Emerging Mechanisms in Initiating and Terminating Autophagy Manuela Antonioli,1,2 Martina Di Rienzo,1,3 Mauro Piacentini,1,3 and Gian Maria Fimia1,4,*

TIBS 1309 No. of Pages 14

Review Emerging Mechanisms in Initiating and Terminating Autophagy Manuela Antonioli,1,2 Martina Di Rienzo,1,3 Mauro Piacentini,1,3 and Gian Maria Fimia1,4,*

Autophagy is a major degradative process activated in a rapid and transient Trends manner to cope with stress conditions. Whether autophagy is beneficial or Autophagy is a catabolic process detrimental depends upon the rate of induction and the appropriateness of essential for the basal turnover of cel- the duration. Alterations in both autophagy initiation and termination predis- lular components. In response to pose the cell to death, and affect the execution of other inducible processes stress stimuli, autophagy is rapidly induced to ensure the restoration of such as inflammation. In this review we discuss how stress signaling path- cellular homeostasis. The duration of ways dynamically control the activity of the autophagy machinery by mediat- autophagy response is also tightly fi regulated to avoid uncontrolled cell ing post-translational modi cations and regulatory protein interactions. In digestion. particular, we highlight the emerging role of TRIM and CULLIN families of ubiquitin ligases which play opposite roles in the autophagy response by Autophagy is mainly regulated at the post-translational level. Phosphoryla- promoting or inhibiting, respectively, the activity of the autophagy initiation tion and ubiquitination are the best- complex. characterized post-translational modifications responsible for the regulation of the autophagy core machinery both Autophagy – A Tightly Regulated Response To Stress at the initiation and termination stages. Macroautophagy, hereafter referred as autophagy (see Glossary), protects cells from stress- fi Recently, two large families of E3 ubi- induced damage by engul ng non-functional cellular components into double-membrane quitin ligases, TRIM and CULLIN, have vesicles called autophagosomes and delivering them to the lysosome for degradation [1].By been identified as crucial regulators of this mechanism, it also maintains an appropriate amount of ‘building elements’ to be recycled for the autophagy response by acting, macromolecular synthesis and energy production when external nutrient supply is limited [1]. respectively, as platforms for the activation of the core machinery and driv- ing its proteasomal degradation to Dysregulated autophagy is associated with several pathological conditions [2]. On the one hand, terminate the process. defective autophagy leads to the accumulation of protein aggregates or dysfunctional organelles that predispose to neurodegeneration, muscular dystrophy, and metabolic syndromes [1,3]. Impairments in autophagy are also associated with increased inflammation and the development 1 of autoimmune pathologies such as Crohn's disease [4]. This is often coupled with accumulation National Institute for Infectious Diseases ‘L. Spallanzani’, Istituto Di of reactive oxygen species (ROS), which can also cause genomic alterations that lead to Ricovero e Cura a Carattere tumorigenesis [1]. On the other hand, an unrestrained response turns autophagy into a cell Scientifico (IRCCS), 00149 Rome, Italy 2 death process as a result of excessive self-digestion. Damage induced by excessive autophagy Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, is observed in myocardial and cerebral ischemia [5]. Freiburg 79104, Germany 3Department of Biology, University of A crucial feature of autophagy is that it is a highly dynamic process able to sense intracellular Rome ‘Tor Vergata’, 00173 Rome, Italy 4Department of Biological and stress within minutes and rapidly mount a response to cope with the damage. This is made Environmental Sciences and possible by the ability of a multitude of unrelated cellular pathways to converge on the autophagy Technologies (DiSTeBA), University of machinery to signal a diversity of stressors [6]. A series of signal transduction kinases were Salento, Lecce 73100, Italy initially identified as crucial switches of autophagy protein activity [6]. It is now emerging that the fi autophagy response to stress also requires the participation of other post-translational modi - *Correspondence: cation enzymes, with a major role played by E3 ubiquitin ligases in controlling autophagy [email protected] (G.M. Fimia).

