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One of the hottest topics in biomedical research is to decipher the functional implications of the Gasdermin (GSDM) protein family in human pathologies. These proteins are the key effectors of a lytic and pro‐inflammatory cell death type termed pyroptosis (also known as "Gasdermin‐mediated programmed cell death"). However, ever‐growing evidence showed that GSDMs can play multiple and complex roles in a context‐dependent manner. In this sense, Gasdermin‐B (GSDMB; the only GSDM gene absent in mice and rats) has been implicated in antibacterial defense, numerous inflammatory pathologies (e.g., asthma, ulcerative colitis), and cancer, but both cell death‐dependent and ‐independent functions have been reported in these diseases, fueling the debate on whether GSDMB has genuine pyroptotic capacity. Recently, a series of seminal papers cast light on the GSDMB multitasking capacity by showing that different GSDMB transcriptional isoforms have distinct biological activities. Nonetheless, there are still obscure areas to be clarified on the precise functional involvement of GSDMB translated variants in physiological and pathological conditions. In this viewpoint, we critically discuss the most recent and exciting data on this topic and propose a series of relevant challenges that need to be overcome before GSDMB‐driven biomedical applications (as a biomarker of disease risk/progression/outcome or as specific therapeutic target) become a reality in clinical settings. [ABSTRACT FROM AUTHOR]

Andrew Wyllie was a key figure in the discovery of the programmed cell death process termed apoptosis. He pioneered the study of apoptosis when this was a little‐known phenomenon and discovered the first biochemical marker of the process, internucleosomal DNA degradation. Andrew recently passed away, aged 78, but leaves a legacy of scientific achievement that opened up a new field of research. [ABSTRACT FROM AUTHOR]

P53 is a master regulator modulating the progression of acute kidney injury (AKI). However, the mechanism underlying p53 regulation in AKI needs further investigation. Mitotic arrest deficient 2 like 2 (MAD2B) is a subunit of DNA polymerase ζ. Its role in AKI remains unclear. Here, we demonstrated that MAD2B acted as an endogenous suppressor of p53. MAD2B conditional knockout augmented the upregulation of p53 in kidneys suffering from cisplatin‐induced AKI, therefore promoting the deterioration of renal function, G1 phase arrest and apoptosis of proximal tubular epithelial cells. Mechanistically, MAD2B deficiency activated the anaphase‐promoting complex/cyclosome (APC/C), which is an inhibitor of the well‐characterized p53‐directed E3 ligase MDM2. The decreased MDM2 diminished the degradation of p53, resulting in the upregulation of p53. The APC/C antagonist proTAME ameliorated cisplatin‐induced AKI and blocked MAD2B knockdown‐induced p53 upregulation and reduced cell cycle arrest and apoptosis in tubular epithelial cells by upregulating MDM2. These results indicate that MAD2B is a novel target for inhibiting p53 and ameliorating AKI. [ABSTRACT FROM AUTHOR]

Oxidative damage‐triggered apoptosis in lens epithelial cells is considered as a main risk factor in the pathogenesis of age‐related cataracts. Ku70 is a key factor in the DNA repair process of double‐strand breaks. In the present study, we aimed to investigate the role of Ku70 and its related E3 ubiquitin ligase in lens epithelial cell apoptosis. The levels of Ku70 in the anterior lens capsules of human cataracts and Emory mice were lower compared to controls. H2O2 treatment resulted in decreased expression of Ku70 through accelerating Ku70 ubiquitination. Parkin, an E3 ubiquitin ligase, could interact with Ku70 and promote the ubiquitination and degradation of this protein. In addition, ubiquitinated Ku70 was regulated by ubiquitin‐proteasome, autophagy‐lysosome and mitophagy pathways. Ectopic expression of Ku70 protected SRA01/04 cells from H2O2‐induced apoptosis, whereas silencing Ku70 exhibited the opposite trend. Co‐transfected with Parkin non‐ubiquitinatable Ku70 mutant could maintain its anti‐apoptosis ability, whereas wild‐type Ku70 failed. Moreover, Ku70 might facilitate mitochondrial fusion by increasing the expression of Mitofusin 1/2. The present study revealed that Parkin‐mediated Ku70 ubiquitination facilitated H2O2‐induced lens epithelial cell apoptosis through alleviating mitochondrial fusion, which could provide potential targets for age‐related cataract treatment. [ABSTRACT FROM AUTHOR]

Hypoxia‐inducible factor‐1, a heterodimer of alpha (HIF‐1α) and beta (HIF‐1β or ARNT) subunits, is a major regulator of the transcriptional response to hypoxia. However, HIF‐1α, the oxygen‐regulated subunit, also exerts nontranscriptional functions through interaction with proteins other than ARNT. We have previously shown that the subcellular localization and protein interactions of HIF‐1α are controlled by ERK‐mediated phosphorylation at Ser641/643. When HIF‐1α is modified at these sites, it is nuclear, binds to ARNT, interacts with nucleophosmin 1 (NPM1) and activates transcription of hypoxia‐target genes. On the contrary, unmodified HIF‐1α is bound by chromosomal region maintenance 1 (CRM1), exits the nucleus and, via its association with mortalin, is targeted to the mitochondria to form an antiapoptotic complex. To further characterize the latter function, recombinant fragments of HIF‐1α and mortalin were used for in vitro binding assays and immunoprecipitation experiments to map the respective binding sites and show that their interaction is direct and functional. We could also show that embelin, a natural product and known inhibitor of the mortalin‐p53 interaction, also disrupts the mortalin‐HIF‐1α association and, furthermore, removes unmodified HIF‐1α from mitochondria. Mitochondrial dissociation of HIF‐1α, either by embelin or overexpression of a HIF‐1α peptide harbouring the mortalin binding site, under stress conditions leads to mitochondrial localization of the pro‐apoptotic protein B‐cell lymphoma 2‐associated X protein (Bax) and induction of apoptosis. We suggest that when ERK activity is low under hypoxia, binding of HIF‐1α to mortalin inhibits mitochondrial recruitment of Bax and protects cells from apoptotic cell death. [ABSTRACT FROM AUTHOR]

The M2 isoform of pyruvate kinase (PKM2) is abundantly expressed in various cancer cells and associated with tumorigenesis, tumour proliferation and tumour progression. However, the role of PKM2 in these oncological processes is not fully understood. In the present study, we depleted PKM2 expression using RNA interference (RNAi), which induced apoptotic cell death and was accompanied by the downregulation of GM130, giantin, and p115 in HeLa and ME‐180 cervical cancer cells. The decreased expression of these proteins caused structural and functional disturbances in the Golgi apparatus, which manifested as the dispersion of the Golgi apparatus and delayed anterograde trafficking from the ER to the Golgi. The transcription factor, TFE3, which functions in the Golgi stress response, was responsible for the expression of GM130, giantin, and p115 that maintained the integrity of the organelle under normal growth conditions. In PKM2‐knockdown cells, the translation of TFE3 was markedly reduced. Knockdown of TFE3 by RNAi resulted in the downregulation of GM130, giantin, and p115, dispersion of the Golgi apparatus, and apoptotic cell death, similar to those observed following PKM2 knockdown. Conversely, the exogenous expression of TFE3 in PKM2 knockdown cells partially mitigated the aforementioned effects. We also demonstrated that PKM2 bound to the 5′ UTR on TFE3 mRNA and promoted translation. This study is the first to identify a new function for PKM2, which activates the basal Golgi stress response to maintain the integrity of the Golgi apparatus through the translation of TFE3 and promote cancer cell survival. [ABSTRACT FROM AUTHOR]

