Figure 1. Molecular basis of mammalian autophagy. Autophagy is a multistep process involving several key ATG proteins and signaling complexes. It requires the formation of double-membrane-containing autophagosomes that sequester proteins, lipids, organelles or invasive microbes and fuse with lysosomes for digestion of content by acidic hydrolases. ULK1, a protein kinase serving as the central initiator of autophagy, is inhibited by the mTORC1 complex that contains mTOR. AMPK serves as a nutrient sensor and negative regulator of mTORC1. Autophagosome biogenesis starts with the formation of an initiation membrane that is derived either from the endoplasmatic reticulum (ER) or from several other cellular membrane sources. Vesicle nucleation is promoted by the BECN1/Vps34 core complex containing the lipid kinase Vps34. Vesicle elongation is regulated by the two ubiquitin-like conjugation systems (UBLs) ATG12-UBL and LC3-UBL that cooperate to catalyze the conjugation of phosphatidylethanolamine (PE) to LC3 and facilitate the conversion of cytosolic LC3-I into a membrane-associated LC3-II that is translocated to the autophagosomal membrane. Following vesicle closure, mature autophagosomes fuse with lysosomes to generate autolysosomes that digest the autophagosomal content by lysosomal proteases for cellular recycling [ 5 ]. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com . In general, autophagy is a pro-survival stress response, for example, autophagy will be activated under situations of nutrient deprivation to ensure supply of basic building blocks for metabolism and survival of the cells/organisms by recycling of non-essential cellular components. Autophagy also serves to remove damaged and potentially harmful organelles, thereby supporting cell survival. On the other hand, there is conclusive evidence that prolonged over activation of the autophagosomal/lysosomal pathway can lead to autophagic celldeath (ACD, type II celldeath). Of note, similar threshold effects on cell survival vs celldeath are commonly observed in various stress responses like the endoplasmatic reticulum (ER) stress response and activation of p53 [ 6 ]. Accordingly, ACD is often described as self-digestion beyond the point allowing cell survival [ 3 , 4 , 6 – 8 ]. Hence, the net effect of autophagy
Cdc48p, which in turn results in lowered protein levels and activity of Sod2p, caus- ing accumulation of ROS in the mitochondria, mitochondrial malfunctioning and ultimately celldeath. This points toward a considerable functional homology be- tween Mga2p and the mammalian NFκB, which is activated by VCP, the mammal- ian homologue of Cdc48p, and is a transcription factor for SOD2. Future experi- ments with overexpression of NFκB in yeast could extend the scope of this similar- ity. Another possibility is that this mechanism for cdc48 S565G -mediated celldeath is
4.5 PS89 as the First Small-Molecule Compound Targeting BAP31 It was now clearly demonstrated that the highly positive interference of PS with the signaling of cytostatics happens at the ER-mitochondrial interface. An implication of BAP in the crosstalk of PS with cytostatics was identified in addition. However, one aspect that made us especially curious about BAP was not addressed so far. It is the fact that BAP was not only mediator, but also a direct target of PS . The binding of PS to BAP was initially recognized by the proteomics target screen and according to the ranking score, BAP was among the three most highly enriched proteins. This was validated by a staining of the PS photo probe with a BAP -specific antibody showing consolidated binding to the same target. In order to understand how the binding of PS influences BAP , a closer examination of the BAP protein complex is required. As already mentioned, Fis was shown to form a tripartite complex with BAP and caspase- CASP . This complex seems to be further under control of the celldeath- inducing p -target protein CDIP as well as the anti-apoptotic proteins Bcl- and Bcl-xL. However, the dynamics which regulate the balance of pro- and anti-apoptotic proteins within the complex have not been clarified yet.
