SummaryAngiogenin (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 stressinduced 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.

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 β-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 regulation of metabolic fluxes is a key process in the cellular response to changes in nutrient availability, and dysregulation of this process may contribute to the development of various diseases 1,2 . The hexosamine biosynthetic pathway (HBP) plays a central role in sensing the nutritional status of the cell, since it integrates molecules coming from carbohydrates, fatty acids, amino acids and nucleotides metabolism 3 . The HBP converts fructose-6-phosphate, a glucose derivative, into UDP-N-acetylglucosamine (UDP-GlcNAc), a central nucleotide sugar acting as a donor substrate for the glycosylation of proteins and lipids. In addition, UDP-GlcNAc is used for the O-GlcNAcylation of proteins, i.e. the addition of N-acetylglucosamine to their serine and threonine residues. Similarly to phosphorylation, O-GlcNAcylation regulates cellular activities, such as signalling and transcription 4 . It has been proposed that O-GlcNAcylated proteins modulate cellular responses to nutrient deprivation and stress, and that their dysregulation is implicated in a wide range of pathologies 5 . Despite these wide-ranging biological effects, the mechanisms underlying the adaptation of the HBP flux to nutrient availability remains poorly understood.The first and rate-limiting enzyme of the HBP, namely glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1), which drives the HBP flux, is involved in various physiopathological processes 6-9 . GFAT1 activity is inhibited by UDP-GlcNAc, the end product of the HBP 10 , and the AMP-activated protein kinase-mediated phosphorylation 11 . In contrary, GFAT1 is stimulated upon phosphorylation by protein kinase A and calcium/ calmodulin-dependent protein kinase II 12 . More recently, it was shown that GFAT1 production is upregulated by a transcription factor of the unfolded protein response (UPR), namely the spliced form of X-box binding protein 1 (XBP1s), resulting in the stimulation of the HBP flux and protein O-GlcNAcylation 13 . These authors unveiled in this way an intrinsic link between the HBP and disruption of the endoplasmic reticulum (ER) homeostasis, i.e. ER stress, which activates the UPR.UPR signalling is mediat...

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.

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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.

Amyloidogenic ‘gain-of-function' mutations in apolipoprotein A-I (ApoA-I) gene (APOA1) result in systemic amyloidosis characterized by aggregate deposition and eventually cell death. However, how amyloidogenic variants of ApoA-I induce cell death is unknown. Here we report that one of the mechanisms by which amyloidogenic ApoA-I induces cell death is through attenuating anti-stress activity of angiogenin (ANG), a homeostatic protein having both pro-growth and pro-survival functions. Under growth conditions, ANG is located in nucleolus where it promotes ribosomal RNA (rRNA) transcription thereby stimulating cell growth. In adverse conditions, ANG is relocated to cytoplasm to promote damage repairs and cell survival. We find that in cells overexpressing the L75P-APOA1 mutant ANG expression is decreased and normal cellular localization of ANG is altered in response to stress and growth signals. In particular, ANG does not relocate to cytoplasm under stress conditions but is rather retained in the nucleolus where it continues promoting rRNA transcription, thus imposing a ribotoxic effect while simultaneously compromising its pro-survival activity. Consistently, we also find that addition of exogenous ANG protects cells from L75P-ApoA-I-induced apoptosis.

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.

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