Regulation of autophagy by amino acids and MTOR-dependent signal transduction

Abstract

Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

Keywords: Glutamine; Leucine; Lysosomes; Mitochondria; Rapamycin.

Fig. 1

Overview of the autophagic machinery. (Macro)autophagy starts with the nucleation of an isolation membrane, named the phagophore, which surrounds a fraction of the cytoplasm destined for degradation. Upon induction of autophagy, e.g., in starvation, the ULK1 complex localizes to a specialized domain of the ER called the omegasome. This privileged site for the biogenesis of the phagophore forms a cradle where the autophagosomal membrane elongates and acts as a template for the spherical form of the autophagosome. Downstream the ULK1 complex is the PIK3C3 complex, which produces phosphatidylinositol 3-phosphate (PI(3)P) to allow the recruitment of the PI(3)P-binding proteins WIPI1/2 and ZFYVE1/DFCP1. Both contribute to the expansion and the closure of the autophagosome together with the ATG12–ATG5-ATG16L complex and the LC3-phosphatidylethanolamine (LC3-PE) conjugate. Whereas ATG12–ATG5-ATG16L only transiently associates with the autophagosomal membrane, LC3–PE constitutes a specific marker of the autophagosome as it remains associated with the autophagosomal inner membrane. The newly formed autophagosome receives input from the endocytic pathway and ultimately fuses with a lysosome, allowing the degradation of autophagic substrates by lysosomal hydrolases. Fusion of the autophagosome with the lysosome requires the small Rab GTPases (such as Rab7, 8B and 24) and the transmembrane lysosomal protein LAMP2. The products of autophagic degradation, such as amino acids, are recycled to the cytosol where they exert a negative feedback on autophagy initiation. In addition to amino acids, autophagy is also controlled by upstream signaling pathways governed by insulin/growth factors, reactive oxygen species (ROS) and the energy status (through AMPK). Most of these factors regulate the two initiation complexes, ULK1 and PIK3C3. As a master regulator of autophagy, MTORC1 integrates multiple of these upstream signals and controls the activity of the ULK1 complex

Fig. 2

Regulation of autophagy by amino acids. Autophagosome formation is regulated by two major modulators of autophagy, the MTORC1 and PIK3C3 complexes, which integrate amino acid signaling. Under fed conditions, when MTORC1 is fully active, MTORC1 downregulates autophagy by phosphorylating ULK1 and ATG13, which inhibits the ULK1 complex. MTORC1 also inhibits the synthesis of ATG proteins and the synthesis of proteins involved in the biogenesis of lysosomes at the transcription level, by preventing the translocation of TFEB to the nucleus. MTORC1 is activated in two ways: first, by insulin/growth factor signaling which involves PIK3C1, PDK1, PKB and TSC1/TSC2 as signaling components, and second, by amino acids through the Rag GTPases. To be active, MTORC1 has to localize at the lysosomal membrane, where its co-activator Rheb^GTP resides. In response to amino acids, Rag promotes the translocation of MTORC1 to the lysosomal membrane and its consecutive activation. Rag proteins are heterodimers of two subunits: RagA/B and RagC/D in which RagA/B is linked to GTP and RagC/D to GDP in the most active form of the dimer. The Rag GTPases are regulated by the v-ATPase, Ragulator and leucyl-tRNA synthetase (LRS). The nucleotide status of RagA/B and of RagC/D is regulated by GATOR and folliculin, respectively (not shown in the figure, for the purpose of clarity; see main text). In response to a rise in the intralysosomal pool of amino acids, the v-ATPase, present in the lysosomal membrane, changes its conformation and recruits Ragulator which displays a guanine nucleotide exchange factor (GEF) activity toward RagA/B. This results in the formation of RagA/B^GTP and the activation of MTORC1. In this mechanism, the transporter PAT1, responsible for the efflux of amino acids from the lysosome, controls the concentration of amino acids in the lysosomal lumen and thus the extent of MTORC1 activation. In the presence of cytosolic leucine, binding of leucine to LRS reveals its GTPase-activating protein (GAP) activity toward RagC/D, resulting in the formation of RagC/D^GDP and activation of MTORC1. The activity of Rag is also promoted by glutamate dehydrogenase (GDH). This mitochondrial enzyme, which plays a central role in amino acid catabolism, is allosterically activated by leucine. The production of 2-oxoglutarate by GDH may stimulate the loading of RagB with GTP. GDH may also activate MTORC1, and inhibit autophagy, through other distinct mechanisms. (1) The production of NAD(P)H by GDH may lead to the reduction of ROS, a potent activator of autophagy which acts through MTORC1-dependent and MTORC1-independent pathways (i.e., by inhibiting MTORC1 and PKB and by activating AMPK and ATG4). In addition to NAD(P)H, 2-oxoglutarate can also act as a scavenger of ROS, which oxidizes 2-oxoglutarate to succinate non-enzymatically. (2) The production of 2-oxoglutarate by GDH replenishes the citric acid cycle intermediates, increases the rate of ATP production and inhibits AMPK. The fall in AMPK activity may inhibit autophagy by MTORC1-dependent and MTORC1-independent mechanisms (i.e., by inhibition of TSC1/TSC2, stimulation of MTORC1 and inhibition of the ULK1 complex and of Beclin1). Probably acting in parallel with the Rag GTPase pathway, MAP4K3 and IPMK are other proteins involved in the regulation of MTORC1 by amino acids. The extracellular pool of amino acids may be sensed by the plasma membrane amino acid receptor T1R1/T1R3, which regulates MTORC1 and autophagy. The other major protein complexes controlling autophagy contain the Beclin1 protein. The core proteins of these two complexes are Beclin1, PIK3C3 and PIK3R4. When associated with ATG14 and AMBRA1, Beclin1 stimulates the early steps of autophagosome formation, downstream of the ULK1 complex. When associated with UVRAG, Beclin1 is mainly involved in the formation and maturation of autophagosomes. In response to amino acids, the protein kinase JNK1 becomes inhibited, leading to the formation of a stable complex between Beclin1 and Bcl-2 which sequesters Beclin1 and results in inhibition of autophagy. Beclin1 is also inhibited by PKB-dependent phosphorylation, which likewise inhibits autophagy. Long-term regulation of autophagy by PKB occurs by phosphorylation of FoxO3, another transcription factor (in addition to TFEB) responsible for the synthesis of ATG proteins. For further details, see main text. For reasons of clarity, the control of the inhibitory acetylation of ATG proteins by mitochondrial amino acid catabolism, which increases the concentration of acetyl CoA in the cytosol, is not indicated in the figure