A second gene which has been connected to celldeath in M. xanthus is the csgA gene. A mutant of csgA did not lyse during development similar to a mazF mutant (Janssen & Dworkin, 1985). The connection between csgA and celldeath was unexpected, because originally, CsgA has been studied intensively because of its role in cell aggregation. The cell surface protein CsgA is proteolytically processed to a smaller form, also known as p17, which acts as an intercellular developmental signal, the C-signal (Lobedanz & Søgaard-Andersen, 2003). p17 is thought to be recognized by an unidentified receptor on a neighboring cell. The C-signal has been proposed to form, together with the chemosensory protein FrzCD and the transcriptional regulator FruA, a positive protein feedback loop, which is thought to control ongoing aggregation during development (Søgaard-Andersen & Kaiser, 1996, Ellehauge et al., 1998, Yoder-Himes & Kroos, 2006; Figure 2). The C-signal has been proposed to activate FruA via phosphorylation, which then stimulates the methylation of FrzCD (Søgaard- Andersen & Kaiser, 1996, Ellehauge et al., 1998). Methylated FrzCD changes the motility mode of the cells to a unidirectional behavior and causes the population to aggregate (Blackhart & Zusman, 1985, Shi et al., 1996). Increased aggregation leads to more cell-cell contact, which again would stimulate the formation of C-signal (Søgaard-Andersen & Kaiser, 1996). Similar to mrpC and csgA mutants, a fruA mutant is unable to aggregate or sporulate (Janssen & Dworkin, 1985, Ellehauge et al., 1998, Sun & Shi, 2001b). In addition to aggregation, FruA has been suggested to control, together with a shorter isoform of MrpC, MrpC2, the cell fate “sporulation” by regulating indirectly the transcription of exoC (Licking et al., 2000, Mittal & Kroos, 2009), which is involved in spore coat polysaccharide synthesis and export) (Ueki & Inouye, 2005).
Neonates are highly susceptible to microbial infections which is partially attributable to fundamental phenotypic and functional diﬀerences between eﬀector cells of the adult and neonatal immune system. The resolution of the inﬂammation is essential to return to tissue homeostasis, but given that various neonatal diseases, such as periventricular leukomalacia, necrotizing enterocolitis, or bronchopulmonary dysplasia, are characterized by sustained in ﬂammation, newborns seem predisposed to a dysregulation of the in ﬂammatory response. Targeted apoptosis of eﬀector cells is generally known to control the length and extent of the inﬂammation, and previous studies have demonstrated that phagocytosis-induced celldeath (PICD), a special type of apoptosis in phagocytic immune cells, is less frequently triggered in neonatal monocytes than in adult monocytes. We concluded that a rescue of monocyte PICD could be a potential therapeutic approach to target sustained in ﬂammation in neonates. The EGFR ligand amphiregulin (AREG) is shed in response to bacterial infection and was shown to mediate cellular apoptosis resistance. We hypothesized that AREG might contribute to the reduced PICD of neonatal monocytes by aﬀecting apoptosis signaling. In this study, we have examined a cascade of signaling events involved in extrinsic apoptosis by using a well-established in vitro E. coli infection model in monocytes from human peripheral blood (PBMO) and cord blood (CBMO). We found that CBMO shows remarkably higher pro-AREG surface expression as well as soluble AREG levels in response to infection as compared to PBMO. AREG increases intracellular MMP-2 and MMP-9 levels and induces cleavage of membrane- bound FasL through engagement with the EGF receptor. Our results demonstrate that loss of AREG rescues PICD in CBMO to the level comparable to adult monocytes. These ﬁndings identify AREG as a potential target for the prevention of prolonged in ﬂammation in neonates.
is abundant in the adult brain and Cdk5 activity increases in neurons during development. p35 can be proteolysed by calpains following changes in calcium homeostasis into a cytosolic C-terminal fragment referred to as p25 that is more stable than p35 and binds more tightly to Cdk5, leading to a hyperactive, mislocalized p25-Cdk5 complex (for review, see Dahavan and Tsai, 2001). Such cleavage of p35 to p25 has been reported in disorders such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (for reviews, see Patzke and Tsai, 2002; Shelton and Johnson, 2004). Furthermore, exposure of primary cortical neurons to various insults like A β peptide, H 2 O 2 or glutamate also leads to p25 formation and celldeath (Patrick et al., 1999; Lee et al., 2000; Kusakawa et al., 2000). However, no mechanisms have been determined by which this kinase complex triggers its neurotoxicity. It is worth noting that, in neurodegenerative disorders and cellular models of neuronal death in which p25-Cdk5 is probably involved, aberrant cell-cycle deregulation has been reported (Vincent et al., 1996; Vincent et al., 1997; Vincent et al., 1998; Nagy et al., 1997; Busser et al., 1998; Copani et al., 1999; Giovanni et al., 1999; Husseman et al., 2000; Zhu et al., 2000; Ranganathan et al., 2001; Yang et al., 2003). In addition, a tight correlation has been established between Cdk5 deregulation and expression of cell cycle regulatory proteins (Nguyen et al., 2002; Nguyen et al., 2003). Nevertheless, pathways linking Cdk5 to cell cycle remain obscure.