complex stability [6]. In this review we provide an integrated view of how the functional interaction Glossary between stress signaling pathways and the core autophagy proteins results in the proper Autophagy: lysosomal degradation initiation and termination of the autophagy response. of intracellular components which is achieved by three different mechanisms, defined as The Core Autophagy Proteins macroautophagy, microautophagy, The autophagy process consists of a sequence of molecular events that lead to the formation of and chaperone-mediated autophagy the autophagosome which enwraps intracellular material and eventually fuses to the lysosome to (CMA). In macroautophagy, degrade its contents [1,7]. Each step is under the control of specific autophagy complexes autophagosomes sequester portions of cytoplasm and deliver them to whose activity is directly or indirectly regulated by stress signaling pathways [6]. lysosomes for degradation. In microautophagy, components are The ULK complex is composed of protein kinases ULK1 and ULK2, in association with the directly sequestrated through FIP200 scaffold protein and the regulatory subunits ATG13 and ATG101, which are thought to invaginations of the endolysosomal membrane. CMA selectively degrades activate ULK proteins by inducing conformational changes [8]. This complex is essential for proteins containing a KFERQ motif, transmitting stress signals to the site where the autophagosome will be formed, mainly in which is recognized by cytosolic nutrient- or energy-deprived conditions, by both mediating activating phosphorylation of down- chaperones and delivered to stream autophagy proteins and playing non-catalytic, scaffolding roles [8,9]. lysosomes via LAMP2A. CULLIN: the largest family of E3 ubiquitin ligases promoting The VPS34 complex I is composed of the class III phosphatidylinositol-3-kinase VPS34, an ubiquitination and degradation of enzyme that also plays a role in endocytosis, together with the autophagy-specific cofactors proteins involved in a variety of BECLIN-1 and ATG14, as well as the VPS15 scaffold protein which is required for the assembly cellular processes. CULLINs are protein complexes composed by four of the catalytic and regulatory subunits [10,11]. This complex allows the formation of phos- elements: the catalytic core (RING- phatidylinositol-3-phosphate (PI3P), a key signal for the recruitment of autophagy factors finger proteins 1 and 2), the scaffolds required for the nucleation of the autophagosome precursor membrane (termed the isolation (CUL1, 2, 3, 4A, 4B, 5, 7, 9), the adaptors (SKP1, elongin B/C, and membrane or phagophore) [7]. PI3P is mainly produced in specialized structures of the DDB1) and the substrate receptors endoplasmic reticulum (ER), known as omegasomes [12], that are preferentially located in (>400 proteins classified on the basis the contact sites of ER with other organelles, such as the ER–mitochondria junction [13] and the of structural domains: F-BOX, SOCS, ER–Golgi intermediate compartment [14]. BTB and WD40). Mechanistic target of rapamycin (mTOR): a Ser/Thr kinase that The phagophore membrane undergoes expansion by a mechanism that involves the recruitment regulates cell growth and metabolism of ATG9 and a large family of mammalian paralogs of yeast Atg8 (mATG8s) that includes the LC3 in response to extracellular signals. and GABARAP subfamilies [7]. Vesicles containing the transmembrane protein ATG9 shuttle mTOR is part of two distinct complexes, TORC1 and TORC2, between the trans-Golgi network, the endosomal compartment, and the phagophore [7,15]. which differ in the accessory proteins Although its exact function remains unclear, ATG9 is thought to deliver lipids to autophagosome they contain, such as RAPTOR and precursors. mATG8 are inserted in the phagophore by covalent attachment to the lipid RICTOR, respectively. mTORC1 phosphatidylethanolamine [16]. The modification of LC3s, the best-characterized mATG8s, controls protein synthesis, ribosomal biogenesis, transcription, and occurs through an ubiquitination-like cascade regulated by the LC3 protease ATG4, the E1 autophagy. mTORC2 mainly activating enzyme ATG7, the E2 conjugating enzyme ATG3, and the E3 ligase complex regulates endocrine signaling, such comprising ATG5/ATG12/ATG16L1 [16]. The LC3 membrane insertion site is determined by as insulin, IGF-1, and leptin. the PI3P interacting protein WIPI2, which binds to ATG16L1 and recruits the E3 ligase to the Tumor necrosis factor receptor- associated factor 6 (TRAF6): an isolation membrane [17]. During autophagosome biogenesis, LC3 carries out membrane E3 ubiquitin ligase acting tethering and hemifusion activities which are thought to control the size of autophagosomes downstream from multiple receptor [18]. Moreover, LC3 interacts with cargo receptors, such as the sequestosome 1/p62-like family families, such as the TNFR superfamily, interleukin-1/Toll-like proteins, which confer selectivity to autophagic degradation [19]. receptor family, tumor growth factor- b receptors, and the T cell receptor. Finally, the fusion of the autophagosome with the lysosome is regulated by proteins that are In this context, TRAF6 activates the largely shared with the endocytic pathway. They include the VPS34 complex II, in which transcription factor NF-kBby catalyzing the non-degradative BECLIN-1, VPS34, and VPS15 are present, while UVRAG replaces ATG14 [7,10], which acts (Lys63-linked) ubiquitination of in cooperation with the small GTPase protein RAB7 [20], the RAB7 effector protein PLEKHM1 various target proteins, including the [21], the SNARE protein syntaxin 17 [22], and the homotypic fusion and vacuole protein sorting TAK1/TAB2/TAB3 complex, complex (HOPS) [23]. Both autophagosome and endosome fusion with the lysosomes are subsequently activating IKKs and MAPKs. negatively regulated by RUBICON, which binds to the VPS34 complex II in a mTORC1- dependent manner and interferes with the activity of RAB7 and HOPS [24].

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Regulation of Autophagy Initiation Tripartite motif proteins (TRIMs): The autophagy machinery is constitutively expressed in all mammalian cells, suggesting that these represent a large family of E3 ubiquitin ligases (>70 members) regulation of autophagy by stress stimuli occurs mainly at the post-translational level. In this characterized by the presence of scenario, a coordinated cascade of phosphorylation and ubiquitination has emerged as a major three domains: RING finger, B-Box- switch to activate the autophagy machinery (Figure 1 provides an overview of the currently type zinc- finger, and a coiled-coil known post-translational modifications of core autophagy proteins, as discussed below). region. Moreover, TRIM proteins are classified into 11 subfamilies, based on their C-terminal domains, which Regulation by Signal Transduction Kinases mediate both regulative protein– ULK1, mTORC1, and Growth Factor-Regulated Kinases protein interactions and substrate The best-characterized stimulus that induces autophagy is nutrient deprivation. Under fed recognition. Ubiquitination: the attachment of conditions, intracellular amino acids and growth factors activate mTORC1, a mechanistic the ubiquitin protein to e-amino target of rapamycin (mTOR) kinase complex that positively regulates anabolic processes and groups of lysine residues of target represses catabolic ones, including autophagy. mTORC1 inhibits the ability of ULK complex to proteins through three different fi activate downstream ATG proteins by phosphorylating ULK1 at Ser638/758 and its partner enzymatic steps. Ubiquitin is rst linked to the E1 ubiquitin-activating ATG13 at Ser258 [25,26]. Interestingly, mTORC1 may redirect rather than fully inhibit ULK1 enzyme and then transferred to the activity. Indeed, it has been observed that type I interferons require ULK1 to activate p38 MAPK E2 ubiquitin-conjugating enzyme. and induce the expression of interferon-responsive genes. In this context, mTORC1 stimulates Finally, ubiquitination linkage to target ULK1 activity through its phosphorylation at Ser758 [27]. proteins is obtained by the association of E2 with an E3 ubiquitin ligase, which confers specificity to In nutrient starvation, inactive mTORC1 is released from ULK1, allowing ULK1 to phosphorylate substrate recognition. Poly- its own complex components and downstream autophagy targets [25]. ULK1 stimulates PI3P ubiquitination is achieved by consecutive linkage of other ubiquitin synthesis by phosphorylating members of the VPS34 complex I, including BECLIN-1 (Ser15/30/ molecules to one of the seven lysines 96/279), VPS34 (Ser249), and ATG14 (Ser29) [28–30]. Moreover, ULK1 activates AMBRA1, a present in its sequence. Depending WD40 protein required for VPS34 complex I stability, by releasing it from different inhibitors such on the type of polyubiquitin chains, as the dynein motor [31] and the E3 ubiquitin ligase CULLIN-4 [32]. ULK1-dependent phos- ubiquitination leads to proteasomal fi degradation (e.g., Lys48-linked) or phorylation sites on AMBRA1 have been identi ed (Ser465/635), although their role has yet to be regulation of signaling pathways (e.g., assessed [29]. Conversely, AMBRA1 activity is suppressed by mTORC1 phosphorylation of Lys63-linked) by modulating protein– Ser52 [33]. protein interactions.

In addition to activating mTORC1, growth factors also inhibit BECLIN-1 activity through AKT- mediated phosphorylation at Ser234/295, which mediates BECLIN-1 interaction with 14-3-3 proteins and its consequent sequestration to the cytoskeleton [34]. Moreover, the epidermal growth factor receptor (EGFR) suppresses autophagy by phosphorylating BECLIN-1 at Tyr229/ 233/352 [35]. These modiﬁcations increase BECLIN-1 binding to the negative regulators BCL-2 and RUBICON, and decrease its association with VPS34. EGFR has a more complex role in autophagy regulation. In fact, unstimulated EGFR acts as a positive regulator of autophagy by forming a complex with LAPTM4b in the endosomal compartment, which is able to displace RUBICON from BECLIN-1, setting the latter free to initiate autophagy [36].

AMP Kinase (AMPK) Energy limitation, either from excessive energy consumption or shortage of energy stores, is another relevant autophagy stimulus. A high AMP/ATP ratio activates the LKB1/AMPK pathway, which stimulates catabolic processes to provide elements for ATP synthesis [37]. In particular, AMPK activates autophagy by phosphorylating ULK1 at multiple sites, including Ser317/555, and BECLIN-1 at Ser91/Ser94 [38–40]. Moreover, AMPK is able to differentially control the endocytic and autophagic pools of VPS34. In fact, AMPK inhibits endosomal VPS34 by phosphorylating it at Thr163/Ser165, thereby shutting down this process when external energy sources become limited. Conversely, the pro-autophagic VPS34 is not phosphorylated by AMPK because of the protective role of ATG14, which instead favors an activating phosphorylation of BECLIN-1 at Ser91/Ser94 by AMPK [41]. AMPK also phosphorylates the scaffold protein RACK1 to induce its binding to BECLIN-1, VPS15, and ATG14, promoting the assembly of VPS34 complex I [40].

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(A) Post-translaonal modiﬁcaons (B) Regulaon via phosphorylaon

Low Low nutrients Nerve injury Bone formaon K162 S317 S555 K606 S637 S758 S1042 T1046 nutrients

ULK1 1 Kinase Ser/ThrULK1/2domain CTD 1050 ER mTORC1 JNK stress S258 ATG13 IRE1 AMBRA1 FIP200

K117 K266 ULK1 BECLIN-1 BCL-2 S93 T108 Y233 K265 S295 K437 S15 S30 S90 S96 T119 Y229 S234 K263 S279 Y352 S409 K430 RAPTOR, TSC2 MST1 BECLIN-1 1 BH3 CCD ECD CTD 450 AMPK DAPK BCL-2 UVRAG VPS34 MEK3/4 ROCK1 DAPK3 ATG14 Low energy

S29 S165 T159 T163 S249 (C) Regulaon via ubiquinaon VPS34 1 C2 Helical Lipid kinase 875 VPS15 BECLIN-1 DEPTOR CULLIN 5 CULLIN 4 RNF216

USP9X K45 S52 S465 S635 USP10

mTORC1 inhibion USP13 AMBRA1 1 WD40 Pro-rich Ser-rich BH3 1298 ULK1 AMBRA1 BECLIN-1 USP19 DDB1 BECLIN-1 ULK1 Dynein BCL-2 WASH ELONGIN-B TRAF6 A20 NEDD4

Figure 1. (A) Post-translational Modiﬁcations of ULK and VPS34 complexes. Domain structures of ULK1, BECLIN-1, VPS34, and AMBRA1 proteins with positions of post-translational modiﬁcations. Abbreviations: ac, acetylation; CCD, coiled-coil domain; CTD, C-terminal domain; ECD, evolutionarily conserved domain; ISG, ISGylation; P, phosphorylation (green, activation by phosphorylation; red, inhibition by phosphorylation); Pro-rich, proline-rich domain; Ser-rich, serine-rich domain; Ser/Thr domain, serine-/threonine-rich domain; U, ubiquitination. (B) Regulation of ULK AND VPS34 complexes via phosphorylation. mTORC1 inhibits autophagy by phosphorylating ULK1 and AMBRA1. mTORC1 activity is inhibited under low nutrient conditions. AMPK, stimulated by low energy levels, activates autophagy by phosphorylating ULK and VPS34 complexes. Activated ULK1 phosphorylates the VPS34 complex I. Other stress signals modulate autophagy via the indicated kinases by phosphorylating either BECLIN-1 or BCL-2. P, phosphorylation (green, activation by phosphorylation; red, inhibition by phosphorylation). (C) Regulation of ULK and VPS34 complexes via ubiquitination. ULK1 is activated by AMBRA1, which mediates ubiquitination by TRAF6; AMBRA1 also indirectly stimulates ULK1 by inhibiting CULLIN-5-mediated degradation of the mTORC1 inhibitor DEPTOR. TRAF6 is also responsible for the non-degradative ubiquitination of BECLIN-1, which is reversed by the deubiquitinase A20. The stability of AMBRA1 and BECLIN-1 proteins is under the control of the E3 ubiquitin ligases CULLIN-4, RNF216, and NEDD4. Indicated deubiquitinating enzymes reverse ubiquitination of BECLIN-1, thereby preventing its degradation. Abbreviation: U, ubiquitination (green, activation by ubiquitination; red, inhibition by ubiquitination); 11, Lys11-linked ubiquitination; 48, Lys48-linked ubiquitination; 63, Lys63-linked ubiquitination.

AMPK indirectly activates autophagy by repressing mTOR activity through inhibition of the mTORC1 component RAPTOR and activation of the mTORC1 inhibitor TSC2 [37]. However, ULK1 activation by AMPK can occur in the absence of mTORC1 inhibition, such as in muscle cells during physical exercise [42].

Importantly, many other stimuli converge on AMPK to activate autophagy. For example, calcium- mobilizing pathways stimulate autophagy via CAMKK2-mediated phosphorylation of AMPK [43].

Stress Kinases Targeting the BECLIN-1/BCL-2 Interaction Under non-stress conditions, the autophagosome-forming activity of BECLIN-1 is inhibited by the interaction of with speciﬁc members of the BCL-2 family: the anti-apoptotic proteins BCL-2, BCL-xL, MCL-1 at the ER [44], and the pro-apoptotic protein BIM at microtubules [45].

Autophagy induction requires the dissociation of these proteins from BECLIN-1, and this is achieved through either signaling kinases or protein–protein displacement (Box 1). JNK was the ﬁrst identiﬁed kinase able to trigger BECLIN-1 activity by releasing BCL-2 binding upon phosphorylation [46]. JNK is activated in nutrient starvation by its recruitment to acetylated tubulin through the JIP1/kinesin complex [47]. The kinase ROCK1 also contributes to this

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Box 1. Regulation of BECLIN-1 Function Through Its BH3 Domain The BH3 domain of BECLIN-1 mediates the inhibitory interaction with the anti-apoptotic members of BCL-2 family. The crucial role of these proteins in the control of autophagy is also underlined by the presence of several viral BCL-2-like proteins which hijack the autophagy inhibitory function of their cellular homologs [91]. For example, alphaherpesvirus-1 ICP34.5, human cytomegalovirus TRS1, Kaposi's sarcoma herpesvirus vBCL-2, and murine gamma herpesvirus 68 M11 inhibit the formation of autophagosomes by binding to BECLIN-1 in infected cells.

In addition to phosphorylation, the BCL-2/BECLIN-1 interaction is disrupted by competitive binding to a series of proteins. Stress-induced expression of BH3-only proteins, such as tBID, BAD, PUMA, NOXA, NIX, and BNIP3, frees BECLIN-1 from BCL-2 family proteins and induces autophagy [92]. AMBRA1 is also able to bind to BCL-2 through a BH3-like domain. Under non-stress conditions, BCL-2 inhibits the pool of AMBRA1 at mitochondria. When autophagy is induced, AMBRA1 dissociates from BCL-2 by an as-yet unknown mechanism. Active AMBRA1 can migrate to the ER where it activates BECLIN-1 by reducing its interaction with BCL-2, or contributes to mitophagy by binding to both the E3 ligase Parkin and LC3 on damaged mitochondria [93,94].

Non-BH3 only proteins may also disrupt BECLIN-1/BCL-2 interaction to induce autophagy, as shown for VMP1, a stress-inducible ER/Golgi transmembrane protein [95], and HMGB1, a chromatin-associated protein that interacts with BECLIN-1 upon nucleus-to-cytosol translocation in a ROS-dependent manner [96]. activation by both phosphorylating BECLIN-1 at Thr119 in the BH3 domain and by stimulating JNK via JIP1 phosphorylation [48]. Several other stimuli induce autophagy via JNK, such as those inducing ER stress via the IRE1/TRAF2/ASK1 pathway [49]. Relevant examples of physiological processes in which autophagy is regulated by JNK are the degradation of unfolded type II collagen in growth plate chondrocytes during bone formation [50], and the breakdown of myelin in Schwann cells after nerve injury [51].

DAPK, a stress kinase activated in response to oxidative and ER stress, is also able to release BCL-2 from BECLIN-1. In this case, binding is disrupted by phosphorylating the BH3 domain of BECLIN-1 (Thr119) [52]. DAPK also activates VPS34 indirectly by phosphorylating protein kinase D, which in turn binds to and phosphorylates VPS34 [53].

Of note, phosphorylation of BECLIN-1 at a different site in the BH3 domain can instead strengthen the BCL-2/BECLIN-1 interaction, as shown for phosphorylation of Thr108 by the pro-apoptotic kinase MST1 [54]. The relevance of this kinase in autophagy regulation has been conﬁrmed analyzing Mst1 transgenic mice, which show cardiac dysfunction associated, on the one hand, with the accumulation of protein aggregates as a consequence of excessive autophagy inhibition by BCL-2 and, on the other hand, on the reduced amount of BCL-2 available to block the pro-apoptotic BH3-only protein BAX [54].

Taken together, these data underline how the BECLIN-1/BCL-2 association is a crucial target of both pro- and anti-autophagic stress kinases. What remains less well characterized is the mechanism by which the BECLIN-1 activity is inhibited by BCL-2. A recent report provided evidence that the inhibitory activity of BCL-2 may reside in its ability to prevent activating phosphorylation of BECLIN-1 at Ser90, which is mediated by the stress kinases MAP- KAPK2/MAPKAPK3 and DAPK3 [55]. Whether this is also the case for other activating mod- iﬁcations of BECLIN-1 and VPS34 remains to be veriﬁed.

Other Stress Kinase-Mediated Regulatory Mechanisms Irrespective of the nature of the initiating stimulus, increasing evidence shows that a series of major stress-response signaling pathways establish a strict crosstalk with autophagy to restore homeostasis [56–58]. Emerging examples are represented by cGAS- and TAK1-regulated pathways that transduce signals from innate and adaptive immune receptors.

The TAK1 kinase and its partners TAB2/TAB3, which are known to control NF-kB transcriptional activity [59], regulate BECLIN-1 activity through direct interactions. In the absence of autophagy

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stimuli, TAB2/TAB3 bind to and inhibit BECLIN-1 [56]. Upon autophagy induction, BECLIN-1 is released from TAB2/TAB3 and engages a stimulatory interaction with TAK1. Activation of TAK1 contributes to autophagy by acting on the AMPK/mTORC1 axis. The important role of TAK1 in autophagy was recently conﬁrmed using liver-speciﬁc knockout mice that show severe autophagy inhibition associated with the development of hepatosteatosis and hepatocarcinoma when challenged with a high-fat diet [60]. Notably, treatment with the mTORC1 inhibitor rapamycin reduced lipid accumulation and hepatocarcinoma by restoring autophagy in the liver of TAK1 knockout mice.

The cytosolic DNA sensor cGAS, which activates the IRF3-mediated expression of type I interferons in response to viral and bacterial infection [61], regulates both ULK1 and BECLIN-1 activity. The direct interaction between cGAS and BECLIN-1 enhances the autophagy response to cytosolic pathogen DNA [57]. In particular, this association leads to the release of RUBICON from BECLIN-1, which allows activation of pathogen-selective autophagy. More- over, induction of autophagy by cGAS has a crucial role in self-limiting its own pathway to avoid excessive inﬂammation. In fact, BECLIN-1 interacts with cGAS and inhibits its activity, while ULK1, also activated by cGAS, mediates inhibitory phosphorylation of STING, a downstream effector of cGAS [58].

Regulation by E3 Ubiquitin Ligases and Deubiquitinases In addition to phosphorylation, autophagy initiation is controlled by ubiquitination of ULK and VPS34 complexes. This post-translational modiﬁcation, mediated by E3 ubiquitin ligases and reversed by deubiquitinases, regulates autophagy by modulating protein activity (non-degradative ubiquitination) or leading to protein degradation (degradative ubiquitination). These different effects depend on the type of poly-ubiquitination, with Lys48-linked chains usually leading to proteasomal degradation and Lys63-linked chains modifying protein interaction properties.

Non-Degradative Ubiquitination The role of non-degradative ubiquitination in stress signaling pathways was first characterized in the regulation of innate immune response, where, together with phosphorylation, it is a crucial covalent modification leading to the activation of the transcription factor NF-kB [62]. Tumor necrosis factor receptor-associated factor 6 (TRAF6), a central E3 ubiquitin ligase of NF-kB signaling pathways [63], also participates in autophagy stimulation by mediating Lys63-linked polyubiquitination of ULK1 [33]. The specific induction of autophagy by TRAF6 is conferred by AMBRA1, which mediates the interaction between TRAF6 and ULK1. Non-degradative ubiquitination by TRAF6 stimulates ULK1 self-association, a prerequisite for its kinase activity.

AMBRA1 also indirectly promotes ULK1 activity by stabilizing the mTOR inhibitor DEPTOR [32]. AMBRA1 blocks DEPTOR degradation by interfering with the E3 ligase CULLIN-5. Because AMBRA1-dependent inhibition of CULLIN-5 requires ULK1 activity, this mechanism acts as a positive ampliﬁcation loop triggered by ULK1 to expedite the decline of mTORC1 activity and allow rapid induction of the autophagy response.

TRAF6 also triggers Lys63-linked ubiquitination of BECLIN-1, which is counteracted by the deubiquitinase A20 [64]. This modiﬁcation, mapping to the BH3 domain (Lys117) of BECLIN-1, is required for autophagy induction by blocking interaction with BCL-2 and favoring protein oligomerization through an ubiquitin-binding domain of BECLIN-1. TRAF6 has a well-estab- lished role in the regulation of both TAK1 and JNK signaling [59], although whether TRAF6 also controls autophagy through these kinases remains largely unexplored.

While the role of AMBRA1 in Lys117-ubiquitination of BECLIN-1 remains unassessed, AMBRA1 has been shown to stimulate K63-linked ubiquitination at Lys437, which promotes

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BECLIN-1/VPS34 association [65]. CULLIN-4 has been proposed as an E3 ligase for Lys437- ubiquitination of BECLIN-1; however, the observed dissociation of CULLIN-4 from AMBRA1 in the early steps of the autophagy response suggests that other E3 ligases may play a direct role in this process. AMBRA1-mediated ubiquitination of BECLIN-1 is negatively regulated by WASH, a factor whose deﬁciency in mice causes embryonic lethality associated with excessive autophagy [65].

Degradative Ubiquitination A further level of regulation of autophagy initiation is represented by the modulation of the protein levels of VPS34 complex I by the ubiquitin–proteasome system. Various types of degradative ubiquitination negatively regulate BECLIN-1 stability. Blocking the BECLIN-1/VPS34 interaction causes Lys11-linked ubiquitination of BECLIN-1 at Lys437 by the E3 ligase NEDD4, which induces its degradation [66]. Moreover, degradation of BECLIN-1 is observed during bacterial infection, and this is mediated by the E3 ligase RNF216 through Lys48-linked ubiquitination [67].

The level of ATG14 is also controlled by ubiquitin-dependent degradation. The E3 ligase responsible for this regulation is the CULLIN-3 complex containing ZBTB16 as the substrate receptor [68]. Upon serum starvation, stability of ATG14 is increased by the kinase GSK3b, which phosphorylates ZBTB16 inducing its self-degradation. Conversely, ATG14 is destabilized by G protein-coupled receptors signaling through GSK3b repression. Importantly, induction of autophagy by pharmacological inhibition of these receptors reduces the accumulation of misfolded proteins in mouse models of Huntington's disease.

Degradative ubiquitination of BECLIN-1 is counteracted by various deubiquitinases, including USP10 and USP13, which are the targets of the autophagy inhibitor spautin [69]. Moreover, BECLIN-1 stability is increased by USP19, which counteracts Lys11-linked ubiquitination at lysine 437 [70]. USP19-mediated BECLIN-1 stabilization regulates not only the autophagy response to nutrient starvation but also the ability of BECLIN-1 to restrict innate immunity by inhibiting the RIG-I/MAVS/TBK1/IRF3 pathway.

Interestingly, deubiquitinases play a role in the repression of BECLIN-1 by BCL-2 family proteins. Indeed, MCL-1 induces BECLIN-1 degradation by displacing it from the deubiquitinase USP9X, as observed in tumor cells that highly express MCL-1, such as melanoma [71].

The Autophagy Initiation Complex Recent evidence points to the association of ULK1 and BECLIN-1 in a common complex as a key requirement for autophagy induction. The assembly of this ‘autophagy initiation complex’ is promoted by speciﬁc scaffold proteins whose gathering activity is under the control of signaling kinases and E3 ubiquitin ligases (Figure 2).