Renal carcinoma is a common malignancy for which the underlying molecular mechanisms warrant further investigation. T cell activation inhibitor mitochondrial (TCAIM), a mitochondrial protein, was found to be expressed to a low extent in renal carcinoma specimens. However, its role in carcinomas remains unclear. In the present study, the role of TCAIM in renal carcinoma was explored through loss‐of‐function and gain‐of‐function experiments. Here, we showed that TCAIM delayed the growth of renal carcinoma cells both in vitro and in vivo by modulating their cell cycle progress. Additionally, TCAIM also enhanced their sensitivity to sunitinib by aggravating apoptosis. Furthermore, TCAIM also decreased the adhesion and migration of renal carcinoma cells. Moreover, the transcription of TCAIM was regulated by vitamin D receptor, which acts as a transcriptional factor. To identify the pathways regulated by TCAIM, 425 unique proteins bound to TCAIM were pulled down through co‐immunoprecipitation and analyzed by mass spectrometry followed by Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. In summary, the present study reveals a tumor‐suppressor role of TCAIM in renal carcinoma cells, as well as its upstream regulation and downstream potential mechanisms. Our study provides theoretical bases and novel insights with respect to the development of new therapeutic strategies for the treatment of renal carcinoma. [ABSTRACT FROM AUTHOR]

The interactions between apoptotic and autophagic proteins via the proteolytic systems are known mechanisms through which autophagy and apoptosis regulate each other. In this issue of The FEBS Journal, Gentle and colleagues propose a mechanism through which autophagy regulates the induction of apoptosis at the level of the TIR‐domain‐containing adaptor‐inducing interferon‐β (TRIF) in TLR signaling. [ABSTRACT FROM AUTHOR]

Neutrophil granulocytes form the first line of host defense against invading pathogens and tissue injury. They are rapidly recruited from the blood to the affected sites, where they deploy an impressive arsenal of effectors to eliminate invading microbes and damaged cells. This capacity is endowed in part by readily mobilizable proteins acquired during granulopoiesis and stored in multiple types of cytosolic granules with each granule type containing a unique cargo. Once released, granule proteins contribute to killing bacteria within the phagosome or the extracellular milieu, but are also capable of inflicting collateral tissue damage. Neutrophil‐driven inflammation underlies many common diseases. Research over the last decade has documented neutrophil heterogeneity and functional versatility far beyond their antimicrobial function. Emerging evidence indicates that neutrophils utilize granule proteins to interact with innate and adaptive immune cells and orchestrate the inflammatory response. Granule proteins have been identified as important modulators of neutrophil trafficking, reverse transendothelial migration, phagocytosis, neutrophil life span, neutrophil extracellular trap formation, efferocytosis, cytokine activity, and autoimmunity. Hence, defining their roles within the inflammatory locus is critical for minimizing damage to the neighboring tissue and return to homeostasis. Here, we provide an overview of recent advances in the regulation of degranulation, granule protein functions, and signaling in modulating neutrophil‐mediated immunity. We also discuss how targeting granule proteins and/or signaling could be harnessed for therapeutic benefits. [ABSTRACT FROM AUTHOR]

zVAD‐fmk is a widely used pan‐caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD‐fmk induces autophagy by inhibiting the N‐glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345 [ABSTRACT FROM AUTHOR]

The proteome of a human cell is partitioned within organelles, such as the nucleus, and other subcellular compartments, such as the cytoplasm, forming a myriad of membrane‐bound and membrane‐free ultrastructures. This compartmentalization allows discrete biochemical processes to occur efficiently in isolation, with relevant proteins localized to appropriate niches to fulfill their biological function(s). Proper delivery and dynamic exchange of proteins between compartments underlie the regulation of many cellular processes, such as cell signaling, division, and programmed cell death. To this end, cells deploy dedicated trafficking mechanisms to ensure correct protein localization, as mis‐localization can result in pathology. In addition to trafficking, variation in the expression, modification, and physical associations of proteins within and between cells can result in biological heterogeneity, motivating the need for single‐cell measurements. In this review, we introduce diverse platform technologies developed for subcellular proteomics and high‐throughput systems biology, with the aim of providing mechanistic insights into fundamental cell biological processes underlying healthy and diseased states, and valuable public data resources. In contrast to the rapidly advancing field of single‐cell genomics, the single‐cell spatial proteomics toolbox remains in its infancy, but is poised to make considerable advances in the coming years. [ABSTRACT FROM AUTHOR]

Myocardial ischemia/reperfusion (I/R) injury is a frequent perioperative threat, with numerous strategies developed to limit and/or prevent it. One interesting axis of research is the anesthetic preconditioning (APc) agent's hypothesis (such as sevoflurane, SEV). However, APc's mode of action is still poorly understood and volatile anesthetics used as preconditioning agents are often not well suited in clinical practice. Here, in vitro using H9C2 cells lines (in myeloblast state or differentiated toward cardiomyocytes) and in vivo in mice, we identified that SEV‐induced APc is mediated by a mild induction of reactive oxygen species (ROS) that activates Akt and induces the expression of the anti‐apoptotic protein B‐cell lymphoma‐extra large (Bcl‐xL), therefore protecting cardiomyocytes from I/R‐induced death. Furthermore, we extended these results to human cardiomyocytes (derived from induced pluripotent stem ‐ IPS ‐ cells). Importantly, we demonstrated that this protective signaling pathway induced by SEV could be stimulated using the antidiabetic agent metformin (MET), suggesting the preconditioning properties of MET. Altogether, our study identified a signaling pathway allowing APc of cardiac injuries as well as a rational for the use of MET as a pharmacological preconditioning agent to prevent I/R injuries. [ABSTRACT FROM AUTHOR]

Apoptosis is a major form of programmed cell death (PCD) that eliminates unnecessary and potentially dangerous cells in all metazoan organisms, thus ensuring tissue homeostasis and many developmental processes. Accordingly, defects in the activation of the apoptotic pathway often pave the way to disease. After several decades of intensive research, the molecular details controlling the apoptosis program have largely been unraveled, as well as the regulatory mechanisms of caspase activation during apoptosis. Nevertheless, an ever‐growing list of studies is suggesting the essential role of caspases and other apoptotic proteins in ensuring nonlethal cellular functions during normal development, tissue repair, and regeneration. Moreover, if deregulated, these novel nonapoptotic functions can also instigate diseases. The difficulty of identifying and manipulating the caspase‐dependent nonlethal cellular processes (CDPs), as well as the nonlethal functions of other cell death proteins (NLF‐CDPs), meant that CDPs and NLF‐CDPs have been only curiosities within the apoptotic field; however, the recent technical advancements and the latest biological findings are assigning an unanticipated biological significance to these nonapoptotic functions. Here, we summarize the various talks presented in the first international conference fully dedicated to discuss CDPs and NFL‐CDPs and named 'The Batsheva de Rothschild Seminar on Non‐Apoptotic Roles of Apoptotic Proteins'. The conference was organized between September 22, 2019, and 25, 2019, by Eli Arama (Weizmann Institute of Science), Luis Alberto Baena‐Lopez (University of Oxford), and Howard O. Fearnhead (NUI Galway) at the Weizmann Institute of Science in Israel, and hosted a large international group of researchers. [ABSTRACT FROM AUTHOR]

Anoikis (detachment‐induced cell death) is a specific type of programmed cell death which occurs in response to the loss of the correct extracellular matrix connections. Anoikis resistance is an important mechanism in cancer invasiveness and metastatic behavior. Autophagy, on the other hand, involves the degradation of damaged organelles and the recycling of misfolded proteins and intracellular components. However, the intersection of these two cellular responses in lung cancer cells has not been extensively studied. Here, we identified that upon matrix deprivation, the lymphocyte lineage‐specific Ets transcription factor SPIB was activated and directly enhanced SNAP47 transcription in certain lung cancer cells. Loss of attachment‐induced autophagy significantly increased anoikis resistance by SPIB activation. Consistent with this function, SPIB depletion by short hairpin RNA abrogated SNAP47 transcriptional activation upon matrix deprivation. Therefore, these data delineate an important role of SPIB in autophagy‐mediated anoikis resistance in lung cancer cells. Accordingly, these findings suggest that manipulating SPIB‐regulated pathways in vivo and evaluating the impact of anoikis resistance warrant further investigation. Database: RNA sequencing and ChIP sequencing data are available in Gene Expression Omnibus database under the accession numbers GSE106592 and GSE125561, respectively. [ABSTRACT FROM AUTHOR]