XIAP's anti-apoptotic activity (Bratton, S. B., Lewis, J. et al., 2002). Interestingly, only the IAPs have been demonstrated to be endogenous repressors of the terminal caspase cascade. In turn, the caspase inhibiting activity of XIAP is negatively regulated by at least two XIAP-interacting proteins, XAF1 and Smac/DIABLO. In addition to the inhibition of caspases, recent discoveries from several laboratories suggest that XIAP is also involved in a number of other biologically significant cellular activities including modulation of receptor-mediated signal transduction and protein ubiquitination. The multiple biological activities of XIAP, its unique translational and post-translational control and the centrality of the caspase cascade make the control of XIAP expression an exceptionally promising molecular target for modulating apoptosis. Therapeutic benefits can be derived from both the suppression of inappropriate celldeath such as in neurodegenerative disorders and ischemic injury (Holcik, M., Gibson, H. et al., 2001). Thus, it seems at least possible that Smac/DIABLO might be a potential target for apoptosis-inhibition, since it selectively blocks the binding of IAP to the apoptosome. Recently, an essential serine protease with pro-apoptotic activity, named HtrA2/Omi, was identified to interact with A in yeast two-hybrid assays. Co-immunoprecipitation assays further confirmed the interaction of A and the protease in cell culture experiments (Park, H. J., Seong, Y. M. et al., 2004). Considerung this fact, HtrA2/Omi-inhibiting substances could be of great interest for AD drug development
Saskia Klutzny 1,2 , Ralf Lesche 1 , Matthias Keck 1 , Stefan Kaulfuss 1 , Andreas Schlicker 1 , Sven Christian 1 , Carolyn Sperl 1 , Roland Neuhaus 1 , Jeffrey Mowat 1 , Michael Steckel 1 , Björn Riefke 1 , Stefan Prechtl 1 , Karsten Parczyk 1 and Patrick Steigemann* ,1 Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer celldeath specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress- response pathways and that hypoxia-specific celldeath is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.
promote pathogen virulence. The mode of action of these effectors is quite diverse, but in general, they interfere with the plant surveillance system or disrupt defense signaling. Bacterial pathogens have developed a type III secretion system (TTSS) to inject effectors proteins into plant cells, and, eventually, suppress MTI (Bent and Mackey, 2007; Jones and Dangl, 2006). The transfer of effectors into plant cells by fungi and oomycetes is less clear. Since pathogens obtained the capacity to suppress host defense, plants have evolved more specialized mechanism to detect microbial effectors or effector activites summarized as effector-triggered immunity (ETI, syn. R gene-mediated resistance) (Bent and Mackey, 2007; Chisholm et al., 2006; Dangl and Jones 2006). R protein activates immune responses after direct binding to effectors or indirectly by monitoring effector action. Direct protein interactions were detected between flax resistance genes and flax rust avirulence genes (Dodds et al., 2006). While there are other cases showing that plant R proteins indirectly recognize pathogen effectors by monitoring the integrity of host cellular targets of these effectors (van der Biezen and Jones, 1998; Jones and Dangl, 2001). This latter principle of effector recognition was defined as “guard hypothesis” (Dangl and Jones, 2006; Bent and Mackey, 2007). Eventually, R-gene mediated resistance is regarded to result in a faster and more efficient initiation of defense signaling than MTI. A hallmark of R-gene mediated defense is the hypersensitive response (HR), a localized programmed celldeath, which occurs at the site of infection and inhibits fungal penetration. During evolution of host-microbe interactions, microbes have found ways to circumvent R gene-mediated defense responses by modifying or eliminating effectors/effector actions resulting in successful plant colonization and microbial propagation. In turn, plants continuously adapt their R protein repertoire. Nowadays, the evolutionary processes in immune signaling are summarized in the zigzag model (Fig. 1-1) (Jones and Dangl, 2006).