Regulation by the Exocyst Complex The first evidence of the presence of an ULK1/BECLIN-1 common complex came from studies on Exocyst, a protein complex originally identified for its role in tethering exocytic vesicles to plasma membrane [72]. More recently, Exocyst has been found to be involved in various physiological processes including inflammation, the cell cycle, cell migration, and tumor invasion [72].

The Exocyst complex is composed of eight subunits: SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84. Two-hybrid screening assays identiﬁed direct interaction between Exocyst proteins and the autophagy factors FIP200, ATG14, and RUBICON [73]. Coimmuno- precipitation and colocalization assays then revealed a dynamic interaction of ULK1, BECLIN-1, and ATG5/ATG12 complexes with distinct Exocyst subcomplexes, depending on nutrient availability. In unstressed conditions, inactive ULK1 and BECLIN-1 associate with a SEC5- positive/EXO84-negative complex of 500 kDa at the Golgi compartment (Figure 2A). This

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(A) Regulaon by Exocyst

ULK VPS34 Innate immune complex complex I TBK1 High signaling nutrients Exocyst Sec5 mTORC1 (C) Regulaon by IRGM U U RalB U U Inacve USP33 STK38 Autophagosome EGFR Pathogens ULK PI3P

LAPTM4B RUBICON complex RalB NOD2 NOD1 RIG-I TLR3 VPS34 Nutrient Phagophore Exocyst Exo84 complex I starvaon TRAF6 ATG5/ATG12/ATG16L1 U U LC3 Omegasome U (B) Regulaon by TRIMosome U AMPK ATG16L1 IRGM • Nutrient starvaon • Basal autophagy ULK VPS34 • HIV infecon complex • IFN-γ complex I α TRIM20 TRIM5 TRIM6 PI3P PI3P Omegasome TRIM17 Complex I

TRIM20 VPS34 TRIMosome TRIM21 TRIM5α TRIM20 TRIM22 Autophagosome TRIM21 TRIM49 TRIM5α Omegasome Phagophore TRIM11 Autophagosome TRIM20 TRIM21 Phagophore Autophagic Cargo

Figure 2. Regulation of the Autophagy Initiation Complex. Three distinct systems promote the interaction between ULK and VPS34 complexes. (A) Exocyst. In high-nutrient conditions, ULK and VPS34 complexes associate with a Sec5-positive Exocyst complex. This complex is also involved in the regulation of mTORC1 activity and the innate immune response. Under nutrient-deprivation conditions, ULK1 and BECLIN-1 associate with an Exo84-positive/Sec5-negative Exocyst complex, which also interacts with ATG5/ATG12 and LC3. Ubiquitination levels of RalB, regulated by USP33, determine which Exocyst complex RALB associates with. The interaction of unstimulated EGFR with LAPTM4B contributes to autophagy induction by binding to the Sec5-positive Exocyst complex and dissociating RUBICON from VPS34 complex I. (B) TRIMosome. A subset of TRIM family proteins (see text) act as a platform to assemble ULK1 and BECLIN-1 into a common complex to promote autophagosome formation. These TRIMs also bind to ATG16L1, mATG8s, and specific cargos to allow target recognition. Specific TRIM proteins are involved in the regulation of autophagy in basal conditions, nutrient deprivation, IFN-g stimulation, or HIV infection (see text) (C) IRGM. Pathogen intracellular sensors activate IRGM through TRAF6-dependent ubiquitination. Activated IRGM interacts with ULK and VPS34 complexes, together with ATG16L1, recruiting them into a single complex. IRGM pro-autophagic activity is also mediated by AMPK. complex is also involved in the regulation of both mTORC1 and innate immune TBK1–IRF3 signaling pathways. Nutrient starvation and pathogen infection both induce the formation of an EXO84-positive/SEC5-negative complex of >700 kDa, characterized by active ULK1 and BECLIN-1 as well as LC3 positivity (Figure 2A). The formation of the ‘pro-autophagic’ EXO84 complex depends on the presence of the GTPase RALB. However, RALB is not exclusive to EXO84 activation because it also stimulates innate immunity through the SEC5 subcomplex [74]. Notably, subcomplex activation is determined by the non-degradative ubiquitination of RALB. Ubiquitinated RALB at Lys47 localizes at the SEC5 subcomplex, while deubiquitination is a prerequisite for RALB interaction with the EXO84 complex. This switch is regulated by the deubiquitinase USP33, while specific E3 ubiquitin ligases targeting RALB remain unknown (Figure 2A).

To date, two signaling kinases have been described as converging on the Exocyst complex to induce autophagy [74]. The EGFR/LAPTM4B complex associates with SEC5 to promote autophagy by releasing RUBICON from BECLIN-1 [36] (Figure 2A). The pro-apoptotic kinase STK38 stimulates the binding of EXO84 with BECLIN-1 and RALB [75] (Figure 2). In addition, interaction with the Exocyst complex determines whether STK38 acts as a pro-autophagic or

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pro-apoptotic factor. In fact, loss of Exocyst activity by RALB inhibition causes STK38 hyper- activation upon autophagy induction, which leads to induction of cell death.

Regulation by the TRIMosome Tripartite motif proteins (TRIMs) proteins are a large family of E3 ubiquitin ligases that have recently been described to regulate the formation of the autophagy initiation complex [76]. Various members of this family participate in autophagosome formation, as shown by the effect of TRIM silencing on both basal and mTORC1-dependent autophagy. Importantly, the majority of the autophagic TRIMs (TRIM5/, TRIM6, TRIM17, TRIM20, TRIM21, TRIM22, TRIM49) bind to both active ULK1 and BECLIN-1, and are required to assemble them into a common complex. In addition, TRIMs contribute to BECLIN-1 activation by favoring the dissociation from the negative regulators BCL-2 and TAB2 [76]. Speciﬁc TRIMs also bind to ATG16L1 (TRIM20) and mATG8s (TRIM5/, TRIM20, TRIM21) [76,77]. Overall, these data indicate that TRIMs act as platforms (TRIMosomes) to allow autophagosome formation (Figure 2B). Evidence that EXO84 interacts with TRIM17, but not with TRIM5 [76], suggests that Exocyst and TRIMosome cooperate in some circumstances to assemble the initiation complex.