Development of human leishmaniasis is dependent on the ability of intracellular Leishmania parasites to spread and enter macrophages. The mechanism through which free promastigotes and amastigotes bind and enter host macrophages has been previously investigated; however, little is known about intracellular trafficking and cell‐to‐cell spreading. In this study, the mechanism involved in the spreading of Leishmania aethiopica and Leishmania mexicana was investigated. A significant increase in phosphatidylserine (PS) exhibition, cytochrome C release, and active caspase‐3 expression was detected (P < 0.05) during L. aethiopica, but not L. mexicana spreading. A decrease (P < 0.05) of protein kinase B (Akt) protein and BCL2‐associated agonist of cell death (BAD) phosphorylation was also observed. The nuclear factor kappa‐light‐chain enhancer of activated B cells (NF‐kB) signaling pathway and pro‐apoptotic protein protein kinase C delta (PKC‐δ) were downregulated while inhibition of caspase‐3 activation prevented L. aethiopica spreading. Overall suggesting that L. aethiopica induces host cell's apoptosis during spreading in a caspase‐3‐dependent manner. The trafficking of amastigotes within macrophages following cell‐to‐cell spreading differed from that of axenic parasites and involved co‐localization with lysosomal‐associated membrane protein 1 (LAMP‐1) within 10 min postinfection. Interestingly, following infection with axenic amastigotes and promastigotes, co‐localization of parasites with LAMP‐1‐positive structures took place at 1 and 4 h, respectively, suggesting that the membrane coat and LAMP‐1 protein were derived from the donor cell. Collectively, these findings indicate that host cell apoptosis, demonstrated by PS exhibition, caspase‐3 activation, cytochrome C release, downregulation of Akt, BAD phosphorylation, NF‐kB activation, and independent of PKC‐δ expression, is involved in L. aethiopica spreading. Moreover, L. aethiopica parasites associate with LAMP‐rich structures when taken up by neighboring macrophages. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are emerging as important regulators of multiple cellular processes such as cell invasion, growth, apoptosis and differentiation. LncRNAs can function as competing endogenous RNAs (ceRNAs) which sponge and sequester microRNA (miRNA) to regulate specific targets. Previously, we found that the target genes of several miRNAs, including FADD, Fas, Casp and Bax, are related to neuronal apoptosis and form a regulatory network. Among several factors, microRNA‐296‐5p expression was found to be negatively correlated with caspase activity and apoptosis. Here, we aimed to investigate the role of miR‐296‐5p in neuroblastoma (NB) cells. By performing quantitative real‐time PCR (qRT‐PCR), western blot and flow cytometry assays we analysed the expression of apoptotic markers in NB cells transfected with miR‐296‐5p mimics or inhibitor. Pathway‐specific PCR array allowed us to identify the target genes of miR‐296‐5p. Using LncBase online tool, we predicted lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) as an upstream regulator of miR‐296‐5p. The binding of KCNQ1OT1 and miR‐296‐5p was validated via RNA immunoprecipitation and Biotin pull‐down assays. We also demonstrate that miR‐296‐5p suppresses apoptosis of NB cells in vitro and in vivo. Mechanistically, miR‐296‐5p directly bound the 3′UTR of Bax mRNA, thus repressing Bax at the mRNA and protein level. Moreover, through bioinformatic analysis and molecular experiments, we showed that KCNQ1OT1 sponged miR‐296‐5p and impaired its effect on NB cell apoptosis. In summary, KCNQ1OT1 is a potent promoting factor of cell apoptosis, which acts by sponging miR‐296‐5p and upregulating Bax. Our findings identify a regulatory axis of cell fate in NB cells. [ABSTRACT FROM AUTHOR]

Apoptosis of vascular endothelial cells (VEC) is the main form of vascular injury that is closely linked to numerous cardiovascular diseases. Therefore, it is important to find new factors that can suppress VEC apoptosis. By using long noncoding RNA (lncRNA) microarray analysis, we found a new read‐through lncRNA,MROH7‐TTC4, which acted as an apoptosis inhibitor in VECs. Furthermore, by using the inhibitor (ABO) of annexin A7 (ANXA7) GTPase, we discovered that ANXA7 translocated into nucleus and interacted with 5′→3′ exoribonuclease (XRN2). The decreased XRN2 phosphorylation induced by ANXA7 GTPase activity inhibition, promoted MROH7‐TTC4 expression. Moreover, T‐cell intracellular antigen‐1 (TIA1), a binding protein of MROH7‐TTC4, processed it into MROH7 and TTC4 that could inhibit VEC apoptosis. Here, we conclude that inhibiting ANXA7 GTPase activity promotes the interaction of ANXA7 and XRN2 in nucleus, which regulates the read‐through transcription of MROH7‐TTC4, and TIA1 is responsible for the process of MROH7‐TTC4 that inhibits apoptosis through MROH7 and TTC4. [ABSTRACT FROM AUTHOR]

The ubiquitin‐proteasome system is an important regulatory machinery involved in proteostasis and cellular signaling. Proteins are ubiquitinated via the concerted action of E1 ubiquitin‐activating enzymes, E2 ubiquitin‐conjugating enzymes, and E3 ubiquitin ligases. Although most of the studies to date focus on the significance of E3 ubiquitin ligases in disease development and therapeutic treatment, recent discoveries suggest that E2 ubiquitin‐conjugating enzymes might also be potential drug targets. The ubiquitin‐conjugating enzyme E2 O (UBE2O), an E3‐independent E2 (i.e. an E2/E3 hybrid enzyme), can directly mediate the ubiquitination of many substrates. These include 5′‐AMP‐activated protein kinase catalytic subunit α2 (AMPKα2), tumor suppressor ubiquitin carboxyl‐terminal hydrolase BAP1, mixed‐lineage leukemia (MLL) protein, SMAD family member 6 (SMAD6), transcription factor c‐Maf and aryl hydrocarbon receptor nuclear translocator‐like protein 1 (ARNTL or BMAL1), and free ribosomal proteins, which are ubiquitinated in distinct ways, thereby associating UBE2O with a variety of biological functions. Furthermore, UBE2O is frequently amplified or mutated in multiple cancers, and its high expression is associated with low survival rate of gastric, lung, breast, and prostate cancer patients. However, the molecular mechanisms by which UBE2O contributes to tumor initiation and progression are not fully elucidated. This review focuses on emerging insights from genetics, biochemistry, and cell biology to explore the biological functions of UBE2O and its therapeutic potential. [ABSTRACT FROM AUTHOR]

Bistability (coexistence of two stable steady states in a dynamical system) is a key mechanism of cellular decision‐making and has been observed in many biochemical reaction networks such as mitogen‐activated protein kinase (MAPK) signaling pathways. Theoretical studies have shown that bistability can arise in a single two‐site MAPK phosphorylation and dephosphorylation cycle. However, the bistable behavior mostly relies on the kinetic mechanisms and parameters of this two‐site modification. In exploring the system‐level properties of MAPK regulation, most models to date focus on two limiting reaction regimes, distributive and processive, and are characterized by high levels of parametric uncertainty. Here, we developed a combined kinetic method which applies a continuous spectrophotometric enzyme‐coupled assay incorporated with the viscosity approach, to perform detailed kinetic analyses of p38α MAPK dual phosphorylation by MKK6. Almost all kinetic rate constants for the first and second phosphorylation steps in p38α activation have been quantitatively determined, supporting that the phosphorylation occurs randomly in the first step, albeit preferring the tyrosine residue. The release rates of monophosphorylated p38α from MKK6, either as the product in the first modification or as the substrate in the second step, were comparable to the respective adjacent phosphoryl transfer steps. These results indicated that dual phosphorylation of p38α by MKK6 involves a random, partially processive mechanism. Based on the experimentally determined models and parameters, dynamics of the p38α‐MKK6‐MKP5 system were explored, demonstrating for the first time that bistability can arise with this model at biologically feasible parameter values. Enzymes: p38α (EC 2.7.11.24); MKK6 (EC 2.7.12.2). Almost all kinetic rate constants for the first and second phosphorylation steps in p38α activation have been quantitatively determined. Dynamics of the p38α‐MKK6‐MKP5 system were explored based on these experimentally determined models and parameters, demonstrating for the first time that bistability (coexistence of two stable steady states in a dynamical system) can arise with this model at biologically feasible parameter values. [ABSTRACT FROM AUTHOR]

The remarkable pro‐apoptotic properties of tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) have led to considerable interest in this protein as a potential anticancer therapeutic. However, TRAIL is largely ineffective in inducing apoptosis in certain cancer cells, and the mechanisms underlying this selectivity are unknown. In colon adenocarcinomas, posttranslational modifications including O‐ and N‐ glycosylation of death receptors were found to correlate with TRAIL‐induced apoptosis. Additionally, mRNA levels of fucosyltransferase 3 (FUT3) and 6 (FUT6) were found to be high in the TRAIL‐sensitive colon adenocarcinoma cell line COLO 205. In this study, we use agonistic receptor‐specific TRAIL variants to dissect the contribution of FUT3 and FUT6‐mediated fucosylation to TRAIL‐induced apoptosis via its two death receptors, DR4 and DR5. Triggering of apoptosis by TRAIL revealed that the low FUT3/6‐expressing cells DLD‐1 and HCT 116 are insensitive to DR5 but not to DR4‐mediated apoptosis. By contrast, efficient apoptosis is mediated via both receptors in high FUT3/6‐expressing COLO 205 cells. The reconstitution of FUT3/6 expression in DR5‐resistant cells completely restored TRAIL sensitivity via this receptor, while only marginally enhancing apoptosis via DR4 at lower TRAIL concentrations. Interestingly, we observed that induction of the salvage pathway by external administration of l‐fucose restores DR5‐mediated apoptosis in both DLD‐1 and HCT 116 cells. Finally, we show that fucosylation influences the ligand‐independent receptor association that leads to increased death inducing signalling complex (DISC) formation and caspase‐8 activation. Taken together, these results provide evidence for the differential impact of fucosylation on signalling via DR4 or DR5. These findings provide novel opportunities to enhance TRAIL sensitivity in colon adenocarcinoma cells that are highly resistant to DR5‐mediated apoptosis. Because of its ability to induce apoptosis in tumour cells, TRAIL [Tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand] has sparked a lot of interest in oncology research. Posttranslational modifications and upregulation of fucosylation were previously found to correlate with TRAIL‐induced apoptosis. This study shows that fucosylation triggers the clustering of death receptors DR4 and DR5, leading to increased cell death. Moreover, apoptosis mediated by DR5 can be stimulated by simple external administration of l‐fucose. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is the pathogen responsible for tuberculosis, a leading cause of illness and death worldwide. Growing evidence suggests that the proinflammatory cytokine IL‐32 plays a major role in host defences against pathogens such as Mtb. IL‐32 exists in six alternatively spliced isoforms, but antituberculosis effects have been reported only for some of them. In this study, we examined the effect of all six IL‐32 isoforms on Mtb replication in the murine macrophage cell line RAW 264.7. Compared with cells transfected with the other isoforms, IL‐32ε‐transfected cells exhibited the strongest antituberculosis effect and the highest rate of Mtb‐induced apoptosis. Of note, this apoptosis pathway was independent of caspase‐3 activation. Instead, N‐Myc interactor (NMI), an inhibitor of Wnt signalling, was a key player in IL‐32ε‐mediated apoptosis by inhibiting Wnt/β‐catenin signalling and thereby activating c‐Myc‐mediated apoptosis. Moreover, we identified two cis‐acting elements that are binding sites for the transcriptional regulators paired box 6 (PAX6) and transcription factor CP2 (TFCP2) in the promoter of NMI and these elements proved essential for IL‐32ε‐induced upregulation of Nmi expression. Furthermore, IL‐32ε‐mediated activation of the mitogen‐activated protein kinase p38 also contributed to NMI upregulation. In conclusion, our results demonstrate that Mtb infection‐induced IL‐32ε‐mediated apoptosis in macrophages plays a key role in host defences against Mtb. In this study we demonstrate that IL‐32ε exhibits antibacterial and pro‐apoptotic effects in RAW264.7 cells infected with Mycobacterium tuberculosis (Mtb). IL‐32ε leads to activation of p38 MAPK, which in turn activates Pax6 and CP2, resulting in upregulation of N‐Myc interactor (NMI) expression. Finally, IL‐32ε represses Wnt/β‐catenin signalling, which results in c‐Myc‐induced apoptosis. Our results contribute to understanding the role of IL‐32 in control of Mtb. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a neurodegenerative genetic disorder. Although described as a brain pathology, there is evidence suggesting that defects in other systems can contribute to disease progression. In line with this, cardiovascular defects are a major cause of death in HD. To date, relatively little is known about the peripheral abnormalities associated with the disease. Here, we applied a range of assays to evaluate cardiac electro‐mechanical properties in vivo, using a previously characterized mouse model of HD (BACHD), and in vitro, using cardiomyocytes isolated from the same mice. We observed conduction disturbances including QT interval prolongation in BACHD mice, indicative of cardiac dysfunction. Cardiomyocytes from these mice demonstrated cellular electro‐mechanical abnormalities, including a prolonged action potential, arrhythmic contractions, and relaxation disturbances. Cellular arrhythmia was accompanied by an increase in calcium waves and increased Ca2+/calmodulin‐dependent protein kinase II activity, suggesting that disruption of calcium homeostasis plays a key part. We also described structural abnormalities in the mitochondria of BACHD‐derived cardiomyocytes, indicative of oxidative stress. Consistent with this, imbalances in superoxide dismutase and glutathione peroxidase activities were detected. Our data provide an in vivo demonstration of cardiac abnormalities in HD together with new insights into the cellular mechanistic basis, providing a possible explanation for the higher cardiovascular risk in HD. Huntington's disease (HD) is a neurodegenerative disorder and the main cause of patients' death is related to cardiovascular defects. Here, we investigated cardiac dysfunction and its mechanisms in 12‐month‐old BACHD mice, which correspond to the late clinical manifestation in humans. Our data provide an in vivo demonstration together with a new evidence of the cellular mechanistic basis in cardiac HD. [ABSTRACT FROM AUTHOR]

Caspase‐2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase‐2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14‐3‐3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase‐2 zymogen. However, the structural details of this interaction and the exact role of 14‐3‐3 in the regulation of caspase‐2 activation remain unclear. Moreover, the caspase‐2 region with both 14‐3‐3‐binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14‐3‐3 binding may regulate the subcellular localization of caspase‐2. Here, we report a structural analysis of the 14‐3‐3ζ:caspase‐2 complex using a combined approach based on small angle X‐ray scattering, NMR, chemical cross‐linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase‐2 and 14‐3‐3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase‐2 are positioned within the central channel of the 14‐3‐3 dimer and stabilized through interactions with the C‐terminal helices of both 14‐3‐3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase‐2 activation by dimerization, is sterically occluded by the 14‐3‐3 dimer, thereby likely preventing caspase‐2 activation. In addition, 14‐3‐3 protein binding to caspase‐2 masks its NLS. Therefore, our results suggest that 14‐3‐3 protein binding to caspase‐2 may play a key role in regulating caspase‐2 activation. Database: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG). Caspase‐2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase‐2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14‐3‐3. Here, we report the structural analysis of the 14‐3‐3ζ:caspase‐2 complex which suggested that 14‐3‐3 binding masks both the nuclear localization sequence and the dimerization interface of caspase‐2. [ABSTRACT FROM AUTHOR]

