A genome-wide RNA interference screen identifies caspase 4 as a factor required for tumor necrosis factor alpha signaling.

Tumor necrosis factor alpha (TNF-α) is a potent inflammatory cytokine secreted upon cellular stress as well as immunological stimuli and is implicated in the pathology of inflammatory diseases and cancer. The therapeutic potential of modifying TNF-α pathway activity has been realized in several diseases, and antagonists of TNF-α have reached clinical applications. While much progress in the understanding of signaling downstream of the TNF-α receptor complex has been made, the compendium of factors required for signal transduction is still not complete. In order to find novel regulators of proinflammatory signaling induced by TNF-α, we conducted a genome-wide small interfering RNA screen in human cells. We identified several new candidate modulators of TNF-α signaling, which were confirmed in independent experiments. Specifically, we show that caspase 4 is required for the induction of NF-κB activity, while it appears to be dispensable for the activation of the Jun N-terminal protein kinase signaling branch. Taken together, our experiments identify caspase 4 as a novel regulator of TNF-α-induced NF-κB signaling that is required for the activation of IκB kinase. We further provide the genome-wide RNA interference data set as a compendium in a format compliant with minimum information about an interfering RNA experiment (MAIRE).

Innate immunity: regulation of caspases by IAP-dependent ubiquitylation.

Caspases are widely known as initiators and executioners of cell death. Full activation of caspases leading to cleavage of many cellular substrates was long considered to be a point-of-no-return in the apoptosis pathway. However, it also has been known that activated caspases do not always have the ability to kill, but instead initiate non-apoptotic processes such as cell differentiation or activation of innate immune responses. In this issue of The EMBO Journal, Meinander et al (2012) explore the contribution of polyubiquitination of Dredd, a known initiator caspase, to the activation of innate immunity. The authors show that infection with gram-negative bacteria leads to DIAP2-dependent ubiquitylation of Dredd which in turn is required for processing of Relish (Rel) and expression of antimicrobial peptide (AMP) genes that are indispensable for fighting the infection.

An RNAi screen identifies USP2 as a factor required for TNF-α-induced NF-κB signaling.

Tumor necrosis factor α (TNF-α) signaling pathways play important roles during tumorigenesis and inflammation. Ubiquitin-dependent processes are central to the regulation of TNF-α and nuclear factor κB (NF-κB) signaling. We performed a targeted siRNA screen for ubiquitin-specific proteases (USPs) and identified USP2 as a modulator of TNF-α-induced NF-κB signaling. We showed that USP2 is required for the phosphorylation of IκB, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes and IL-8 secretion. Our study also provides evidence for isoform-specific functions of USP2. The immunohistochemical analysis of breast carcinomas revealed that USP2 expression is frequently downregulated. Together, our results implicate USP2 as a novel positive regulator of TNF-α-induced NF-κB signaling and show that its expression is altered in tumor cells.

High-throughput RNAi screening to dissect cellular pathways: a how-to guide.

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.

TRAIL and other TRAIL receptor agonists as novel cancer therapeutics.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo2L, is a member of the TNF superfamily (TNFSF) of cytokines. TRAIL gained much attention during the past decade due to the demonstration of its therapeutic potential as a tumor-specific apoptosis inducer. TRAIL was identified as a protein with high homology to other members of the TNF cytokine family, especially to the ligand of Fas/Apo-1 (CD95), CD95L (FasL/APO-1L). TRAIL has been shown to induce apoptosis selectively in many tumor cell lines without affecting normal cells and tissues, making TRAIL itself as well as agonists of the two human receptors of TRAIL which can submit an apoptotic signal, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), promising novel biotherapeutics for cancer therapy. An increasing number of publications now shows that TRAIL resistance in primary human tumor cells will have to be overcome and that sensitization to TRAIL-induced apoptosis will be required in many cases. Therefore, it will also be instrumental to develop suitable diagnostic tests to identify patients who will benefit from TRAIL-based novel anticancer therapeutics and those who will not. Interestingly, the first clinical results even in monotherapy with TRAIL as well as various agonistic TRAIL receptor-specific antibodies have shown encouraging results. This chapter provides a compact overview on the biochemistry of the TRAIL/TRAIL-R system, the physiological role of TRAIL and its receptors and the results of clinical trials with TRAIL and various TRAIL-R agonistic antibodies.

Following TRAIL's path in the immune system.

