The GCN2 kinase is required for activating autophagy in response to indispensable amino acid deficiencies

ORGANIZING COMMITTEEChairs: Didier Attaix - Lydie Combaret - Daniel TaillandierDaniel Béchet - Agnès Claustre - Cécile Coudy-Gandilhon - Christiane Deval - Gérard Donadille - Cécile PolgeSCIENTIFIC COMMITTEEDidier Attaix - Lydie Combaret - Alfred L. Goldberg - Ron Hay - Germana Meroni - Marco Sandri - Daniel Taillandier - Keiji Tanaka - Simon S. WingPoster Session 3 - AutophagyImbalances in dietary amino acid (AA) supply, including deficits in one or more indispensable amino acids (IAA), are stressful conditions for the organism that needs to modulate a number of physiological functions to adapt to this situation. In particular, since there is no system dedicated for storing AA in the body, the release of free AA occurs by proteolysis at the expense of functional proteins, notably in the liver by up-regulating autophagy. This process can be rapidly mobilized within the cell in response to a number of stresses, by post-translational regulations of autophagy-related proteins already present in the cytosol. The protein kinase GCN2 is activated upon IAA scarcity in order to promote cell adaptation to a nutritional stress condition. In response to IAA limitation, GCN2 couples the accumulation of uncharged transfer RNAs to the phosphorylation of eIF2a on serine 51. By this mean, GCN2 diminishes the overall protein synthesis rate, while simultaneously activating a gene expression program mediated by the translational upregulation of the transcription factor ATF4. Our recent work has shown that the GCN2/p-eIF2a/ATF4 signaling pathway plays an essential role in the induction of transcription of a number of autophagy-related genes involved in the maintenance of the autophagic process in response to an IAA deficiency (B’chir et al., 2013). In the present study we sought to determine whether GCN2 could play a role in regulating the early stages of autophagy. The most upstream complex for triggering the autophagic process (initiation complex) is notably composed of the ULK kinase and the ATG13 bridging protein, and is classically viewed to be controlled by mTORC1. Indeed, the activity of the autophagy initiation complex has been shown to be modulated according to AA availability by the activity of mTORC1, which phosphorylates different sites in ULK. Here, by using a GCN2 knock-out mouse model we investigated the role of GCN2 in the upregulation of autophagy in the first hour of an IAA deficiency. Our results show that 1) GCN2 is required for upregulating liver autophagy in response to an IAA-deficient diet, which is confirmed in cell culture model; 2) this early activation of the autophagic process does not require the transcription factor ATF4; 3) moreover, while this effect can occur without concomitant inhibition of mTORC1 activity, our results suggest that ULK/ATG13 couple is involved in the GCN2-dependent activation of autophagy. Our results demonstrate that in the particular model of an IAA deficiency GCN2 plays a preponderant role in triggering the adaptive autophagy upregulation, a mechanism which can operate without concomitant inhibition of mTORC1 activit

Deriving the number of jobs in proximity services from the number of inhabitants in French rural municipalities

We use a minimum requirement approach to derive the number of jobs in proximity services per inhabitant in French rural municipalities. We first classify the municipalities according to their time distance to the municipality where the inhabitants go the most frequently to get services (called MFM). For each set corresponding to a range of time distance to MFM, we perform a quantile regression estimating the minimum number of service jobs per inhabitant, that we interpret as an estimation of the number of proximity jobs per inhabitant. We observe that the minimum number of service jobs per inhabitant is smaller in small municipalities. Moreover, for municipalities of similar sizes, when the distance to the MFM increases, we find that the number of jobs of proximity services per inhabitant increases.Comment: 6 pages, 5 figure

Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

The TNF-related cytokine TWEAK promotes skeletal muscle atrophy that is associated with classical disuse syndromes

Subversion of Cellular Autophagosomal Machinery by RNA Viruses

Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells

Potential antiproteolytic effects of L-leucine: observations of in vitro and in vivo studies

The purpose of present review is to describe the effect of leucine supplementation on skeletal muscle proteolysis suppression in both in vivo and in vitro studies. Most studies, using in vitro methodology, incubated skeletal muscles with leucine with different doses and the results suggests that there is a dose-dependent effect. The same responses can be observed in in vivo studies. Importantly, the leucine effects on skeletal muscle protein synthesis are not always connected to the inhibition of skeletal muscle proteolysis. As a matter of fact, high doses of leucine incubation can promote suppression of muscle proteolysis without additional effects on protein synthesis, and low leucine doses improve skeletal muscle protein ynthesis but have no effect on skeletal muscle proteolysis. These research findings may have an important clinical relevancy, because muscle loss in atrophic states would be reversed by specific leucine supplementation doses. Additionally, it has been clearly demonstrated that leucine administration suppresses skeletal muscle proteolysis in various catabolic states. Thus, if protein metabolism changes during different atrophic conditions, it is not surprising that the leucine dose-effect relationship must also change, according to atrophy or pathological state and catabolism magnitude. In conclusion, leucine has a potential role on attenuate skeletal muscle proteolysis. Future studies will help to sharpen the leucine efficacy on skeletal muscle protein degradation during several atrophic states

EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on Dietary Reference Values for protein

This opinion of the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) deals with the setting of Dietary Reference Values (DRVs) for protein. The Panel concludes that a Population Reference Intake (PRI) can be derived from nitrogen balance studies. Several health outcomes possibly associated with protein intake were also considered but data were found to be insufficient to establish DRVs. For healthy adults of both sexes, the average requirement (AR) is 0.66 g protein/kg body weight per day based on nitrogen balance data. Considering the 97.5th percentile of the distribution of the requirement and assuming an efficiency of utilisation of dietary protein for maintenance of 47 %, the PRI for adults of all ages was estimated to be 0.83 g protein/kg body weight per day and is applicable both to high quality protein and to protein in mixed diets. For children from six months onwards, age-dependent requirements for growth estimated from average daily rates of protein deposition and adjusted by a protein efficiency for growth of 58 % were added to the requirement for maintenance of 0.66 g/kg body weight per day. The PRI was estimated based on the average requirement plus 1.96 SD using a combined SD for growth and maintenance.For pregnancy, an intake of 1, 9 and 28 g/d in the first, second and third trimesters, respectively, is proposed in addition to the PRI for non-pregnant women. For lactation, a protein intake of 19 g/d during the first six months, and of 13 g/d after six months, is proposed in addition to the PRI for non-lactating women. Data are insufficient to establish a Tolerable Upper Intake Level (UL) for protein. Intakes up to twice the PRI are regularly consumed from mixed diets by some physically active and healthy adults in Europe and are considered safe

Évolution de la qualité des emplois dans les services : stabilité et rémunération des postes entre 1997 et 2007

Contrairement à une opinion répandue, la qualité des emplois des secteurs des services, au sens de la rémunération et de la stabilité, a connu une évolution plutôt favorable entre 1997 et 2007. Dans la majorité de ces secteurs, y compris parmi ceux recourant fortement au travail non qualifié, on assiste à une augmentation de la rémunération horaire réelle des postes. La stabilité des emplois progresse dans le quart des secteurs des services et reste constante dans la moitié des secteurs. Cependant, les salariés ayant effectué un passage d’un poste de l’industrie à un poste des services ou de l’intérim ont généralement subi à cette occasion une dégradation de la stabilité et de la rémunération horaire de l’emploi. Entre le début et la fin de la période, l’ampleur de cet impact défavorable est restée globalement stable pour les transitions industrie-services mais s’est sensiblement accentuée pour les transitions industrie-intérim. Elle n’a, par ailleurs, pas évolué de la même façon pour toutes les catégories de salariés, les différences tendant néanmoins à s’atténuer.Mordier Bénédicte. Évolution de la qualité des emplois dans les services : stabilité et rémunération des postes entre 1997 et 2007. In: Economie et statistique, n°454, 2012. pp. 43-77

Introduction de cadrage. Les sans-domicile en France : caractéristiques et principales évolutions entre 2001 et 2012

Plus de 140 000 personnes sont sans domicile en France en 2012, soit une augmentation de plus de 50 % en onze ans. En particulier, les sans-domicile nés à l’étranger, venant pour beaucoup des anciennes colonies françaises, sont nettement plus nombreux qu’en 2001. Parmi les adultes sans domicile dans les agglomérations de 20 000 habitants ou plus, si la population masculine reste majoritaire, c’est parmi les femmes et les enfants que la progression a été la plus forte. L’accueil en centre reste le mode d’hébergement principal des sans-domicile, mais l’hébergement en hôtel s’est particulièrement développé pour accueillir les couples sans domicile toujours plus nombreux. Le nombre d’adultes hébergés y a plus que triplé. Concernant leur situation vis-à-vis du marché du travail, un quart des adultes sans domicile de moins de 65 ans travaillent et tirent des ressources de leur activité. Près d’un sur deux se dit «chômeur » et un sur dix n’est pas autorisé à travailler (notamment demandeur d’asile ou en congé maladie de plus de trois mois). La ressource la plus souvent perçue par les sans-domicile est le revenu de solidarité active (RSA), puis les revenus issus du travail. L’allocation logement et les allocations familiales sont fréquemment citées par les sans-domicile francophones alors que l’allocation temporaire d’attente est une ressource fréquente pour les non-francophones. L’état de santé perçu des sans-domicile est moins bon que celui des personnes vivant dans un logement ordinaire. Les non-francophones se disent moins souvent en bonne santé que les autres sans-domicile, ils sont également moins nombreux à disposer d’une couverture maladie.Mordier Bénédicte. Introduction de cadrage. Les sans-domicile en France : caractéristiques et principales évolutions entre 2001 et 2012. In: Economie et statistique, n°488-489, 2016. Les sans-domicile - Les écarts de rémunération entre les hommes et les femmes dans la fonction publique. pp. 25-35