One of the classic hallmarks of cancer is disruption of cell death signalling. Inhibition of cell death promotes tumour growth and metastasis, causes resistance to chemo‐ and radiotherapies as well as targeted agents, and is frequently due to overexpression of antiapoptotic proteins rather than loss of pro‐apoptotic effectors. FLIP is a major apoptosis‐regulatory protein frequently overexpressed in solid and haematological cancers, in which its high expression is often correlated with poor prognosis. FLIP, which is expressed as long (FLIP(L)) and short (FLIP(S)) splice forms, achieves its cell death regulatory functions by binding to FADD, a critical adaptor protein which links FLIP to the apical caspase in the extrinsic apoptotic pathway, caspase‐8, in a number of cell death regulating complexes, such as the death‐inducing signalling complexes (DISCs) formed by death receptors. FLIP also plays a key role (together with caspase‐8) in regulating another form of cell death termed programmed necrosis or 'necroptosis', as well as in other key cellular processes that impact cell survival, including autophagy. In addition, FLIP impacts activation of the intrinsic mitochondrial‐mediated apoptotic pathway by regulating caspase‐8‐mediated activation of the pro‐apoptotic Bcl‐2 family member Bid. It has been demonstrated that FLIP can not only inhibit death receptor‐mediated apoptosis, but also cell death induced by a range of clinically relevant chemotherapeutic and targeted agents as well as ionizing radiation. More recently, key roles for FLIP in promoting the survival of immunosuppressive tumour‐promoting immune cells have been discovered. Thus, FLIP is of significant interest as an anticancer therapeutic target. In this article, we review FLIP's biology and potential ways of targeting this important tumour and immune cell death regulator. FLIP is a key regulator of apoptosis through its ability to regulate procaspase‐8 dimerization, processing and activation. In addition, FLIP is involved in other critical cellular processes such as autophagy and necroptosis. Here, we provide an up‐to‐date review on the function and regulation of FLIP and discuss strategies for therapeutic targeting of this important cell death regulator in cancer. [ABSTRACT FROM AUTHOR]

Craniofacial malformations are common congenital birth defects and usually caused by abnormal development of the cranial neural crest cells. Some nucleolar ribosome biogenesis factors are implicated in neural crest disorders also known as neurocristopathies. However, the underlying mechanisms linking ribosome biogenesis and neural crest cell (NCC) development remain to be elucidated. Here we report a novel zebrafish model with a CRISPR/Cas9‐generated esf1 mutation, which exhibits severe NCC‐derived pharyngeal cartilage loss and defects in the eyes, brain, and heart. The expression of several typical NCC markers, including sox10, dlx2a, nrp2b, crestin, vgll2a, and sox9a, was reduced in the head of the esf1 mutants, which indicates that esf1 plays a role in the development of zebrafish NCCs. We demonstrate that, similar to the yeast, loss of esf1 in zebrafish leads to defects in 18S rRNA biogenesis and ribosome biogenesis. We also show strong upregulation of p53 signaling as well as apoptosis, and poor proliferation in mutants. Inactivation of p53 rescues the early tissue defects and pharyngeal cartilage loss observed in esf1 mutants, indicating that increased cell death and pharyngeal cartilage defects observed in esf1 mutants are mediated via upregulated p53 signaling pathways. Based on transplantation analysis, we found esf1 functions in NCC in a cell autonomous fashion. Together, our results suggest that esf1 is required for NCC development and pharyngeal cartilage formation. These studies provide a potential model for investigating the relationship between ribosome biogenesis defects and craniofacial neurocristopathies. [ABSTRACT FROM AUTHOR]

The cytotoxic synapse formed between cytotoxic T lymphocytes or natural killer cells expressing CD95L and target cells with CD95 on their surface is a key pathway for apoptosis induction by the immune system. Despite similarities with the immune synapse in antigen presenting cells, little is known about the role of the spatiotemporal organization of agonistic proteins/receptor interactions for CD95 signaling. Here, we have developed an artificial cytotoxic synapse to examine how mobility and geometry of an anti‐CD95 agonistic antibody affect receptor aggregation and mobility, ie the first step of receptor activation. By measuring the distribution, diffusion coefficient, and fraction of immobile CD95 receptor in living cells, we show that at short times, the initial activation of CD95 occurs locally and is limited to the contact region of the cytotoxic synapse. This anisotropic activation of apoptotic signaling supports a role for confined interactions on the efficiency of signal transduction that may have implications for biomedical applications of extrinsic apoptosis induction. [ABSTRACT FROM AUTHOR]

Ischemic stroke caused by an embolus or local thrombosis results in neural tissue damage (an infarct) in the territory of the occluded cerebral artery. Decades of studies have increased our understanding of the molecular events during cerebral infarction; however, translation of these discoveries to druggable targets for ischemic stroke treatment has been largely disappointing. Interleukin‐4 (IL‐4) is a multifunctional cytokine that exerts its cellular activities via the interleukin‐4 receptor α (IL‐4Rα). This cytokine receptor complex is associated with diverse immune and inflammatory responses. Recent studies have suggested a role of the cytokine IL‐4 in long‐term ischemic stroke recovery, involving immune cell activity. In contrast, the role of the receptor, IL‐4Rα especially in the acute phase of infarction is unclear. In this study, we determined that IL‐4Rα is expressed on neurons and that during the early phases of cerebral infarction (24 h) levels of this receptor are increased to regulate cellular apoptosis factors through activation of STAT6. In this context, we show a neuroprotective role for IL‐4Rα in an in vivo surgical model of cerebral ischemia and in ex vivo brain slice explants, using both genetic knockout of this receptor and RNAi‐mediated gene knockdown. IL‐4Rα may therefore represent a novel target and pathway for therapeutic development in ischemic stroke. [ABSTRACT FROM AUTHOR]

Overexpression of BCLX and BFL1/A1 has been reported in various human malignancies and is associated with poor prognosis and drug resistance, identifying these prosurvival BCL2 family members as putative drug targets. We have generated transgenic mice that express human BFL1 or human BCLX protein throughout the haematopoietic system under the control of the Vav gene promoter. Haematopoiesis is normal in both the Vav‐BFL1 and Vav‐BCLX transgenic (TG) mice and susceptibility to spontaneous haematopoietic malignancies is not increased. Lymphoid cells from Vav‐BCLX TG mice exhibit increased resistance to apoptosis in vitro while most blood cell types form Vav‐BFL1 TG mice were poorly protected. Both transgenes significantly accelerated lymphomagenesis in Eμ‐MYC TG mice and, surprisingly, the Vav‐BFL1 transgene was the more potent. Unexpectedly, expression of transgenic BFL1 RNA and protein is significantly elevated in B lymphoid cells of Vav‐BFL1/Eμ‐MYC double‐transgenic compared to Vav‐BFL1 mice, even during the preleukaemic phase, providing a rationale for the potent synergy. In contrast, Vav‐BCLX expression was not notably different. These mouse models of BFL1 and BCLX overexpression in lymphomas should be useful tools for the testing the efficacy of novel human BFL1‐ and BCLX‐specific inhibitors. [ABSTRACT FROM AUTHOR]

