Widespread mitochondrial depletion via mitophagy does not compromise necroptosis

Programmed necrosis (or necroptosis) is a form of cell death triggered by the activation of receptor interacting protein kinase-3 (RIPK3). Several reports have implicated mitochondria and mitochondrial reactive oxygen species (ROS) generation as effectors of RIPK3-dependent cell death. Here, we directly test this idea by employing a method for the specific removal of mitochondria via mitophagy. Mitochondria-deficient cells were resistant to the mitochondrial pathway of apoptosis, but efficiently died via tumor necrosis factor (TNF)-induced, RIPK3-dependent programmed necrosis or as a result of direct oligomerization of RIPK3. Although the ROS scavenger butylated hydroxyanisole (BHA) delayed TNF-induced necroptosis, it had no effect on necroptosis induced by RIPK3 oligomerization. Furthermore, although TNF-induced ROS production was dependent on mitochondria, the inhibition of TNF-induced necroptosis by BHA was observed in mitochondria-depleted cells. Our data indicate that mitochondrial ROS production accompanies, but does not cause, RIPK3-dependent necroptotic cell death

Mechanism of inactivation of NF-κΒ by a viral homologue of ΙκΒα. Signal-induced release of ΙκΒα results in the binding of the viral homologue to NF-κΒ

Activation of the nuclear factor κB plays a key role in viral pathogenesis, resulting in inflammation and modulation of the immune response. We have previously shown that A238L, an open reading frame from African swine fever virus (ASFV), encoding a protein with 40% homology to porcine IκBα exerts a potent anti-inflammatory effect in host macrophages, where it down-regulates NF-κB-dependent gene transcription and proinflammatory cytokine production. This paper reveals the mechanism of suppression of NF-κB activity by A238Lp. A238Lp is synthesized throughout infection as two molecular mass forms of 28 and 32 kDa, and vaccinia-mediated expression of A238L demonstrated that both proteins are produced from a single gene. Significantly, the higher 32-kDa form of A238L, but not the 28-kDa form, interacts directly with RelA, the 65-kDa subunit of NF-κB, indicating that the binding is dependent on a post-translational modification. Immunoprecipitation analysis shows the NF-κB p65-A238L p32 heterodimer is a separate complex from NF-κB-IκBα, and it resides in the cytoplasm. Moreover, we show that ASFV infection stimulates the NFκB signal transduction pathway, which results in the rapid degradation of endogenous IκBα, although both forms of A238Lp are resistant to stimulus-induced degradation. Using the proteasome inhibitor MG132, we show that when degradation of IκBα is inhibited, A238Lp binding to NF-κB p65 is reduced. The results suggest that the virus exploits its activation of the NF-κB pathway to enable its own IκB homologue to bind to NF-κB p65. Last, we show that synthesis of IκBα is increased during ASFV infection, indicating RelA-independent transcription of the IκBα gene

Mitochondria and cell signalling

Mitochondria have long been considered as crucial organelles, primarily for their roles in biosynthetic reactions such as ATP synthesis. However, it is becoming increasingly apparent that mitochondria are intimately involved in cell signalling pathways. Mitochondria perform various signalling functions, serving as platforms to initiate cell signalling, as well as acting as transducers and effectors in multiple processes. Here, we discuss the active roles that mitochondria have in cell death signalling, innate immunity and autophagy. Common themes of mitochondrial regulation emerge from these diverse but interconnected processes. These include: the outer mitochondrial membrane serving as a major signalling platform, and regulation of cell signalling through mitochondrial dynamics and by mitochondrial metabolites, including ATP and reactive oxygen species. Importantly, defects in mitochondrial control of cell signalling and in the regulation of mitochondrial homeostasis might underpin many diseases, in particular age-related pathologies

Mitochondria and cell signalling

Mitochondria have long been considered as crucial organelles, primarily for their roles in biosynthetic reactions such as ATP synthesis. However, it is becoming increasingly apparent that mitochondria are intimately involved in cell signalling pathways. Mitochondria perform various signalling functions, serving as platforms to initiate cell signalling, as well as acting as transducers and effectors in multiple processes. Here, we discuss the active roles that mitochondria have in cell death signalling, innate immunity and autophagy. Common themes of mitochondrial regulation emerge from these diverse but interconnected processes. These include: the outer mitochondrial membrane serving as a major signalling platform, and regulation of cell signalling through mitochondrial dynamics and by mitochondrial metabolites, including ATP and reactive oxygen species. Importantly, defects in mitochondrial control of cell signalling and in the regulation of mitochondrial homeostasis might underpin many diseases, in particular age-related pathologies

Ionizing radiation modulates the TRAIL death-inducing signaling complex, allowing bypass of the mitochondrial apoptosis pathway

In many tumor cell types, ionizing radiation (IR) or DNA-damaging anticancer drugs enhance sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, which is of great clinical interest. We have investigated the molecular mechanism underlying the response to combined modality treatment in p53-mutant Jurkat T leukemic cells overexpressing Bcl-2. These cells are largely resistant to individual treatment with TRAIL or IR, but sensitive to combined treatment, in vitro as well as in vivo. We demonstrate that IR and DNA-damaging anticancer drugs enable TRAIL receptor-2 and CD95/Fas to bypass the mitochondrial pathway for effector caspase activation. This was validated by RNA interference for Bax and Bak and by overexpression of dominant-negative Caspase-9. Improved effector caspase activation was neither caused by altered expression of proapoptotic components nor by impaired activity of inhibitor of apoptosis proteins or nuclear factor-κB signaling. Rather, we found that pretreatment of cells with IR caused quantitative and qualitative changes in death receptor signaling. It strongly improved the capacity of ligand-bound receptors to recruit FADD and activate Caspase-8 and -10 in the death-inducing signaling complex, while c-FLIPL levels were unaffected

Resistance to caspase-independent cell death requires persistence of intact mitochondria

During apoptosis, mitochondrial outer membrane permeabilization (MOMP) is often a point-of-no-return; death can proceed even if caspase activation is disrupted. However, under certain conditions, resistance to MOMP-dependent, caspase-independent cell death is observed. Mitochondrial recovery represents a key process in this survival. Live cell imaging revealed that during apoptosis not all mitochondria in a cell necessarily undergo MOMP. This incomplete MOMP (iMOMP) was observed in response to various stimuli and in different cell types regardless of caspase activity. Importantly, the presence of intact mitochondria correlated with cellular recovery following MOMP, provided that caspase activity was blocked. Such intact mitochondria underwent MOMP in response to treatment of cells with the Bcl-2 antagonist ABT-737, suggesting that the resistance of these mitochondria to MOMP lies at the point of Bax or Bak activation. Thus, iMOMP provides a critical source of intact mitochondria that permits cellular survival following MOMP

Characterization of cytoplasmic Caspase-2 activation by induced proximity

Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90α as a key negative regulator of heat shock-induced caspase-2 activatio

A unified model of mammalian BCL-2 protein family interactions at the mitochondria

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two “modes” whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins (“MODE 1”) or by binding active BAX and BAK (“MODE 2”). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics