Autophagy Processes and Mechanisms

By Rohan Dhiman and Sujit K. Bhutia

Autophagy Processes and Mechanisms details the process of autophagy and its significance in diseases and aging. It provides insights into autophagy mechanisms and processes to broaden our understanding. By collecting recent progress on several aspects of the autophagy process, it provides a more integrative perspective and serves as a resource that can influence future research initiatives in the field. This new book is appropriate for basic and applied researchers in cell biology, biologists and those working in the pharmaceutical sciences.

• Includes cutting-edge knowledge on autophagy processes as well as methodologies of research
• Integrates knowledge from the perspectives of basic biological science, bioinformatics, clinical research and the pharmaceutical sciences
• Provides an educational resource for students and investigators with an interest in autophagy, but who are not currently working in the field

• Language: English
• Publisher: Academic Press
• Release date: Sep 14, 2023
• ISBN: 9780323901437

Rohan Dhiman

Rohan Dhiman is Associate Professor in the Department of Life Science at Rourkela, Odisha, India. He completed his Ph.D. at Institute of Microbial Technology, Chandigarh before moving to University of Texas Health Science Centre at Tyler for post-doctorate training. After four years there with virulent mycobacteria to study the role of NK cells, regulatory T-cells and monocyte heterogeneity in tuberculosis, he returned to India and joined as a Research Scientist in Translational Health Science and Technology Institute where he started studying different facets of pathways related to autophagy and post-translational modification regulated by mycobacterium in infected macrophages. After joining in current institute in 2014, he started his independent lab and currently focusing on screening some libraries w.r.t. autophagy induction ability of the molecules and Calcimycin is one of the compounds that his lab found through this approach. He has published 35 papers till now and co-authored different book chapters.

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Chapter 1: mTOR signaling and autophagy regulation

Amruta Singh, and Sujit Kumar Bhutia     Cancer and Cell Death Laboratory, Department of Life Science, National Institute of Technology, Rourkela, Odisha, India

Abstract

The mechanistic target of rapamycin (mTOR) pathway integrates a range of cellular and environmental signals for the growth and development of eukaryotic cells. For over two and half decades, studying mTOR signaling has revealed it as a central regulator of diverse anabolic and catabolic pathways, including biogenesis, metabolism, and autophagy. mTOR inhibition induces autophagy for the recycling of cellular components to counteract cellular stress. The mTOR kinase blocks autophagy, including direct inhibition of the ULK1 complex and the control of lysosomal biogenesis and degradative ability. Although the physiological function is incompletely understood, the complex molecular connection and crosstalk between the mTOR pathway and autophagy are significantly documented at the cellular level to develop novel therapeutics to control different metabolic diseases. This chapter highlights an overview of mTOR structure and signaling pathway in autophagy regulation and focuses on different mTOR modulators to regulate autophagy signaling.

Keywords:

Autophagy; Lysosome; Metabolism; mTOR; ULK1 complex

1. Introduction

Autophagy is an evolutionary conserved intracellular regulatory process for nutrient recycling that involves lysosomal degradation of dysfunctional unwanted proteins and organelles. Unlike the ubiquitinated proteasomal system that degrades short-lived ubiquitinated proteins by deubiquitinating enzymes, this lysosomal-based pathway is quite complex. The traditional model dictates that autophagy activation occurs under starvation conditions, but basal-level autophagy also occurs under nutrient-rich conditions. Under nutrient starvation or stress, the cells rewire their cellular metabolism from growth toward autophagy induction. Autophagy, as an intracellular regulatory mechanism, detects the energy need of the cell and manages the nutrient supply for survivability. This detection involves mTOR (mechanistic or mammalian targets of rapamycin) pathway that responds to growth factors, energy levels, and nutrients. The protein kinase mTOR stands to be a central regulator in autophagy induction. It acts as a primary sensor of nutrients in cells and maintains cellular homeostasis in response to physiological and environmental stress. In mammals, the mTOR gene encodes this highly conserved protein, belonging to the family of PIKK (phosphatidylinositol-3 kinase-related kinase). The mTOR protein was initially named FRAP1 (FK506 binding protein 12-rapamycin associated protein 1) encoded by the FRAP1 gene. The protein was discovered through independent studies carried out for the naturally found compound, rapamycin (Heitman et al., 1991). These studies reported mTOR as a central regulator in different signaling networks. The TOR protein kinase is known to be involved in a broad range of cellular activities, which are generally seen to be dysfunctional in many disorders. For example, in neurodegenerative disorders such as Alzheimer's, the PI3K/Akt/mTOR pathway is hyperactivated, leading to impairment in autophagy. Similarly, in Parkinson's disease, the Lewy bodies so formed are aggregates of misfolded α-synuclein, that are not degraded by the cellular clearance mechanism. Also, the mTOR protein is reported to be hyper-activated in the case of insulin resistance, leading to type 2 diabetes. Moreover, chronically active mTOR stimulates cell proliferation and increases metabolism in cancerous cells, leading to tumor progression. In this chapter, we highlight the structure of mTOR and its associated signaling in controlling autophagy. Further, we focus on different mTOR modulators to regulate autophagy signaling, which could have a potential therapeutic benefit in curing a wide range of disorders.

2. Structure of mTORC1

mTOR is the master regulator for maintaining cellular homeostasis arising due to changes in energy level, growth factors, and nutrient balance. The TOR protein is a serine-threonine kinase, initially discovered as the target of rapamycin, which in mammals exists as two different types of multimeric protein complex, mTORC1 and mTORC2 (Yang et al., 2013). The mTORC1 complex has a prime regulatory role in autophagy, ribosomal biogenesis, and protein translation. This complex primarily constitutes three core subunits, including raptor (Hara et al., 2002), mTOR, and mLST8 (mammalian lethal with SEC13 protein 8) (Kim et al., 2003). mTOR provides the catalytic site for binding of mLST8 that leads to downstream recruitment of substrates through raptor. Additionally, studies reveal mLST8 as an essential component for stabilization of the kinase activation loop (Yang et al., 2013), whereas raptor is necessary for subcellular localization of the complex (Nojima et al., 2003). Apart from the three core components, PRAS40 (Haar et al., 2007; Sancak et al., 2007) and DEPTOR (Peterson et al., 2009) are the two inhibitory subunits essential for modulating the regulation of mTORC1. Cryo-electron microscopy reveals mTORC1 as a dimer having a rhomboidal shape with a cavity in the middle. The mTOR kinase is a 2549 amino acid long bilobed structure having an amino-terminal lobe, a large carboxyl-terminal lobe, and a cleft in-between them for binding with ATP. In mammals, unlike yeasts, the mTOR kinase in both complexes has the same TOR gene, a functional homolog of the yeast TOR2 gene. The mTOR kinase starts at the N-terminus consisting of tandem HEAT (Huntington's, Elongation factor 3, PP2A, and TOR1 yeast kinase) repeats made of α-helix, which involves multimerization and binding of raptor (Aylett et al., 2016; Yip et al., 2010). It is followed by the FAT (FRAP, ATM, TRAP) domain made of α-helices for interacting with the kinase domain by clamping to it. This conformation helps to regulate the mTOR kinase activity by bringing stability to the activation loop. The FAT domain present toward the C-terminus is the FRB domain (FKBP12–rapamycin-binding), where the FKBP12 (FRB-FK506 binding protein 12)–rapamycin complex binds and drastically reduces the accessibility of substrates to the catalytic site. The C-terminus lobe constitutes the FATC (C-terminal FAT) domain with the mLST8 binding site called the LBE domain. mLST8 is understood to be an activator of mTORC1 by indirectly influencing the conformation of the active site. The FATC forms an integral part of the kinase domain as it interacts with and stabilizes the activation loop structure by forming the LBE-FATC-activation loop spine. To prevent random substrate binding, the activation site is highly guarded by the FRB domain as well as by an inhibitory helix in the catalytic site, thus facilitating privileged binding to specific substrates (Yang et al., 2013). Overall, the structural reconstitution of the complex discloses it as a highly restricted but intrinsically active kinase with a mechanism similar to canonical protein kinases (Fig. 1.1).

Figure 1.1  (A) Domain structure of mTOR with its binding site for the respective subunit. (B) Components of mTORC1 and cellular processes they regulate.