The main function of the A kinase‐anchoring proteins (AKAPs) is to target the cyclic AMP‐dependent protein kinase A (PKA) to its cellular substrates through the interaction with its regulatory subunits. Besides anchoring of PKA, AKAP8 participates in regulating the histone H3 lysine 4 (H3K4) histone methyltransferase (HMT) complexes. It is also involved in DNA replication, apoptosis, transcriptional silencing of rRNA genes, alternative splicing, and chromatin condensation during mitosis. In this study, we focused on the interaction between AKAP8 and the core subunit of all known H3K4 HMT complexes—DPY30 protein. Here, we demonstrate that the PKA‐binding domain of AKAP8 and the C‐terminal domain of DPY30, also called Dpy‐30 motif, are crucial for the interaction between these proteins. We show that a single amino acid substitution in DPY30 L69D affects its dimerization and completely abolishes its interaction with AKAP8 and another DPY30‐binding partner brefeldin A‐inhibited guanine nucleotide‐exchange protein 1 (BIG1), which is also AKAP domain‐containing protein. We further demonstrate that AKAP8 interacts with DPY30 and the RII alpha regulatory subunit of PKA both in the interphase and in mitotic cells, and we show evidences that AKAP8L, a homologue of AKAP8, interacts with core subunits of the H3K4 HMT complexes, which suggests its role as a potential regulator of these complexes. The results presented here reinforce the analogy between AKAP8–RII alpha and AKAP8–DPY30 interactions, postulated before, and improve our understanding of the complexity of the cellular functions of the AKAP8 protein. [ABSTRACT FROM AUTHOR]

Inhibitory PAS domain protein ( IPAS) is a bifunctional protein that downregulates hypoxic gene expression and exerts proapoptotic activity by preventing prosurvival activity of Bcl- xL and its related factors. Proapoptotic activity of IPAS is attenuated by the activation of the PINK1-Parkin pathway, and involved in neuronal degeneration in an experimental mouse model of Parkinson's disease. The current study shows that phosphorylation of IPAS at Ser184 by MAPK-activated protein kinase 2 ( MK2 or MAPKAPK2) enhances the proapoptotic function of IPAS. Perinuclear clustering of mitochondria and activation of caspase-3 caused by the transient expression of EGFP- IPAS were increased by UVB irradiation. The C-terminal region of IPAS mediated the UVB susceptibility of IPAS. Increase in IPAS-induced mitochondrial clustering by UVB was completly inhibited by the p38 MAPK inhibitor SB203580. Mass spectrometry analysis of UVB-activated IPAS identified several phosphorylation sites in the C-terminal region containing p38 MAPK consensus phosphorylation sites at Ser219 and Ser223, and an MK2 consensus site at Ser184. Although mutations of Ser219 and Ser223 to Ala did not suppress the UVB-induced mitochondrial clustering, replacement of Ser184 with Ala blocked it. A phosphomimetic substitution at Ser184 enhanced mitochondrial clustering and activation of caspase-3 without UVB exposure. Furthermore, binding affinity to Bcl- xL was increased by the mutation. Treatment of PC12 cells with CoCl2 caused activation of MK2 and mitochondrial clustering. IPAS-dependent cell death induced by CoCl2 in PC12 cells was decreased by the treatment with the MK2 inhibitor MK2 inhibitor III and by si RNA-directed silencing of MK2. [ABSTRACT FROM AUTHOR]

The formation of amyloid-like protein structures has recently emerged as a feature in signal transduction, particularly in innate immunity. These structures appear to depend on defined domains for their formation but likely also require dedicated ways to terminate signalling. We, here, define the innate immunity protein/Toll-like receptor adaptor TIR-domain-containing adapter-inducing interferon-β ( TRIF) as a novel platform of fibril formation and probe signal initiation through TRIF as well as its termination in Toll-like receptor 3 ( TLR3)-stimulated melanoma cells. A main signalling pathway triggered by TLR3 caused apoptosis, which was controlled by inhibitor of apoptosis proteins and was dependent on RIPK1 and independent of TNF. Using correlative electron/fluorescence microscopy, we visualised fibrillar structures formed through both Toll/interleukin-1 receptor and RIP homotypic interacting motif regions of TRIF. We provide evidence that these fibrillary structures are active signalling platforms whose activity is terminated by autophagy. TRIF-signalling enhanced autophagy, and fibrillary structures were partly contained within autophagosomes. Inhibition of autophagy increased levels of pro-apoptotic TRIF complexes, leading to the accumulation of active caspase-8 and enhanced apoptosis while stimulation of autophagy reduced TRIF-dependent death. We conclude that pro-death signals through TRIF are regulated by autophagy and propose that pro-apoptotic signalling through TRIF/ RIPK1/caspase-8 occurs in fibrillary platforms. [ABSTRACT FROM AUTHOR]

Cancer cells require both energy and material to survive and duplicate in a competitive environment. Nutrients, such as amino acids ( AAs), are not only a caloric source, but can also modulate cell metabolism and modify hormone homeostasis. Our hypothesis is that the environmental messages provided by AAs rule the dynamics of cancer cell life or death, and the alteration of the balance between essential amino acids ( EAAs) and non-essential amino acids ( NEAAs) (lower and higher than 50%, respectively) present in nutrients may represent a key instrument to alter environment-dependent messages, thus mastering cancer cells destiny. In this study, two AA mixtures, one exclusively consisting of EAAs and the other consisting of 85% EAAs and 15% NEAAs, were tested to explore their effects on the viability of both normal and cancer cell lines and to clarify the molecular mechanisms involved. Both mixtures exerted a cell-dependent anti-proliferative, cytotoxic effect involving the inhibition of proteasome activity and the consequent activation of autophagy and apoptosis. These results, besides further validating the notion of the peculiar interdependence and extensive crosstalk between the ubiquitin-proteasome system (UPS) and autophagy, indicate that variation in the ratio of EAAs and NEAAs can deeply influence cancer cell survival. Consequently, customization of dietary ratios among EAAs and NEAAs by specific AA mixtures may represent a promising anticancer strategy able to selectively induce death of cancer cells through the induction of apoptosis via both UPS inhibition and autophagy activation. [ABSTRACT FROM AUTHOR]

Atherosclerosis predisposes patients to cardiovascular diseases, such as myocardial infarction and stroke. Instigation of vascular injury is triggered by retention of lipids and inflammatory cells in the vascular endothelium. Whereas these vascular lesions develop in young adults and are mostly considered harmless, over time persistent inflammatory and remodeling processes will ultimately damage the arterial wall and cause a thrombotic event due to exposure of tissue factors into the lumen. Evidence from human tissues and preclinical animal models has clearly established the role of cathepsin cysteine proteases in the development and progression of vascular lesions. Hence, understanding the function of cathepsins in atherosclerosis is important for developing novel therapeutic strategies and advanced point of care diagnostics. In this review we will describe the roles of cysteine cathepsins in different cellular process that become dysfunctional in atherosclerosis, such as lipid metabolism, inflammation and apoptosis, and how they contribute to arterial remodeling and atherogenesis. Finally, we will explore new horizons in protease molecular imaging, which may potentially become a surrogate marker to identify future cardiovascular events. [ABSTRACT FROM AUTHOR]

Organismal development and function requires multiple and accurate signal transduction pathways to ensure that proper balance between cell proliferation, differentiation, inactivation, and death is achieved. Cell death via apoptotic caspase signal transduction is extensively characterized and integral to this balance. Importantly, the view of apoptotic signal transduction has expanded over the previous decades. Subapoptotic caspase signaling has surfaced as mechanism that can promote the adoption of a range of cellular fates. An emerging mechanism of subapoptotic caspase signaling is the activation of the caspase-activated DNase ( CAD) through controlled cleavage of the inhibitor of CAD ( ICAD). CAD-induced DNA breaks incite a DNA damage response, frequently invoking p53 signaling, that transduces a change in cell fate. Cell differentiation and senescence are fates demonstrated to arise from CAD-induced DNA breaks. Furthermore, an apparent consequence of CAD activity is also emerging, as a potential source of oncogenic mutations. This review will discuss the mechanisms underlying CAD-induced DNA breaks and highlight how CAD activity promotes diverse cell fates. [ABSTRACT FROM AUTHOR]

