ATF4-Dependent NRF2 Transcriptional Regulation Promotes Antioxidant Protection during Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress generates reactive oxygen species (ROS) that induce apoptosis if left unabated. To limit oxidative insults, the ER stress PKR-like endoplasmic reticulum Kinase (PERK) has been reported to phosphorylate and activate nuclear factor erythroid 2-related factor 2 (NRF2). Here, we uncover an alternative mechanism for PERK-mediated NRF2 regulation in human cells that does not require direct phosphorylation. We show that the activation of the PERK pathway rapidly stimulates the expression of NRF2 through activating transcription factor 4 (ATF4). In addition, NRF2 activation is late and largely driven by reactive oxygen species (ROS) generated during late protein synthesis recovery, contributing to protecting against cell death. Thus, PERK-mediated NRF2 activation encompasses a PERK-ATF4-dependent control of NRF2 expression that contributes to the NRF2 protective response engaged during ER stress-induced ROS production.

Display of the peroxiredoxin Bcp1 of Sulfolobus solfataricus on probiotic spores of Bacillus megaterium.

Bacterial spores displaying heterologous proteins have been proposed as a safe and efficient method for delivery of antigens and enzymes to animal mucosal surfaces. Initial studies have been performed using Bacillus subtilis spores, but other spore forming organisms have also been considered. B. megaterium spores have been shown capable of displaying large amounts of a model heterologous protein (Discosoma red fluorescent protein mRFP) that in part crossed the exosporium to localize in the space between the outer coat layer and the exosporium. Here, B. megaterium spores have been used to adsorb Bcp1 (bacterioferritin comigratory protein 1), a peroxiredoxin of the archaeon Sulfolobus solfataricus, known to have an antioxidant activity. The spores were highly efficient in adsorbing the heterologous enzyme which, once adsorbed, retained its activity. The adsorbed Bcp1 localized beneath the exosporium, filling the space between the outer coat and the exosporium. This unusual localization contributed to the stability of the enzyme-spore interaction and to the protection of the adsorbed enzyme in simulated intestinal or gastric conditions.

Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress.

Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells.

Nutrient shortage triggers the hexosamine biosynthetic pathway via the GCN2-ATF4 signalling pathway.

The hexosamine biosynthetic pathway (HBP) is a nutrient-sensing metabolic pathway that produces the activated amino sugar UDP-N-acetylglucosamine, a critical substrate for protein glycosylation. Despite its biological significance, little is known about the regulation of HBP flux during nutrient limitation. Here, we report that amino acid or glucose shortage increase GFAT1 production, the first and rate-limiting enzyme of the HBP. GFAT1 is a transcriptional target of the activating transcription factor 4 (ATF4) induced by the GCN2-eIF2α signalling pathway. The increased production of GFAT1 stimulates HBP flux and results in an increase in O-linked ß-N-acetylglucosamine protein modifications. Taken together, these findings demonstrate that ATF4 provides a link between nutritional stress and the HBP for the regulation of the O-GlcNAcylation-dependent cellular signalling.

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants.

Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival.

Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production. By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.

Enhanced cytotoxic activity of a bifunctional chimeric protein containing a type 1 ribosome-inactivating protein and a serine protease inhibitor.

Both ribosome-inactivating proteins (RIPs) and plant proteinase inhibitors, belong to protein families known to regulate cellular homeostasis and likely involved in plant defense. Nevertheless the interest in these protein classes is due to their potential use for the treatment of several important human diseases such as cancer. Thus, in the present study, type 1 ribosome-inactivating protein and wheat subtilisin/chymotrypsin inhibitor, were engineered into a chimeric protein with cytotoxic action selective for murine tumor cells, while lacking any appreciable toxicity on murine normal cells. This chimeric protein selectively sensitizes to apoptotic death cells derived from Simian-virus-40-transformed mouse fibroblasts (SVT2 cells). The cytotoxicity of this new recombinant product has been detected also on three different human malignant cells. Therefore action on tumor cells of this protein could represent a potentially very attractive novel tool for anticancer drug design.