Purified vascular endothelial cell (EC) growth factor receptor-2 (VEGFR2) auto-phosphorylates upon VEGF-A occupation in vitro, arguing that VEGR2 confers its mitotic and viability signaling in and of itself. Herein, we show that, in ECs, VEGFR2 function requires concurrent C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 co-signaling. C3ar1/C5ar1 or IL-6R blockade totally abolished VEGFR2 auto-phosphorylation, downstream Src, ERK, AKT, mTOR and STAT3 activation, and EC cell cycle entry.

IjBa resides in the cytosol where it retains the inducible transcription factor NF-jB. We show that IjBa also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF-jB that accumulate high amounts of mitochondrial IjBa as a NF-jB target gene. 3T3 IjBa À/À cells also become protected from apoptosis when IjBa is specifically reconstituted at the OMM. Using various IjBa mutants, we demonstrate that apoptosis inhibition and NF-jB inhibition can be functionally and structurally separated. At mitochondria, IjBa stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IjBa is reduced in tumour cells with constitutively active NF-jB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IjBa in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF-jB.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.

No abstract

Double‐stranded RNA (ds RNA ) is a potent proinflammatory signature of viral infection and is sensed primarily by RIG ‐I‐like receptors ( RLR s). Oligomerization of RLR s following binding to cytosolic ds RNA activates and nucleates self‐assembly of the mitochondrial antiviral‐signaling protein ( MAVS ). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self‐propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super‐resolution light microscopy that MAVS activation by ds RNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context, MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling, and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon ds RNA sensing.

The permeability transition pore (PT-pore) mediates cell death through the dissipation of the mitochondrial membrane potential (DY m ). Because the exact composition of the PT-pore is controversial, it is crucial to investigate the actual molecular constituents and regulators of this complex. We found that mitochondrial creatine kinase-1 (CKMT1) is a universal and functionally necessary gatekeeper of the PT-pore, as its depletion induces mitochondrial depolarization and apoptotic cell death. This can be inhibited efficiently by bongkrekic acid, a compound that is widely used to inhibit the PT-pore. However, when the 'classical' PTpore subunits cyclophilin D and VDAC1 are pharmacologically inhibited or their expression levels reduced, mitochondrial depolarization by CKMT1 depletion remains unaffected. At later stages of drug-induced apoptosis, CKMT1 levels are reduced, suggesting that CKMT1 downregulation acts to reinforce the commitment of cells to apoptosis. A novel high-molecular-mass CKMT1 complex that is distinct from the known CKMT1 octamer disintegrates upon treatment with cytotoxic drugs, concomitant with mitochondrial depolarization. Our study provides evidence that CKMT1 is a key regulator of the PT-pore through a complex that is distinct from the classical PT-pore.

1 Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. 2Oligomerization of RIG-I-like receptors on cytosolic dsRNA nucleates self-assembly of the 3 mitochondrial antiviral signaling protein (MAVS). In the current signaling model, the caspase 4 recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote 5 signaling complex assembly. However, there is no conclusive evidence that MAVS forms 6 fibrils in cells or with the transmembrane anchor present. We show here with super-resolution 7 light microscopy that MAVS activation by dsRNA induces mitochondrial membrane 8 remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in 9 the cellular context MAVS signaling complexes and the fibrils within them are smaller than 80 10 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon 11 signaling and proapoptotic activities. We conclude that membrane tethering of MAVS restrains 12 its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA 13 sensing. 14 Introduction 15Recognition of viral nucleic acids by innate immune receptors is one of the most conserved 16 and important mechanisms for sensing viral infection. Many viruses deliver or generate double-17 stranded RNA (dsRNA) in the cytosol of the host cell. Cytosolic dsRNA is a potent 18 proinflammatory signal in vertebrates. Endogenous dsRNAs are modified or masked through 19 various mechanisms to prevent autoimmune signaling, and genetic deficiencies in these dsRNA 20 modification pathways can cause autoimmune disorders [1][2][3]. Cytosolic dsRNA is primarily 21 sensed by the RIG-I-like receptors (RLRs) RIG-I (DDX58), MDA5 (IFIH1) and LGP2 22 polymerization of MAVS CARD fibrils with amyloid-like (or prion-like) properties including 1 resistance to detergents and proteases [19][20][21]. MAVS polymerization is required for signaling, 2 and the spontaneous elongation of MAVS fibrils following nucleation is thought to provide a 3 signal amplification mechanism [21]. MAVS fibrils then recruit proteins from the TRAF and 4 TRIM families to form multimeric signaling platforms, or signalosomes [21]. MAVS is 5 localized primarily on the outer mitochondrial membrane [5] but can also migrate via the 6 mitochondria-associated membrane (MAM) to peroxisomes [22], which function as an 7 alternative signaling platform to mitochondria [23]. MAVS signalosomes activate both type I 8 interferon (through IRF3) and NF-kB-dependent inflammatory responses [11, 13,21]. 9Overexpression of MAVS induces apoptotic cell death, and this proapoptotic activity is mitochondrial membrane (via the TM of each MAVS molecule in the fibril). Here, we address 1 the question of how the current model of MAVS signaling can be reconciled with these physical 2 constraints, and the requirement of the TM, for cell signaling. Imaging of MAVS signaling 3 complexes by super-resolution light microscopy with effective optical resolutions of up to 32 4 nm reveal that...

ORCTL3 is a member of a group of genes, the so-called anticancer genes, that cause tumour-specific cell death. We show that this activity is triggered in isogenic renal cells upon their transformation independently of the cells’ proliferation status. For its cell death effect ORCTL3 targets the enzyme stearoyl-CoA desaturase-1 (SCD1) in fatty acid metabolism. This is caused by transmembrane domains 3 and 4, which are more efficacious in vitro than a low molecular weight drug against SCD1, and critically depend on their expression level. SCD1 is found upregulated upon renal cell transformation indicating that its activity, while not impacting proliferation, represents a critical bottleneck for tumourigenesis. An adenovirus expressing ORCTL3 leads to growth inhibition of renal tumours in vivo and to substantial destruction of patients’ kidney tumour cells ex vivo. Our results indicate fatty acid metabolism as a target for tumour-specific apoptosis in renal tumours and suggest ORCTL3 as a means to accomplish this.