Abnormal lipid metabolism, renal lipid accumulation and lipotoxicity are associated with the pathological features of glomerulopathy. However, the mechanisms by which lipid accumulation leads to the development or progression of this disease have not been fully elucidated. In this work, we have identified a role for the rate-limiting enzyme in lipolysis, adipose triglyceride lipase ( ATGL; also called patatin-like phospholipase domain-containing protein 2), in renal lipid metabolism and kidney disease. ATGL-deficient ( Atgl(−/−)) mice displayed albuminuria, accompanied by ectopic deposition of fat in the kidney. Magnetic resonance imaging demonstrated that the contrast agent gadopentetic acid was retained in kidney tissue, suggesting defects in the glomerular filtration barrier. Furthermore, transmission electron microscopy revealed lipid deposits in the podocyte, along with foot process fusion and morphological changes suggestive of apoptosis. Indeed, shRNA-mediated depletion of ATGL promoted podocyte apoptosis, accompanied by increased levels of intracellular reactive oxygen species ( ROS) and F-actin fibre redistribution. These effects could be partially reversed by treatment with the antioxidant N-acetylcysteine. These data suggest that ATGL deficiency induces renal lipid accumulation, proteinuria and glomerular filtration barrier dysfunction and implicate increased intracellular ROS levels in inducing podocyte F-actin rearrangement, foot process fusion and apoptosis that underlie these pathological features. Enzymes Adipose triglyceride lipase, EC3.1.1.3. [ABSTRACT FROM AUTHOR]

It is now well recognized that mutations, deregulated expression, and aberrant recruitment of epigenetic readers, writers, and erasers are fundamentally important processes in the onset and maintenance of many human tumors. The molecular, biological, and biochemical characteristics of a particular class of epigenetic erasers, the histone deacetylases ( HDACs), have been extensively studied and small-molecule HDAC inhibitors ( HDACis) have now been clinically approved for the treatment of human hemopoietic malignancies. This review explores our current understanding of the biological and molecular effects on tumor cells following HDACi treatment. The predominant responses include induction of tumor cell death and inhibition of proliferation that in experimental models have been linked to therapeutic efficacy. However, tumor cell-intrinsic responses to HDACi, including modulating tumor immunogenicity have also been described and may have substantial roles in mediating the antitumor effects of HDACi. We posit that the field has failed to fully reconcile the biological consequences of exposure to HDACis with the molecular events that underpin these responses, however progress is being made. Understanding the pleiotrophic activities of HDACis on tumor cells will hopefully fast track the development of more potent and selective HDACi that may be used alone or in combination to improve patient outcomes. [ABSTRACT FROM AUTHOR]

Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation. DNA-damage associated with differentiation is accompanied by phosphorylation of Histone 2 AX, an event normally catalysed by kinases ATR, ATM or DNA- PK. However, the kinase responsible for phosphorylation during differentiation is not known. Here we show that inhibition of DNA- PK, but not of ATR or ATM, blocked histone phosphorylation during differentiation. We also show that DNA- PK inhibition and si RNA-mediated DNA- PK knockdown blocked cell fusion. These data implicate a new role for DNA- PK in myogenic differentiation. [ABSTRACT FROM AUTHOR]

Cells. They assemble, thrive, and cooperate to compose an organism, simple or complex. And like any living thing, they die. They die by catastrophe, they become sabotaged by condition, or they remove themselves on command from within or without. Each small life is followed by a death, to the benefit or the harm of the whole. Our story, here, is not of how each quietus occurs, but instead, of our ongoing effort to understand these tiny demises, to manipulate them, and to some day control them. [ABSTRACT FROM AUTHOR]

This review aims to highlight the proteases required for regulated cell death mechanisms in animals and plants. The aim is to be incisive, and not inclusive of all the animal proteases that have been implicated in various publications. The review also aims to focus on instances when several publications from disparate groups have demonstrated the involvement of an animal protease, and also when there is substantial biochemical, mechanistic and genetic evidence. In doing so, the literature can be culled to a handful of proteases, covering most of the known regulated cell death mechanisms: apoptosis, regulated necrosis, necroptosis, pyroptosis and NETosis in animals. In plants, the literature is younger and not as extensive as for mammals, although the molecular drivers of vacuolar death, necrosis and the hypersensitive response in plants are becoming clearer. Each of these death mechanisms has at least one proteolytic component that plays a major role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes. Some similarities are found among animal and plant cell death proteases but, overall, the pathways that they govern are kingdom-specific with very little overlap. [ABSTRACT FROM AUTHOR]

Hepatic injury provoked by cold storage is a major problem affecting liver transplantation, as exposure to cold induces apoptosis in hepatic tissues. Long noncoding RNAs (lncRNAs) are increasingly understood to regulate apoptosis, but the contribution of lncRNAs to cold-induced liver injury remains unknown. Using RNA-seq, we determined the differential lncRNA expression profile in mouse livers after cold storage and found that expression of the lncRNA TUG1 was significantly down-regulated. Overexpression of TUG1 attenuated cold-induced apoptosis in mouse hepatocytes and liver sinusoidal endothelial cells LSECs, in part by blocking mitochondrial apoptosis and endoplasmic reticulum (ER) stress pathways. Moreover, TUG1 attenuated apoptosis, inflammation, and oxidative stress in vivo in livers subjected to cold storage. Overexpression of TUG1 also improved hepatocyte function and prolonged hepatic graft survival rates in mice. These results suggest that the lncRNA TUG1 exerts a protective effect against cold-induced liver damage by inhibiting apoptosis in mice, and suggests a potential role for TUG1 as a target for the prevention of coldinduced liver damage in liver transplantation. [ABSTRACT FROM AUTHOR]

Inhibitor of apoptosis ( IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c- IAP) proteins. The c- IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c- IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c- IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c- IAPs illustrate their importance as intracellular signaling intermediates. [ABSTRACT FROM AUTHOR]

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression. [ABSTRACT FROM AUTHOR]

In the neuronal system, polysialic acid (polySia) is known to be involved in several cellular processes such as the modulation of cell-cell interactions. This highly negatively-charged sugar moiety is mainly present as a posttranslational modification of the neural cell adhesion molecule (NCAM). More than 20 years ago, differently glycosylated forms of NCAM were detected in the ovaries. However, the exact isoform of NCAM, as well as its biological function, remained unknown. Our analysis revealed that granulosa cells of feline tertiary follicles express the polysialylated form of NCAM-140. Unexpectedly, polySia was only expressed in the granulosa layers of atretic follicles and not of healthy follicles. By contrast, only the un-polysialylated form of NCAM was present on the membrane of granulosa cells of healthy follicles. To study a possible cellular function of poly- Sia in feline follicles, a primary granulosa cell culture model was used. Interestingly, loss of polySia leads to a significant inhibition of apoptosis, demonstrating that polySia is involved during atretic processes in granulosa cells. Thus, polySia might not only directly influence regeneration processes as shown, for example, in the neuronal system, but also apoptosis. [ABSTRACT FROM AUTHOR]