3. mTOR signaling in ULK1 activation to induce autophagy

As a central nutrient regulator, mTOR plays a crucial role in maintaining cellular homeostasis, especially by controlling intensive catabolic pathways such as autophagy (Chan et al., 2007; Jung et al., 2009). For the regulation of autophagy, ULK1 (Unc 51 like kinase 1) (Ganley et al., 2009; Jung et al., 2009) and VPS34 (Vacuolar protein sorting 34) act as primary mTORC1 targets. The mTOR kinase modulates the autophagy pathway by mediating inhibitory phosphorylation of its effector protein, ULK1, and transmitting the upstream nutrient signals to the downstream targets. The serine/threonine kinase is a structural and functional homolog of yeast ATG1 (Autophagy related gene 1) (Kuroyanagi et al., 1998), whose activation is essential and an indispensable step in the early stages of autophagy. Along with ULK1, the complex constitutes FIP200/RB1CC1, ATG 13, and ATG101 subunits that play a major role in autophagosome formation (Dunlop et al., 2011; Ganley et al., 2009; Jung et al., 2009). Under nutrient-rich conditions, raptor directly binds to ULK1 and phosphorylates it, resulting in the inhibition of the complex, whereas a nutrient-starved state leads to the dissociation of raptor, leading to the formation of autophagosome (Dunlop et al., 2011). Consequently, this results in the mTOR-dependent ULK1 activation in response to nutrients. Moreover, the mTORC1 activity negatively correlates with the binding of phosphorylated FIP200 to ULK1 in an ATG13-dependent manner (Dunlop et al., 2011). The FIP200 interacts with proteins, including FAK, Pyk2, TSC1, ASK1, and TRAF2, which maintain the microtubule dynamics and cell growth, thus stabilizing the complex (Gan & Guan, 2008). Along with nutrient deficiency, stress and pharmacological inhibition also function as major factors for mTOR inhibition and initiation of autophagy. Autophagy is initiated when mTOR-dependent phosphorylation is impeded, which promotes the interaction of ULK1 with AMPK. Under glucose deficiency, this interaction leads to the phosphorylation of Ser317 and Ser777 residues of ULK1. However, Ser757 phosphorylation leads to ULK1 inhibition, thus concluding that this synchronized phosphorylation is highly crucial in regulating the autophagy mechanism (Kim et al., 2011). The downstream kinase activity of the complex on the autophagosomal surface mainly depends on the phosphorylation of ULK1, which is brought about after the association of ULK1 binding proteins. Once activated, the ULK1 complex further promotes the phosphorylation of Beclin1 resulting in the ER translocation of VPS34 from the dynein complex (Russell et al., 2013). The VPS34 on the pre-autophagosome membrane recruits PI as its effector and gives rise to PI3P-enriched membrane, which aids in the nucleation of the autophagosome.

Along with ULK1, DAP1 (death-associated protein 1) is also a downstream effector of mTORC1. This proline-rich 102 residues long protein has a highly conserved sequence that transcribes a 2.4-kb mRNA. Under amino acid starvation, DAP1 showed an increase in dephosphorylation at Ser3 and Ser51, which increased its electrophoretic mobility (Koren et al., 2010). These characteristic alterations appear similar to the downstream effectors of mTOR, the S6K. Further, the knockdown of DAP1 under amino acid starvation led to an increase in autophagic flux, thus suggesting its inhibitory role in autophagy machinery (Koren et al., 2010). Additional studies on DAP1 mutants and knockdowns established mTOR as the specific kinase for DAP1 (Ramchandani et al., 2020; Yahiro et al., 2014). Nonetheless, autophagy modulated by DAP1 at a molecular level still needs to be investigated.

