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

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

Induction of CHOP expression by amino acid limitation requires both ATF4 expression and ATF2 phosphorylation

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression

Amino acid sensig: The EIF2A signaling in health and disease

National audienc

Differences in the molecular mechanisms involved in the transcriptional activation of the CHOP and asparagine synthetase genes in response to amino acid deprivation or activation of the unfolded protein response

A promoter element called the amino acid response element (AARE), which is essential for the induction ofCHOP (a CCAAT/enhancer-binding protein-related gene) transcription by amino acid depletion, has been previously characterized. Conversely, the human asparagine synthetase (AS) promoter contains two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and NSRE-2) that are required to activate the gene by either amino acid deprivation or the endoplasmic reticulum stress response. The results reported here document the comparison between CHOP and AStranscriptional control elements used by the amino acid pathway. We first establish that the AS NSRE-1 sequence shares nucleotide sequence and functional similarities with theCHOP AARE. However, we demonstrate that theCHOP AARE can function independently, whereasAS NSRE-1 is functionally weak by itself and instead requires the presence of NSRE-2. Furthermore, AS NSRE-2 can confer endoplasmic reticulum stress responsiveness to theCHOP AARE. Using activating transcription factor-2-deficient mouse embryonic fibroblasts, we also show that lack of this transcription factor does not abolish the amino acid inducibility of AS transcription, but this transcription factor is necessary to obtain the full AS response to amino acid starvation. Collectively, these results document that there are significant differences in the molecular mechanisms involved in the transcriptional activation of CHOP and AS by amino acid limitation

Role of the repressor JDP2 in the amino acid-regulated transcription of CHOP

International audienceThe transcriptional activation of CHOP (C/EBP-homologous protein) by amino acid deprivation involves ATF2 and ATF4 binding at the amino acid response element within the promoter. In this report, we investigate the role of JDP2 (Jun Dimerization Protein 2) in the amino acid control of CHOP transcription following amino acid starvation. Our results show that JDP2 binds to the CHOP AARE in unstimulated cells and that its binding decreases following amino acid starvation. We demonstrate that JDP2 acts as a repressor and suggest that it could be functionally associated with HDAC3 to inhibit CHOP transcription

Adaptation to the availability of essential amino-acids: role of GCN2/eIF2α/ATF4 pathway

In mammals, metabolic adaptations are required to overcome nutritional deprivation in amino-acids/proteins as well as episodes of malnutrition. GCN2 protein kinase, which phosphorylates the α subunit of the translation initiation factor eIF2, is a sensor of amino-acid(s) deficiencies. On one hand, this review briefly describes the main features of amino-acid metabolism. On the other hand, it describes the role of GCN2 in regulating numerous physiological functionsChez les mammifères, des systèmes d’adaptation du métabolisme sont nécessaires afin de surmonter les privations nutritionnelles en acides aminés/protéines, ainsi que certains épisodes de malnutrition. La protéine kinase GCN2, qui phosphoryle la sous-unitéα du facteur d’initiation de la traduction eIF2, est un détecteur des déficits en acides aminés. Dans un premier temps, cette revue décrit brièvement les principales caractéristiques du métabolisme des acides aminés. Dans un deuxième temps, elle décrit le rôle de GCN2 dans la régulation de nombreuses fonctions physiologique

L’activation de la voie eIF2α-ATF4, une réponse adaptative au stress cellulaire

L’activation de la voie eIF2α-ATF4, une réponse adaptative au stress cellulair

Cellular adaptation to amino acid availability: mechanisms involved in the regulation of gene expression

International audienceIn mammals, the impact of nutrients on gene expression has become an important area of research. Nevertheless, the current understanding of amino acid-dependent control of gene expression is limited. Amino acids have multiple and important roles, so their homeostasis has to be finely maintained. However, the blood amino acid content can be affected by certain nutritional conditions or various forms of pathology. It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating expression of numerous genes. The aim of this review is to examine the role of amino acids in regulating mammalian gene expression and physiological functions. A limitation for several individual amino acids strongly increases the expression of target genes such as insulin-like growth factor-binding protein1 (IGFBP-1), C/EBP homologous protein (CHOP) and asparagine synthetase (ASNS) genes. The molecular mechanisms involved in the regulation of CHOP and ASNS gene transcription in response to amino acid starvation have been partly identified. In particular, a signalling pathway requiring the protein kinase general control non-derepressive 2 (GCN2) and the activating transcription factor 4 (ATF4) has been described as sensing the amino acid limitation. In the case of an amino acid-imbalanced food source, this pathway has been shown to decrease food intake by activating a neuronal circuit. Taken together, the results discussed in this review demonstrate that amino acids by themselves can act as 'signal' molecules, with important roles in the control of gene expression and physiological functions