Enhancer Clusters Drive Type I Interferon-Induced TRAIL Overexpression in Cancer, and Its Intracellular Protein Accumulation Fails to Induce Apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine produced and secreted by immune cells in response to an infection, often in response to interferon (IFN) stimulation. In cancer, it has also been shown that IFN stimulates the production of TRAIL, and it has been proposed that this TRAIL can induce apoptosis in an autocrine or paracrine manner in different cancer cells. Yet, the mechanism mediating TRAIL upregulation and the implications of TRAIL as an apoptotic molecule in cancer cells are still poorly understood. We show here that in certain cancer cells, TRAIL is upregulated by enhancer clusters, potent genomic regulatory regions containing densely packed enhancers that have combinatorial and additive activity and that are usually found to be associated with cancer-promoting genes. Moreover, we found that TRAIL upregulation by IFNα is mediated by these enhancer clusters in breast and lung cancer cells. Surprisingly, IFNα stimulation leads to the intracellular accumulation of TRAIL protein in these cancer cells. Consequently, this TRAIL is not capable of inducing apoptosis. Our study provides novel insights into the mechanism behind the interferon-mediated upregulation of TRAIL and its protein accumulation in cancer cells. Further investigation is required to understand the role of intracellular TRAIL or depict the mechanisms mediating its apoptosis impairment in cancer cells

Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family

<p>Abstract</p> <p>Background</p> <p>Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.</p> <p>Results</p> <p>The crystal structure of a putative AhpD from <it>Pseudomonas aeruginosa </it>has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from <it>Mycobacterium tuberculosis </it>despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the <it>P. aeruginosa </it>protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in <it>M. tuberculosis </it>AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of <it>P. aeruginosa </it>have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from <it>P. aeruginosa </it>exhibits a weak ability to reduce H₂O₂as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents.</p> <p>Conclusion</p> <p>Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.</p

Proteomic Profile of Reversible Protein Oxidation Using PROP, Purification of Reversibly Oxidized Proteins

Signal transduction pathways that are modulated by thiol oxidation events are beginning to be uncovered, but these discoveries are limited by the availability of relatively few analytical methods to examine protein oxidation compared to other signaling events such as protein phosphorylation. We report here the coupling of PROP, a method to purify reversibly oxidized proteins, with the proteomic identification of the purified mixture using mass spectrometry. A gene ontology (GO), KEGG enrichment and Wikipathways analysis of the identified proteins indicated a significant enrichment in proteins associated with both translation and mRNA splicing. This methodology also enabled the identification of some of the specific cysteine residue targets within identified proteins that are reversibly oxidized by hydrogen peroxide treatment of intact cells. From these identifications, we determined a potential consensus sequence motif associated with oxidized cysteine residues. Furthermore, because we identified proteins and specific sites of oxidation from both abundant proteins and from far less abundant signaling proteins (e.g. hepatoma derived growth factor, prostaglandin E synthase 3), the results suggest that the PROP procedure was efficient. Thus, this PROP-proteomics methodology offers a sensitive means to identify biologically relevant redox signaling events that occur within intact cells

Mini Review: An Unexplored Role for Peroxiredoxin in Exercise-induced Redox Signalling?

Peroxiredoxin (PRDX) is a ubiquitous oxoreductase protein with a conserved ionised thiol that permits catalysis of hydrogen peroxide (H2O2) up to a million times faster than any thiol-containing signalling protein. The increased production of H2O2 within active tissues during exercise is thought to oxidise conserved cysteine thiols, which may in turn facilitate a wide variety of physiological adaptations. The precise mechanisms linking H2O2 with the oxidation of signalling thiol proteins (phosphates, kinases and transcription factors) are unclear due to these proteins’ low reactivity with H2O2 relative to abundant thiol peroxidases such as PRDX. Recent work has shown that following exposure to H2O2 in vitro, the sulfenic acid of the PRDX cysteine can form mixed disulphides with transcription factors associated with cell survival. This implicates PRDX as an ‘active’ redox relay in transmitting the oxidising equivalent of H2O2 to downstream proteins. Furthermore, under oxidative stress, PRDX can form high molecular weight structures that can be secreted into the extracellular space, potentially acting as an extracellular ‘stress’ signal. There is extensive literature assessing non-specific markers of oxidative stress in response to exercise, however the PRDX catalytic cycle may offer a more robust approach for measuring changes in redox balance following exercise. This review discusses studies assessing PRDX-mediated cellular signalling and integrates the recent advances in redox biology with investigations that have examined the role of PRDX during exercise in humans and animals. Future studies should explore the role of PRDX as a key regulator of peroxide mediated-signal transduction during exercise in humans

(Homo)glutathione Deficiency Impairs Root-knot Nematode Development in Medicago truncatula

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes

Bactericidal Activity of the Human Skin Fatty Acid cis-6-Hexadecanoic Acid on Staphylococcus aureus

Human skin fatty acids are a potent aspect of our innate defenses, giving surface protection against potentially invasive organisms. They provide an important parameter in determining the ecology of the skin microflora, and alterations can lead to increased colonization by pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new avenue of exploration in the generation of control measures against drug-resistant organisms. Despite their importance, the mechanism(s) whereby skin fatty acids kill bacteria has remained largely elusive. Here, we describe an analysis of the bactericidal effects of the major human skin fatty acid cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen S. aureus. Several C6H concentration-dependent mechanisms were found. At high concentrations, C6H swiftly kills cells associated with a general loss of membrane integrity. However, C6H still kills at lower concentrations, acting through disruption of the proton motive force, an increase in membrane fluidity, and its effects on electron transfer. The design of analogues with altered bactericidal effects has begun to determine the structural constraints on activity and paves the way for the rational design of new antistaphylococcal agents

ROS-dependent signaling pathways in plants and algae exposed to high light: Comparisons with other eukaryotes

Abstract Like all aerobic organisms, plants and algae co-opt reactive oxygen species (ROS) as signaling molecules to drive cellular responses to changes in their environment. In this respect, there is considerable commonality between all eukaryotes imposed by the constraints of ROS chemistry, similar metabolism in many subcellular compartments, the requirement for a high degree of signal specificity and the deployment of thiol peroxidases as transducers of oxidizing equivalents to regulatory proteins. Nevertheless, plants and algae carry out specialised signaling arising from oxygenic photosynthesis in chloroplasts and photoautotropism, which often induce an imbalance between absorption of light energy and the capacity to use it productively. A key means of responding to this imbalance is through communication of chloroplasts with the nucleus to adjust cellular metabolism. Two ROS, singlet oxygen (1O2) and hydrogen peroxide (H2O2), initiate distinct signaling pathways when photosynthesis is perturbed. 1O2, because of its potent reactivity means that it initiates but does not transduce signaling. In contrast, the lower reactivity of H2O2 means that it can also be a mobile messenger in a spatially-defined signaling pathway. How plants translate a H2O2 message to bring about changes in gene expression is unknown and therefore, we draw on information from other eukaryotes to propose a working hypothesis. The role of these ROS generated in other subcellular compartments of plant cells in response to HL is critically considered alongside other eukaryotes. Finally, the responses of animal cells to oxidative stress upon high irradiance exposure is considered for new comparisons between plant and animal cells