Altered metabolism is a hallmark of cancer that opens new therapeutic possibilities. 2-deoxyglucose (2- DG) is a non-metabolizable glucose analog tested in clinical trials and is frequently used in experimental settings to mimic glucose starvation. However, in the present study, conducted in a rhabdomyosarcoma cell line, we find that 2- DG induces classical nuclear apoptotic morphology and caspase-dependent cell death, whereas glucose deprivation drives cells toward necrotic cell death. Necrosis induced by glucose deprivation did not resemble necroptosis or ferroptosis and was not prevented by antioxidants. Both stimuli promote endoplasmic reticulum stress. Moreover, the transcription factor ATF4 is found to mediate both the apoptosis induced by 2- DG and the glycosylation inhibitor tunicamycin, as well as the necrosis provoked by glucose withdrawal. Several hexoses partially prevented glucose deprivation-induced necrosis in rhabdomyosarcoma, although only mannose prevented apoptosis induced by 2- DG. In both cases, a reduction of cell death was associated with decreased levels of ATF4. Our results confirm previous data indicating the differential effects of these two forms with respect to inhibiting glucose metabolism, and they place endoplasmic reticulum stress as the critical mediator of glucose starvation-induced cell death. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand ( TRAIL) is a member of the tumor necrosis factor superfamily, whose members are capable of inducing apoptosis and inflammation. Endoplasmic reticulum stress ( ERS) plays a key role in immune surveillance in macrophages. TRAIL mRNA and protein expression have previously been detected in macrophages; however, whether ERS has any effects on TRAIL expression in macrophages has not yet been determined. Here, we demonstrate that thapsigargin ( TG) and tunicamycin ( TM), two ERS inducers activated macrophages were able to increase TRAIL mRNA and protein expression in RAW264.7 macrophages, the culture supernatant of THP-1 cells, and mouse peritoneal macrophages, indicating that ERS as a potent inducer of TRAIL transcription and expression in macrophages. This effect was blocked by the specific JNK inhibitor SP600125 and transcription factor AP-1 inhibitor SR 1130. Interestingly, at the molecular level, regulation of TRAIL expression by ERS was accompanied by a significant decrease in cytokine signaling suppressor 3 ( SOCS3). SOCS3 si RNA clearly increased the expression of TRAIL mRNA and protein under ERS by activating the AP-1 components phosphorylated c-Jun and phosphorylated c-Fos in RAW264.7 cells. In contrast, over-expression of SOCS3 reversed ERS-induced TRAIL expression. These findings provide in vitro evidence that SOCS3 plays a critical negative role in the regulation of ERS-induced TRAIL expression via the Jun N-terminal kinase/ AP-1 signaling pathway in macrophages. [ABSTRACT FROM AUTHOR]

When intracellular damage accumulates in proliferating somatic cells, the cell cycle usually arrests in G1 or G2 in a checkpoint-dependent manner, either to repair the damage or to die by apoptosis. In contrast, early embryonic cells lack checkpoint-mediated cell-cycle arrest, and it is not clear whether apoptosis in early embryonic cells occurs at a specific cell cycle stage or at random points. Here, we examined the functional molecular link between the embryonic cell cycle and apoptosis using Xenopus egg extracts. When apoptosis was induced in egg extracts by addition of exogenous cytochrome c during cell-cycle progression, cyclin B accumulation was inhibited, Cdc2 was not activated, and the cell cycle arrested at interphase. However, addition of recombinant cyclin B failed to activate Cdc2 due to the strong inhibitory phosphorylation of Cdc2 Tyr15 in apoptotic egg extracts. We found that endogenous Cdc25C, which activates the Cdc2-cyclin B complex by dephosphorylating Cdc2 Tyr15, was inactivated by caspase-mediated cleavage at two sites in the N-terminal regulatory domain. When the hyperactive Cdc25A catalytic fragment was added together with recombinant cyclin B to artificially dephosphorylate Cdc2 Tyr15, M-phase induction was restored in apoptotic egg extracts, indicating that the blockage of cyclin B accumulation and the caspase-mediated inactivation of Cdc25C dually inhibited Cdc2 activation. Apoptosis induction in cytostatic factor-arrested metaphase egg extracts resulted in inactivation of Cdc2 without cyclin B degradation. These results suggest that apoptotic inactivation of Cdc25C plays an important role in arresting the embryonic cell cycle at interphase during apoptosis. [ABSTRACT FROM AUTHOR]

Hepatitis B virus ( HBV) enters the host and survives by using several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. Previous reports have shown that micro RNA (mi RNA)-30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that overexpression of mi RNA-30a could inhibit HBV-induced autophagosome formation in hepatic cells. To study this, both HepG2 cells and HepG2.2.1.5 cells ( HBV-expressing stable cell line) were transfected with mi RNA-30a, and the cells were collected either for RNA isolation or protein isolation after 72 h of transfection. Beclin-1 expression was significantly higher in untransfected HepG2.2.1.5 cells than in HepG2 cells. Western blots showed that mi RNA-30a overexpression resulted in a significant decrease in beclin-1 expression (eight-fold and four-fold in HepG2 and HepG2.2.1.5 cells, respectively) and c-myc expression, whereas the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ( TUNEL)-positive cells were increased. In contrast, overexpression of HBV X protein ( HBx) in HepG2 cells resulted in the enhancement of beclin-1 (six-fold increase as compared with the empty vector-transfected cells) and c-myc expression, whereas the numbers of TUNEL-positive cells were reduced. To confirm these findings, HBx and mi RNA-30a were coexpressed in HepG2 cells, and the results showed significant inhibition of autophagosome formation and beclin-1 and c-myc expression, whereas apoptosis increased. These data demonstrate that HBx induces autophagosome formation via beclin-1 expression, whereas mi RNA-30a overexpression could successfully inhibit the beclin-1 expression induced by HBx, thereby modulating autophagosome formation in hepatic cells. [ABSTRACT FROM AUTHOR]

Cell survival depends on the maintenance of mitochondrial integrity controlled by a well-balanced interplay between anti- and pro-apoptotic B cell lymphoma 2 (Bcl2) family members. Given their frequent deregulation in human pathologies, including autoimmunity and cancer, significant research efforts have increased our molecular understanding of how Bcl2 proteins control cell death. This has fostered the development of small non-peptidic compounds, so-called BH3-mimetics, that show excellent prospects of passing clinical trials and entering daily use for targeted therapy. Possible limitations in clinical application may, to a certain degree, be predicted from loss-of-function phenotypes gathered from studies using gene-modified mice that we attempt to summarize and discuss in this context. [ABSTRACT FROM AUTHOR]

Fernández‐Marrero and colleagues show in their work that the proapoptotic BCL‐2 family member BOK can form large, stable pores in artificial liposomes. This can be enhanced by the proapoptotic protein cBID and is unaffected by the antiapoptotic BCL‐XL. Although BOK can bind to isolated mitochondria, it is unable to cause cytochrome c release even with the help of cBID. [ABSTRACT FROM AUTHOR]

For many years, transcription factor Sp1 was viewed as a basal transcription factor and relegated to a role in the regulation of so-called housekeeping genes. Identification of Sp1's role in recruiting the general transcription machinery in the absence of a TATA box increased its importance in gene regulation, particularly in light of recent estimates that the majority of mammalian genes lack a TATA box. In this review, we briefly consider the history of Sp1, the founding member of the Sp family of transcription factors. We review the evidence suggesting that Sp1 is highly regulated by post-translational modifications that positively and negatively affect the activity of Sp1 on a wide array of genes. Sp1 is over-expressed in many cancers and is associated with poor prognosis. Targeting Sp1 in cancer treatment has been suggested; however, our review of the literature on the role of Sp1 in the regulation of genes that contribute to the 'hallmarks of cancer' illustrates the extreme complexity of Sp1 functions. Sp1 both activates and suppresses the expression of a number of essential oncogenes and tumor suppressors, as well as genes involved in essential cellular functions, including proliferation, differentiation, the DNA damage response, apoptosis, senescence and angiogenesis. Sp1 is also implicated in inflammation and genomic instability, as well as epigenetic silencing. Given the apparently opposing effects of Sp1, a more complete understanding of the function of Sp1 in cancer is required to validate its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]