integrations obtained by infecting the interleukin-3 (IL-3)-dependent hematopoietic cell line - FLOXIL3 - with U3Cre gene trap virus, we have isolated 125 individual clones that converted to factor independence upon IL-3 withdrawal. Of 102 cellular sequences adjacent to U3Cre integration sites, 17% belonged to known genes, 11% matched single expressed sequence tags (ESTs) or full cDNAs with unknown function and 72% had no match within the public databases. Most of the known genes recovered in this analysis encoded proteins with survival functions. Conclusions: We have shown that hematopoietic cells undergoing apoptosis after withdrawal of IL-3 activate survival genes that impede celldeath. This results in reduced apoptosis and improved survival of cells treated with a transient apoptotic stimulus. Thus, apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals, rather than a simple death by default.
2 Jurkat cells were transiently transfected with scrambled (scr) or Apaf-1 siRNA oligonucleotides and treated with helenalin (Hel; 20 µM, 24 h). Celldeath was quantified by Nicoletti assay. Apaf-1 protein levels in transfected cells were analyzed by Western blot. B, Western blot analysis of caspase activation in Bcl-2 Jurkat cells after incubation with helenalin (Hel; 20 µM) for the indicated times. Arrows indicate proforms of caspase-9 and -3 and location of their active, cleaved forms, respectively. C and D, Determination of capase-3 like and caspase-8 activity in Bcl-2 Jurkat cells. Cells were treated with helenalin (Hel; 20 µM) or β-phenylethyl isothiocyanate (PEITC; 20 µM) for the indicated time points and caspase activity was determined. E, Bcl-2 Jurkat cells were treated with β-phenylethyl isothiocyanate (PEITC; 20 µM) or helenalin (Hel; 20 µM) for 16 h and celldeath was measured by PI exclusion. If indicated, cells were pre-incubated with the pan-caspase inhibitor Q-VD-OPh (10 µM) for 1 h. Data are expressed as mean ± SEM (n=3). *, p < 0.001 (ANOVA, Bonferroni). Experiments for Figure 10 A and B were performed by N. López Antón.
Further studies have shown a key role of 12/15-LOX in different models of neuronal celldeath [133, 134]. Recent work in models of cerebral ischemia exposed LOX as a potential target for neuroprotective strategies in stroke treatment. In these studies, genetic 12/15-LOX deletion significantly reduced the infarct size in a mouse model of transient cerebral ischemia as compared to wild type mice, and similar protective effects against ischemic brain damage were achieved by treating wild type mice with the LOX inhibitor baicalein . It is important to note that baicalein is supposed to inhibit 12/15-LOX but it has been shown that it also has antioxidant properties, which may partly contribute to the protective effects achieved in vitro and in vivo where baicalein was applied in very high doses. Consequently, in the present study the selective 12/15-LOX inhibitor PD146176 was chosen for the experiments.
Activated p53 can induce apoptosis, directing cells to die. Apoptosis means “falling off” in Greek language, as leaves from a tree or petals from flowers . It is a pro- grammed celldeath, through which cells destroy themselves in a highly regulated and controlled way . Cells undergoing apoptosis display characteristic morphological changes , including cell shrinkage, chromatin condensation (pykosis), nuclear frag- mentation (karyorrhexix) and membrane blebbing with formation of apoptotic bodies . Finally, they are engulfed and “eaten” by neighboring cells or macrophages, without leaking any cellular contents or causing a deleterious inflammatory response. Apoptosis eliminates cells when they are no longer needed, irreversibly injured, or turn to be a threat to the organism  . In the human embryonic development, the individual fingers and toes separate because apoptosis of cells between them oc- curs. In homeostatic adult tissues, celldeath and cell division must be kept in bal- ance. Both excessive and insufficient apoptosis are associated with pathological pro- cesses: while accelerated apoptosis contributes to acute and degenerative disease, deficient apoptosis can give rise to tumorigenesis or autoimmune disorders . The underlying biochemical mechanism for apoptosis is a caspase-mediated proteo- lytic cascade (Figure 5). Caspases are intracellular proteases that can cleave specific sequences in proteins, thereby activating signal transduction for celldeath. There are initiator caspases (caspase 8,9) and executioner caspases (caspase 3,6,7), both normally exist as inactive precursors in cells and only activated during apoptosis  .