mTORC1 also assists in the translocation of transcription factors including TFE3 (transcription factor E3), TFEB (transcription factor EB), and MITF (microphthalmia-associated transcription factor) that are essential in lysosomal biogenesis. Lysosomes are acidic vesicles with hydrolases for the breakdown and recycling of essential nutrients for the maintenance of cellular homeostasis. Many cellular functions are regulated through the lysosomal degradation pathway ranging from autophagy to phagocytosis, and endocytosis. The lysosomal biogenesis is mainly controlled by the gene network called CLEAR (coordinated lysosomal expression and regulation), consisting of the master regulators TFEB, TFE3, and MITF (Palmieri et al., 2011; Sardiello et al., 2009; Settembre et al., 2011). Amino acid abundancy leads to activation of the Rag heterodimer, resulting in the recruitment of mTORC1 to the surface of the lysosome membrane where Rheb GTPase aids in activating the complex (Durán et al., 2012; Kern et al., 2015). Simultaneously, the active Rag GTPases recruit the transcription factors TFEB, TFE3, and MITF to lysosome, where they get phosphorylated by mTORC1, triggering their binding to the 14-3-3 proteins. This interaction inhibits their translocation to the nucleus. thus retaining them in the cytosol (Martina et al., 2012; Napolitano & Ballabio, 2016). On the contrary, Rag GTPase's inactivation due to amino acid deficiency leads to suppression of mTORC1 activity along with calcineurin-dependent dephosphorylation of TFEB causing its subcellular localization from cytoplasm to the nucleus where it triggers the transcription of autophagy- and lysosomal-related genes (Medina et al., 2015). Along with autophagy, mTORC1 also mediates the phosphorylation of two major substrates: S6K1 (40S ribosomal S6 protein Kinase 1) and 4E-BP1 (eIF-4E binding protein 1) essential for mRNA translation and vital for protein translation (Schalm et al., 2003) (Fig. 1.2).

4. mTOR upstream signaling in autophagy

mTOR balances anabolic and catabolic pathways which must only be turned on or off under highly regulated conditions. The oscillation in nutrient levels, predominantly growth factors, including insulin and amino acids (leucine, arginine, and glutamine), plays a vital role in mTOR activity. The G proteins, Rheb and Rag GTPases, act as transmitters of upstream signals for modulating the kinase activity and intracellular localization of mTOR. The kinase domain is stimulated by Rheb only when it is localized on the lysosome in a GTP-bound state. Still, simultaneously the heterodimer Rag must aid in the recruitment of mTOR onto the lysosomal surface leading to activation of the mTOR pathway required for growth and development.

4.1. Growth factors as mTORC1 modulators

Growth factor signals involving insulin and IGF1 (insulin-like growth factor 1) generally inhibit autophagy through the insulin-PI3KC1-Akt-TSC-mTOR mediated pathway (Harrington et al., 2004; Meijer & Codogno, 2009). This involves the positive mTOR regulators, including Rheb and PDK1, and negative mTOR regulators TSC2 and PTEN. The PI3KC1 complex is made of a catalytic, and a regulatory subunit gets activated upon growth factor stimulation. The PI3K, upon activation, phosphorylates PI(4,5)P2 to PI(3,4,5)P3, which aids in recruiting proteins containing the PH (Pleckstrin homolog) domain (Dibble & Cantley, 2015). The Akt is a serine-threonine kinase with a PH domain that gets recruited to the plasma membrane in a PI(3,4,5)P3-dependent manner (Ebner et al., 2017). The PH domain of Akt is activated at its T loop consisting of Thr308 and Ser435. At its Thr308 residue, Akt gets phosphorylated by the PDK1, which also gets recruited to the membrane in a PI(3,4,5)P3-dependent manner (Wei et al., 2019). Hence, the PI(3,4,5)P3 has an essential role in the PI3K-Akt pathway, as its dephosphorylation by PTEN leads to a decrease in PI3K recruitment. Further, the activated Akt causes autophagy inhibition through TSC-dependent and TSC-independent pathways. The TSC-independent pathway involves the direct phosphorylation of Akt at the PRAS40 subunit of the mTORC1 complex and, in turn, causes mTOR activation (Huang & Porter, 2005; Inoki, Zhu et al., 2003; Inoki, Li et al., 2003). Whereas the TSC-dependent pathways involve the heterodimerization of TSC1-TSC2 to form a complex, which have GAP (GTPase Binding Protein) activity for small G-proteins like Rheb GTPase. The Rheb protein interacts with the catalytic site of mTOR, and the conversion of RhebGTP to RhebGDP renders the mTOR complex inactive (Inoki, Zhu et al., 2003; Inoki, Li et al., 2003). However, the insulin binding activates Akt1 through the PI3K, which in turn inhibits TSC2 by phosphorylating it. This causes the active RhebGTP to bind with the catalytic domain of mTOR, leading to activation of the complex, thus inhibiting autophagy (Inoki et al., 2002). However, mTOR is inhibited under growth factor scarcity due to a decrease in the number of nutrient receptors on the cell surface, thus leading to a decrease in the cellular nutrient pool. This triggers autophagy to maintain the nutrient and energy supply of cells for survival