The role of TRIMs in autophagy is not limited to the induction of the process, and they directly contribute to selective recognition of autophagy cargos by cooperating with mATG8s and p62 family members. Identiﬁed targets are HIV ENV protein through TRIM5/ [76],IFN-inducing transcription factor IRF3 through TRIM21 [77],andvariouscomponentsoftheinﬂammasome such as NLRP3, NLRP1, and pro-caspase 1 through TRIM20 [77], as well as AIM2 through TRIM11 [78]. Importantly, the early and late autophagic functions of TRIMs appear to be interdependent because TRIM5/ mutants unable to bind LC3 are also defective in activating ULK1 [76].

Finally, the recent identiﬁcation of TRIM31 as an inducer of autophagy in intestinal epithelial cells independently of BECLIN-1, ATG5, and ATG7 suggests that TRIM may also be involved in the regulation of non-canonical forms of autophagy [79].

Regulation by IRGM IRGM is a member of a large family of GTPases dedicated to a cell-autonomous defense response to infections [80]. The crucial role of IRGM in immunity is suggested by the association between IRGM mutations and susceptibility to tuberculosis and Crohn's disease [80]. IRGM activates xenophagy, a selective form of autophagy for pathogens, when stimulated by several pathogen intracellular sensors including NOD2, NOD1, RIG-I, and TLR3 [81] (Figure 2C). IRGM interacts with ULK1 and BECLIN-1, and favors their assembly in a common complex together with ATG16L1 (Figure 2C). Notably, the E3 ligase TRAF6 ubiquitinates IRGM, and promotes its oligomerization and consequent formation of the autophagy initiation complex (Figure 2C). Moreover, in the absence of IRGM, the proteins ULK1, ATG14, AMBRA1, and ATG16L1 undergo proteasomal degradation, suggesting a role for IRGM in controlling degradative ubiquitination. IRGM also stimulates autophagy via AMPK, and this is presumably related to the ability of IRGM to modulate mitochondrial function [82].

Taken together, these studies highlight the presence of a higher-order assembly of autophagy proteins during autophagy initiation. Importantly, this level of regulation relies on ‘hub’ proteins shared by multiple inducible processes, indicating that different cellular activities may be directly coordinated in response to stress.

Regulation Of Autophagy Termination Although it has long been known that autophagy is self-inhibited to avoid death by unrestrained cell digestion, the mechanisms that regulate autophagy termination have begun to be elucidated only recently (Figure 3).

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Ub Ub RNF2 Ub Ub AMBRA1 p BECLIN-1 CUL4CUL4 Ub Ub48 ProteasomalProte p AMBRA1AMBRA1 DDB1DDB1 Ub Vps34 Ub degradaondegra Ub Ub48 Ub Ub

p AMBRA1AMBRA1 CUL3CUL3 KLHL20KLHL20 BECLIN-1BECLIN-1 Vps34Vps34 Ub Ub48 p p Ub Ub Ub Ub ULK1ULK1 Ub UbUb CUL3CUL3 KLHL20KLHL20 ULK1ULK1 p ULK1ULK1 Ub mTORC1mTORC1 Ub48 Ub Ub RRagulatoraagulator v-ATPase Autophagosome Autolysosome

Autophagy levels RAGsRAGs Lysosome mTORC1mTORC1 LysosomeLysosome RagsRags mTORC1 reacvaonr mTORC1 RaRagulatorgulator Phagophore (high AA) inhibion v-ATPase (low AA)

Iniaon Execuon Terminaon

Figure 3. Temporal Regulation of Autophagy Response in Nutrient Starvation. Nutrient starvation leads to mTORC1 inactivation and dissociation from lysosomal membrane, which promotes the activation of ULK and VPS34 complexes via a multitude of post-translational modiﬁcations (autophagy initiation). Once activated, ULK and VPS34 complexes allow the execution of the autophagy response. Autophagy termination is subsequently triggered by degradative ubiquitination (Ub) of active ULK and BECLIN-1 complexes performed by the indicated E3 ubiquitin ligases. In addition, amino acids produced by lysosomal digestion reactivate mTORC1, which in turn inhibits autophagy. Abbreviation: AA, amino acid levels.

Regulation by mTORC1 The expected termination signal is resolution of the stress that triggered autophagy. Consistent with this assumption, mTORC1 localizes to the lysosome surface where it senses the increased availability of amino acids following lysosomal degradation [83]. Stimulation of mTORC1 by amino acids requires an interaction with Rag GTPases, a family of Ras-related GTP-binding proteins whose activity is regulated by a series of amino acid binding proteins such as lysosomal permeases, aminoacyl-tRNA synthetases, and sestrins. mTORC1 reactivation may thus contribute to shutting down autophagy when nutrients are replenished [84] (Figure 3). Moreover, mTORC1 reactivation plays a crucial role in the regeneration of ‘ready-to-use’ lysosomes from ‘end-point’ autolysosomes which takes place via membrane tubulation [84].

Regulation by CULLINs It is now evident that, in addition to mTORC1 reactivation, autophagy is self-inhibited independently of stress resolution because prolonged autophagy in persistent stress conditions can be more detrimental than the causative stress. The main mechanism determining the duration of the autophagy response is the proteasomal degradation of ULK and VPS34 complex members, which occurs shortly after their activation.

CULLINs are the main E3 ubiquitin ligases responsible for triggering degradation of autophagy proteins to terminate the process (Figure 3). AMBRA1 was the ﬁrst autophagy protein to be

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identiﬁed as a target for CULLINs [32]. In particular, AMBRA1 stability is regulated by CULLIN-4 in a time-dependent manner. In nutrient-rich conditions, CULLIN-4 association limits AMBRA1 abundance. ULK1 activation by nutrient deprivation causes a rapid release of AMBRA1 from CULLIN-4 and consequent AMBRA1 protein stabilization. Several hours later, CULLIN-4 reas- sociates with AMBRA1 and triggers its degradation, initiating autophagy termination. Expression of a WD40-domain mutant of AMBRA1, which cannot bind to CULLIN-4, results in a prolonged autophagy response. The E3 ligase RNF2 has also been reported to induce AMBRA1 degradation to terminate autophagy [85]. Whether or not CULLIN-4 and RNF2 work together remains to be determined.