Physiologically, XC - transports glutamate out of the cell in exchange for cystine  (Figure 3). However, upon high extracellular glutamate concentrations or direct inhibition of the transporter, this exchange is shut off thereby reducing cysteine pools required for glutathione (GSH) synthesis as part of the cell’s redox defense [2,184]. GSH depletion in turn leads to compromised function of glutathione peroxidase 4 (GPX4), which is essentially responsible for the maintenance of a proper redox state by reduction of hydrogen peroxide, organic hydroperoxides as well as lipid peroxides as products of 12/15 lipoxygenases (LOX) (e.g. oxidized polyunsaturated fatty acids: PUFA-OOH and phospholipids: PL-OOH) at the expense of reduced glutathione [4,45,114]. In addition, GPX4 inhibition is correlated to enhanced 12/15-LOX activity through accumulated ROS giving rise to a chain reaction of massive soluble and lipid ROS production , which can be blocked using the antioxidants vitamin E , Trolox and the LOX inhibitors baicalein or PD146176 [106,186]. Subsequent to increased ROS formation, the dynamin-related protein 1 (DRP1) and the pro-apoptotic BCL-2 family protein BID translocate to the OMM, where they mediate fission of the mitochondrial network, mitochondrial ROS production, and loss of mitochondrial membrane potential and ATP production through electron leakage from the electron transport chain [103,186]. Notably, depending on their subtype and differentiation state, neurons exclusively inherit an alternatively spliced, BH3 domain-only form of BAK (N-BAK) with strong translational arrest of the mRNA, thus not being a substitute for BAX or a target for BID at the mitochondrial site during oxidative celldeath [86,189]. Sequential release of pro-apoptotic proteins, for instance the mitochondrial flavoprotein AIF anchored at the inner mitochondrial membrane, mediate final celldeath execution upon translocation to the nucleus [72,186], nuclear condensation and DNA cleavage  presumably through interaction with cytosolic CypA  or MIF nuclease . Despite this pro-death signaling upon mitochondrial release, AIF is believed to play an important role in the regulation of mitochondrial morphology and mitochondrial energy metabolism . In this context, AIF was shown to stabilize mitochondrial complex I and vice
Although little is known about the signalling cascade during PCD in plants, recent studies have shown that activation of phospholipase C (PLC) and phospholipase D (PLD) is required during camptothecin- and cadmium- induced PCD in tomato suspension cells (Yakimova et al., 2006; Woltering et al., 2007). As PLD and PLC have also been described to be activated during salt stress caused by NaCl or KCl in the unicellular green alga Chlamydomonas moewusii (Munnik et al., 2000; Meijer et al., 2002; Arisz et al., 2003) a similar signalling mechanism could play a pivotal role during salt stress-iduced PCD in Micrasterias. In summary, it has been demonstrated that Micrasterias shows PCD hallmarks like autophagy, vacuolization, ultra- structural changes, and DNA laddering upon salt stress. As the iso-osmotic sorbitol treatment does not result in these effects, the ionic, rather than the osmotic component of salt stress seems to lead to PCD in Micrasterias. The ap- pearance of these changes was accompanied by an active metabolism measured by viability assay, pigment composi- tion, photosynthesis, and respiration pointing towards a programmed celldeath and not to a necrotic, accidental celldeath event. Our data also reveal that KCl has more pronounced effects on viability and on ultrastructural changes when compared to NaCl. This suggests that Micrasterias can cope better with NaCl than with KCl. A possible explanation could be that salt stress in nature is usually caused by NaCl and not by KCl (Ramos et al., 2004). The physiological, biochemical, and ultrastructural changes observed in Micrasterias cells during salt stress- induced PCD differ in several features from those described after H 2 O 2 induction (see Dahrehshouri et al., 2008). As Morel and Dangle (1997) suggested, the diversity of morphologies during celldeath (including PCD) probably reflects different ways in which cells may die. In addition, our results show that different inducers may lead to dif- ferent celldeath pathways in one and the same organism.