The members of the tumour necrosis factor (TNF) superfamily of cytokines play important roles in the regulation of various immune-cell functions. Likewise, induction of cell death by apoptosis is indispensable for the normal functioning of the immune system. There are two major pathways of apoptosis induction. The intrinsic, or mitochondrial, pathway is regulated by the activation and interaction of members of the Bcl-2 family. The extrinsic, or death receptor, pathway is triggered by certain TNF family members when they engage their respective cognate receptors on the surface of the target cell. Hence, cell-to-cell-mediated death signals are induced by activation of these death receptor-ligand systems. Besides TNF itself and the CD95 (Fas/APO-1) ligand (FasL/Apo1L), the TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) belongs to the subfamily of ligands that is responsible for extrinsic induction of cell death. Depending on their status of stimulation, TRAIL can be expressed by various cells of the immune system, amongst them natural killer (NK) cells, T cells, natural killer T cells (NKT cells), dendritic cells and macrophages. TRAIL has been implicated in immunosuppressive, immunoregulatory and immune-effector functions. With respect to pathological challenges, TRAIL and its receptors have been shown to play important roles in the immune response to viral infections and in immune surveillance of tumours and metastases. In this review we summarize the current knowledge on the role of TRAIL and its receptors in the immune system and, based on this, we discuss future directions of research into the diverse functions of this fascinating receptor-ligand system.

Norvaline is accumulated after a down-shift of oxygen in Escherichia coli W3110.

BACKGROUND: Norvaline is an unusual non-proteinogenic branched-chain amino acid which has been of interest especially during the early enzymological studies on regulatory mutants of the branched-chain amino acid pathway in Serratia marcescens. Only recently norvaline and other modified amino acids of the branched-chain amino acid synthesis pathway got attention again when they were found to be incorporated in minor amounts in heterologous proteins with a high leucine or methionine content. Earlier experiments have convincingly shown that norvaline and norleucine are formed from pyruvate being an alternative substrate of alpha-isopropylmalate synthase, however so far norvaline accumulation was not shown to occur in non-recombinant strains of E. coli. RESULTS: Here we show that oxygen limitation causes norvaline accumulation in E. coli K-12 W3110 during grow in glucose-based mineral salt medium. Norvaline accumulates immediately after a shift to oxygen limitation at high glucose concentration. On the contrary free norvaline is not accumulated in E. coli W3110 in aerobic cultures. The analysis of medium components, supported by transcriptomic studies proposes a purely metabolic overflow mechanism from pyruvate into the branched chain amino acid synthesis pathway, which is further supported by the significant accumulation of pyruvate after the oxygen downshift. The results indicate overflow metabolism from pyruvate as necessary and sufficient, but deregulation of the branched chain amino acid pathway may be an additional modulating parameter. CONCLUSION: Norvaline synthesis has been so far mainly related to an imbalance of the synthesis of the branched chain amino acids under conditions were pyruvate level is high. Here we show that simply a downshift of oxygen is sufficient to cause norvaline accumulation at a high glucose concentration as a consequence of the accumulation of pyruvate and its direct chain elongation over alpha-ketobutyrate and alpha-ketovalerate.Although the flux to norvaline is low, millimolar concentrations are accumulated in the cultivation broth, which is far above the level which has been discussed for being relevant for misincorporation of norvaline into recombinant proteins. Therefore we believe that our finding is relevant for recombinant protein production but also may even have implications for the physiology of E. coli under oxygen limitation in general.

TRAIL signalling: decisions between life and death.

The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumour cells. This property has made TRAIL and agonistic antibodies against its death inducing receptors (TRAIL-R1 and TRAIL-R2) to some of the most promising novel biotherapeutic agents for cancer therapy. Here we review the signalling pathways initiated by the apoptosis- as well as the non-apoptosis-inducing receptors, TRAIL-R3 and TRAIL-R4. The TRAIL "death-inducing signalling complex" (DISC) transmits the apoptotic signal. DISC formation leads to activation of a protease cascade, finally resulting in cell death. The TRAIL death receptor-mediated "extrinsic" pathway and the "intrinsic" pathway, which is controlled by the interaction of members of the Bcl-2 family, interact with each other in the decision about life or death of a cell. Apoptotic and non-apoptotic signalling is influenced by the NF-kappaB, PKB/Akt and the MAPK signalling pathways. In this review we intend to summarise the most important findings on the TRAIL signalling network and the interplay in the decisions between life and death of a tumor cell.

Transient increase of ATP as a response to temperature up-shift in Escherichia coli.

SUMMARY: BACKGROUND: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift. RESULTS: The current study shows that a temperature up-shift is followed by a very fast significant transient increase of the cellular ATP concentration within the first minutes. This increase is connected to a longer lasting elevation of the cellular respiration and glucose uptake. Also the mRNA level of typical heat shock genes increases within only one minute after the heat-shock. CONCLUSION: The presented data prove the very fast response of E. coli to a heat-shock and that the initial response includes the increase of the ATP pool which is important to fulfil the need of the cell for new syntheses, as well as for the function of chaperones and proteases.