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

Hypothalamic eIF2α signaling regulates food intake.

The reversible phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is a highly conserved signal implicated in the cellular adaptation to numerous stresses such as the one caused by amino acid limitation. In response to dietary amino acid deficiency, the brain-specific activation of the eIF2α kinase GCN2 leads to food intake inhibition. We report here that GCN2 is rapidly activated in the mediobasal hypothalamus (MBH) after consumption of a leucine-deficient diet. Furthermore, knockdown of GCN2 in this particular area shows that MBH GCN2 activity controls the onset of the aversive response. Importantly, pharmacological experiments demonstrate that the sole phosphorylation of eIF2α in the MBH is sufficient to regulate food intake. eIF2α signaling being at the crossroad of stress pathways activated in several pathological states, our study indicates that hypothalamic eIF2α phosphorylation could play a critical role in the onset of anorexia associated with certain diseases.This work was supported by grants from “Fondation pour la Recherche Médicale,” “Société Française de Nutrition,” Ajinomoto Amino Acid Research Program (3ARP), and “Agence Nationale pour la Recherche.”This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2014.01.00

Impairing the production of ribosomal RNA activates mammalian target of rapamycin complex 1 signalling and downstream translation factors

Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all these processes. Here, we show that, in human cells, impaired rRNA processing, caused by expressing an interfering mutant of BOP1 or by knocking down components of the PeBoW complex elicits activation of mTORC1 signalling. This leads to enhanced phosphorylation of its substrates S6K1 and 4E-BP1, and stimulation of proteins involved in translation initiation and elongation. In particular, we observe both inactivation and downregulation of the eukaryotic elongation factor 2 kinase, which normally inhibits translation elongation. The latter effect involves decreased expression of the eEF2K mRNA. The mRNAs for ribosomal proteins, whose translation is positively regulated by mTORC1 signalling, also remain associated with ribosomes. Therefore, our data demonstrate that disrupting rRNA production activates mTORC1 signalling to enhance the efficiency of the translational machinery, likely to help compensate for impaired ribosome production

Genetic changes in human pluripotent stem cells: implications for basic biology and regenerative medicine

Chronic tissue and organ failure caused by an injury, disease, ageing or congenital defects represents some of the most complex therapeutic challenges and poses a significant financial healthcare burden. Regenerative medicine strategies aim to fulfil the unmet clinical need by restoring the normal tissue function either through stimulating the endogenous tissue repair or by using transplantation strategies to replace the missing or defective cells. Stem cells represent an essential pillar of regenerative medicine efforts as they provide a source of progenitors or differentiated cells for use in cell replacement therapies. Whilst significant leaps have been made in controlling the stem cell fates and differentiating them to cell types of interest, transitioning bespoke cellular products from an academic environment to off-the-shelf clinical treatments brings about a whole new set of challenges which encompass manufacturing, regulatory and funding issues. Notwithstanding the need to resolve such issues before cell replacement therapies can benefit global healthcare, mounting progress in the field has highlighted regenerative medicine as a realistic prospect for treating some of the previously incurable conditions

Amino Acid Availability Controls TRB3 Transcription in Liver through the GCN2/eIF2α/ATF4 Pathway

In mammals, plasma amino acid concentrations are markedly affected by dietary or pathological conditions. It has been well established that amino acids are involved in the control of gene expression. Up to now, all the information concerning the molecular mechanisms involved in the regulation of gene transcription by amino acid availability has been obtained in cultured cell lines. The present study aims to investigate the mechanisms involved in transcriptional activation of the TRB3 gene following amino acid limitation in mice liver. The results show that TRB3 is up-regulated in the liver of mice fed a leucine-deficient diet and that this induction is quickly reversible. Using transient transfection and chromatin immunoprecipitation approaches in hepatoma cells, we report the characterization of a functional Amino Acid Response Element (AARE) in the TRB3 promoter and the binding of ATF4, ATF2 and C/EBPβ to this AARE sequence. We also provide evidence that only the binding of ATF4 to the AARE plays a crucial role in the amino acid-regulated transcription of TRB3. In mouse liver, we demonstrate that the GCN2/eIF2α/ATF4 pathway is essential for the induction of the TRB3 gene transcription in response to a leucine-deficient diet. Therefore, this work establishes for the first time that the molecular mechanisms involved in the regulation of gene transcription by amino acid availability are functional in mouse liver

Mécanismes moléculaires impliqués dans la régulation de l'expression des gènes par les acides aminés chez les mammifères. Exemples de chop et d'IGFBP1

*INRA, Centre de Recherches de Clermont-Ferrand/Theix. Unité de Nutrition Cellulaire et Moléculaire 63122 St Genès Champanelle Diffusion du document : INRA, Centre de Recherches de Clermont-Ferrand/Theix. Unité de Nutrition Cellulaire et Moléculaire 63122 St Genès Champanelle Diplôme : Dr. d'Universit

Mécanismes moléculaires impliqués dans la régulation de l'expression des gènes par les acides aminés chez les Mammifères (exemples de CHOP et d'IGFBP1)

CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Leucine limitation regulates myf5 and myoD expression and inibits myoblast differentiation

absen

Adaptation to Amino Acid Availability: Role of GCN2 in the Regulation of Physiological Functions and in Pathological Disorders

International audienc

Cassette d’expression inductible et ses utilisations

La présente invention concerne une cassette d'expression comprenant un gène d'intérêt sous le contrôle d'un promoteur inductible caractérisé en ce que ledit promoteur inductible comprend au moins une séquence régulatrice CARE (C/EBP- ATF responsive element) et un promoteur minimal. L'invention concerne aussi un vecteur et une cellule hôte, ainsi qu'une composition pharmaceutique comprenant une telle cassette et leur utilisation pour le traitement des maladies par thérapie génique