96 fragmentation and the loss of MMP in HT22 cells as demonstrated in this study and previously shown by others (Figure 10 and 11; 71,72 ). Lipid peroxidation occurred in a biphasic manner after glutamate toxicity. An initial moderate increase appeared after 6-8 h of glutamate challenge followed by a second much more pronounced boost after 8-18 h arising from the mitochondria 72 . This work demonstrated that delivering of siCypA in HT22 cells before glutamate exposure abrogated the second boost of lipid peroxidation, while the initial increase was not affected (Figure 17). The inhibition of lipid peroxidation by siCypA was accompanied by decreased mitochondrial fragmentation and preserved MMP, which is in accordance with previous results showing that AIF release from the mitochondria was prohibited (Summary of results are depicted in Figure 39). However, the mechanism through which CypA silencing provides protective effects is still unknown. One possible mechanism could be through regulating cell cycle arrest and cell cycle transition. PPIases are known to be involved in the modulation of cellular pathways such as cell cycle regulation. In particular Pin1 regulates the stability of p53 and thereby gives rise to cell cycle arrest 210 . Cyp18, another member of the PPIase family, was identified as another interaction partner of p53. The impairment of the Cyp18-p53 interaction results in an accumulation of cells in the G2/M cell cycle phase. Moreover, Cyp18 knockout cells underwent increased p53-dependent apoptosis, suggesting an anti-apoptotic potential of Cyp18-p53 interaction 163 . In tumor lung cancer cells, CypA was identified to induce the up-regulation of cyclin D1 and cyclin-dependent kinase 4 (cdk4), whose activity is required for cell cycle G1/S transition, which resulted in cell cycle progression 189 . In fact, these findings revealed a pro-survival role for PPIases by modulating cell cycle phases, however, in the case of glutamate-induced celldeath in HT22 cells there is no evidence that CypA has an influence at all on cell cycle arrest/progression/transition (Figure 21).
Flow cytometric celldeath analysis in t-BuOOH-treated NIH3T3, HaCaT and Caco-2 cells additionally provided first evidence for t-BuOOH to be a so far unknown inducer of ferroptosis – a yet recognized RN subroutine, which is selectively inhibited by FS (Dixon et al. 2012; Friedmann Angeli et al. 2014; Skouta et al. 2014; Yang et al. 2014; Dong et al. 2015). These findings were strengthened by further experiments in our lab, demonstrating inhibition of t-BuOOH-induced necrosis in NIH3T3 cells by other agents that have been already described to block ferroptotic celldeath through (i) an antioxidative defense mechanism as mediated by FS against cytosolic and lipid ROS formation (e.g., liproxstatin-1, LX; α-tocopherol), or by (ii) iron chelation (e.g., deferoxamine) (Dixon et al. 2012; Skouta et al. 2014; Dong et al. 2015; Kabiraj et al., 2015; Zilka et al., 2017; Wenz et al., 2018). Besides lipid ROS formation and iron overload, GSH depletion or inhibition of GPX4 activity, have been also described to be crucial mediators of ferroptosis in vitro and in vivo (Yang & Stockwell, 2008; Dixon et al. 2012; Linkermann et al., 2013; Dixon et al., 2014; Friedmann Angeli et al., 2014; Linkermann et al., 2014; Xie et al., 2016; Yang & Stockwell 2016; Yang et al., 2016; Yang & Stockwell, 2016; Yu et al., 2017; Galluzzi et al., 2018). In the course of this, t-BuOOH-induced ferroptosis was also inhibited by the presence of NAC – a widely known ROS scavenger, which enhances the GSH de novo synthesis by supply of cysteine, and thus being yet described to block ferroptosis upon prevention of lipid ROS-mediated cellular damage and GSH depletion (Aruoma et al., 1998; Yang et al., 2014; Gao et al., 2015; Lörincz et al., 2015; Xie et al., 2016; Yu et al., 2017; Li et al., 2018; Wu et al., 2018).
