domain of eIF4AI when RNA hairpin is present•Hippuristanol locks eIF4AI in the closed conformation and inhibits RNA unwinding
Sun et al. use single molecule fluorescence to monitor the conformational dynamics of a key eukaryotic initiation factor eIF4AI. During RNA unwinding in the presence of eIF4H, eIF4AI transitions between two conformations, which facilitate substrate binding, RNA duplex unzipping, and a partial substrate release.
Drugs targeting various pro-survival BCL-2 family members (''BH3 mimetics'') have efficacy in hemopoietic malignancies, but the non-targeted pro-survival family members can promote resistance. ...Pertinently, the sensitivity of some tumor cell lines to BH3 mimetic ABT737, which targets BCL-2, BCL-XL, and BCL-W but not MCL-1, is enhanced by 2-deoxyglucose (2DG). We found that 2DG augmented apoptosis induced by ABT737 in 3 of 8 human hemopoietic tumor cell lines, most strongly in pre-B acute lymphocytic leukemia cell line NALM-6, the focus of our mechanistic studies. Although 2DG can lower MCL-1 translation, how it does so is incompletely understood, in part because 2DG inhibits both glycolysis and protein glycosylation in the endoplasmic reticulum (ER). Its glycolysis inhibition lowered ATP and, through the AMPK/mTORC1 pathway, markedly reduced global protein synthesis, as did an ER integrated stress response. A dual reporter assay revealed that 2DG impeded not only cap-dependent translation but also elongation or cap-independent translation. MCL-1 protein fell markedly, whereas 12 other BCL-2 family members were unaffected. We ascribe the MCL-1 drop to the global fall in translation, exacerbated for mRNAs with a structured 5' untranslated region (5'UTR) containing potential regulatory motifs like those in MCL-1 mRNA and the short half-life of MCL-1 protein. Pertinently, 2DG downregulated two other short-lived oncoproteins, MYC and MDM2. Thus, our results support MCL-1 as a critical 2DG target, but also reveal multiple effects on global translation that may well also affect its promotion of apoptosis.
Intrinsic apoptosis resulting from BAX/BAK-mediated mitochondrial membrane damage is regarded as immunologically silent. We show here that in macrophages, BAX/BAK activation results in inhibitor of ...apoptosis (IAP) protein degradation to promote caspase-8-mediated activation of IL-1β. Furthermore, BAX/BAK signaling induces a parallel pathway to NLRP3 inflammasome-mediated caspase-1-dependent IL-1β maturation that requires potassium efflux. Remarkably, following BAX/BAK activation, the apoptotic executioner caspases, caspase-3 and -7, act upstream of both caspase-8 and NLRP3-induced IL-1β maturation and secretion. Conversely, the pyroptotic cell death effectors gasdermin D and gasdermin E are not essential for BAX/BAK-induced IL-1β release. These findings highlight that innate immune cells undergoing BAX/BAK-mediated apoptosis have the capacity to generate pro-inflammatory signals and provide an explanation as to why IL-1β activation is often associated with cellular stress, such as during chemotherapy.
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•MCL-1 and BCL-XL prevent BAX/BAK-induced IL-1β activation and death in macrophages•BAX/BAK-induced IL-1β requires the downstream effector caspases, caspase-3 and -7•Caspase-3/-7 cause potassium efflux to activate the NLRP3 inflammasome and IL-1β•BAX/BAK-induced caspase-3/-7 and IAP loss also trigger caspase-8 to cleave IL-1β
BAX/BAK-mediated apoptosis is considered immunologically silent. Vince et al. show that in macrophages, MCL-1 and BCL-XL restrain BAX/BAK-induced pro-inflammatory IL-1β activation. IAP degradation and activation of caspase-3 and -7 downstream of BAX/BAK triggers caspase-8-mediated cleavage and activation of IL-1β and cause potassium ion efflux to trigger NLRP3 inflammasome formation.
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