The barley powdery mildew fungus Bgh is an obligate biotrophic pathogen that attacks epidermal cells of barley (Hordeum vulgare L.). The crucial step of fungal invasion is the penetration of the cell wall followed by the establish- ment of a haustorium that does not destroy PM integrity. During penetration, superoxide radical anions ( O2 ) are produced around the site of successful penetration and haustorium establishment (HuÈckelhoven and Kogel, 1998). Resistance to the powdery mildew fungus is mediated by major genes such as the powdery mildew resistance genes a x , MIa x , or by loss of powdery mildew resistance gene o (MLO)-function in Mlo-mutant genotypes (e.g. mlo5-barley, Jorgensen, 1994). The latter is expressed exclusively via penetration resistance, which is accompanied by accumu- lation of hydrogen peroxide but not by detectable O 2 generation (HuÈckelhoven and Kogel, 2003; Schulze-Lefert and Vogel, 2000). The wild-type MLO protein is a seven- transmembrane-protein reminiscent of G-protein-coupled receptors in animals and fungi (Devoto et al., 1999). It could be excluded that MLO signalling in susceptibility to Bgh depends on heterotrimeric G-proteins (Kim et al., 2002). However, HvRACB, a small monomeric G-protein of the RAC/ROP family, may be linked to the MLO-signalling path- way because the transient knock down by HvRacB-dsRNA interference strongly enhanced penetration resistance to Bgh in susceptible barley but not in lines bearing the required for mlo-speci®ed resistance (ror)1-2 mutant allele (Schultheiss et al., 2002), which was discovered as a sup- pressor allele of mlo-mediated penetration resistance (Freial- denhoven et al., 1996). This puts HvRACB as an upstream antagonist of the hypothetical ROR1 protein that was not yet identi®ed. Interestingly, recent studies revealed that over- expression of barley BAX (BCL-2 associated X protein; BCL-2: B-cell lymphoma protein-2) Inhibitor 1, a putative celldeath inhibitor without sequence similarity to MLO, is able to suppress mlo penetration resistance (HuÈckelhoven et al., 2003). This underscores a possible link of penetration resis- tance, RAC/ROPs and celldeath regulation.
Extensive attention has been given to the presence of the above-mentioned proteins known to interfere in tumor growth or apoptosis induction such as the tumor suppressor protein p53, caspases or members of the bcl-2-family (including Bak and Bax). These proteins are typically mutated in tumor cells (e.g. 50% of cancers exhibit mutated p53 (Harrod-Kim 2006) ). Thus, the outcome of treatments like PDT, interfering with cell processes involving these proteins, can be expected to depend on the mutation status of the latter. Astonishingly, experiments provided evidence that, although their presence may favor a certain celldeath pathway, the overall PDT-induced cell-killing does not seem to depend on them (Miller et al. 2007). Lately too, besides the mentioned proteins, mitochondrial phospholipids were found as possible early targets of PDT (Miller et al. 2007).
induced inﬂammation, importantly beyond TNF as the only endogenous inducer of this celldeath.
HOIP and HOIL-1 are essential to maintain skin homeostasis. To understand the role of LUBAC in the skin, we generated mice that lack HOIP or HOIL-1 selectively in epidermal keratinocytes by crossing Hoip- and Hoil-1-ﬂoxed mice with mice expressing the Cre recombinase under the control of the human keratin 14 (K14) promoter. The genotype of the mice was conﬁrmed by PCR (Supplementary Fig. 1a). At the protein level, deletion of HOIP or HOIL-1 in keratinocytes was veriﬁed by western blot and immunohistochemistry (Supplementary Fig. 1b, c). As expected, HOIP deﬁciency abrogated linear ubiquitination at the TNFR1- SC (Supplementary Fig. 1d) and reduced TNFR1-mediated NF- κB activation in primary murine keratinocytes (PMKs) without preventing it (Supplementary Fig. 1e). Mice homozygous for keratinocyte-speciﬁc deletion of HOIP or HOIL-1 (Hoip E-KO and Hoil-1 E-KO mice, respectively) were born at the expected Men- delian frequencies and were macroscopically indistinguishable from littermates up to postnatal day (P) 2 (data not shown). From this day onwards, however, both Hoip E-KO and Hoil-1 E-KO mice developed severely damaged and scaly skin, which, invariably, resulted in the death of these mice between P4 and P6 (Fig. 1 a). No Hoip or Hoil-1 gene dosage effect was observed as Hoip ﬂ/wt K14Cre + and Hoil-1 ﬂ/wt K14Cre + mice developed nor- mally into adulthood without showing any signs of skin disease (data not shown).