Recent research has highlighted how other members of the ULK and VPS34 complexes are targeted by CULLINs for proteasomal degradation at the end of the autophagy response [86] (Figure 3). Speciﬁcally, ULK1 autophosphorylation at Ser1042/Thr1046 triggers its interaction with CULLIN-3 via the receptor KLHL20 and consequent Lys48-linked ubiquitination. KLHL20 also binds to and ubiquitinates active BECLIN-1 and VPS34. Other members of the ULK and VPS34 complexes, such as ATG13 and ATG14, are degraded during starvation in a KLHL20- dependent but indirect manner because no direct ubiquitination by KLHL20 was observed. Interestingly, KLHL20 also controls the stability of the pro-autophagic kinase DAPK when activated by type I IFN signaling [87], suggesting that BECLIN-1 kinases could also be degraded to terminate autophagy.

Importantly, preventing autophagy termination during nutrient starvation by blocking either CULLIN-3- or CULLIN-4-mediated degradation of autophagy regulators increases susceptibility to cell death stimuli. This observation conﬁrms that, for the pro-survival role of autophagy, the timely switching off of the autophagy response is as crucial as its rapid induction [32,86].

What remains poorly characterized is whether the identiﬁed mechanisms of autophagy termination upon nutrient starvation also apply to other type of stress stimuli. Recent evidence shows that CULLIN-1 is responsible for terminating autophagy after induction by DNA damaging agents [88]. In particular, CULLIN-1 targets VPS34 for proteasomal degradation through binding to its receptor FBXL20. Two regulatory events are required for this degradation: (i) VPS34 phosphorylation by CDK1 at Thr159, which occurs during mitotic arrest induced by DNA damage agents, and (ii) transcriptional induction of FBXL20 by the tumor-suppressor protein p53. Importantly, in this system degradation of VPS34 by DNA damage does not exclusively affect autophagy and also affects the activity of VPS34 in endocytosis [88].

Finally, degradation of autophagy proteins by the caspase and calpain proteases has been shown to be responsible for autophagy termination by apoptotic stimuli (Box 2).

Box 2. Autophagy Termination by Apoptosis An alternative and irreversible form of autophagy termination is executed by the apoptotic machinery [92,94]. Autophagy represents the primary cellular strategy to adapt to and cope with stress. However, when stress exceeds a crucial threshold and duration, apoptosis needs to be activated to allow damaged cells to die in a controlled manner to avoid harming the surrounding healthy cells. At the point of no return, the autophagy machinery is rapidly degraded by the apoptotic executioner proteases, such as caspases and calpains, as shown for ATG5, BECLIN-1, AMBRA1, ATG3, ATG4D, and ATG16L1 [6]. Notably, a Thr300Ala substitution in ATG16L1, which is associated with increased risk of developing Crohn's disease, leads to a reduced ATG16L1 protein level as a result of enhanced degradation by CASPASE-3 upon death-receptor activation or starvation [97].

Interestingly, apoptotic cleavage products of ATG5, ATG4D, BECLIN-1, and AMBRA1 acquire a pro-apoptotic function by translocating to mitochondria and interfering with BCL-2/BCL-xL [6,98]. During inﬂammation, BECLIN-1 and ATG5 cleavage by calpains can be prevented by the interaction with HMGB1. This protective binding has been shown to contribute to the autophagy-dependent resistance of different tumor cells to chemotherapy [99].

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Box 3. Regulation of Autophagy by Acetyltransferases/Deacetylases Outstanding Questions Acetylation has more recently emerged as an important post-translational modiﬁcation that controls autophagy by A multifaceted combination of post- targeting autophagy regulators in either direct or indirect mechanisms [100]. translational modiﬁcations regulates both the initiation and termination of ULK1 is positively regulated by acetylation at Lys162/606, which is mediated by the acetyltransferase TIP60 under the autophagy response to stress. conditions of serum starvation. TIP60 is activated by GSK3b-mediated phosphorylation at Ser86 [101]. However, the What are the mechanisms that ensure details on how ULK1 is activated by acetylation remain to be determined. basal autophagy? Does stress activate a different autophagy mechanism? In particular, are the high basal levels of By contrast, BECLIN-1 and ATG proteins involved in autophagosome expansion are negatively regulated by acetylation. autophagy observed in tumor cells reg- The protein deacetylases sirtuins act as important players in this context by modulating autophagy in transcriptional- ulated by different mechanisms? Is it dependent and -independent manner [100]. SIRT1 directly activates BECLIN-1 by reversing inhibitory acetylation at possible to target basal and induced Lys430/437 carried out by the acetyltransferase p300 [102]. SIRT1-mediated deacetylation is stimulated by casein autophagy independently? kinase 1-mediated phosphorylation of BECLIN-1 at Ser409. Acetylation impairs BECLIN-1 activity by promoting interaction with the autophagy inhibitor RUBICON. Three different components, the Exo- cyst, the TRIMosome, and IRGM, pro- SIRT1 also interacts and positively regulates the activity of ATG5, ATG7, and LC3. In particular, it has been shown that, in mote the assembly of the ULK1/ unstressed conditions, a consistent fraction of LC3 has a nuclear localization dependent on acetylation at Lys49 and Lys51 BECLIN-1 initiation complex, a crucial [103]. Autophagy induction stimulates SIRT1-mediated deacetylation of LC3, which allows its nucleus-to-cytoplasm step for autophagy induction. Do these translocation and binding to ATG7 that is required for lipidation. Interestingly, under conditions of glucose starvation, AMPK proteins act in a cooperative manner? drives SIRT1 activation by phosphorylating GAPDH, which translocates to the nucleus and displaces the SIRT1 repressor Are they differently involved depending DBC1 [104]. It must be emphasized that p300 has both pro- or anti-autophagic activities depending on its subcellular on the stress and cell/tissue localization. Indeed, BAG6/BAT3-regulated translocation of p300 from cytoplasm to nucleus impedes negative acetylation characteristics? of autophagy proteins, while it stimulates autophagy gene transcription in a p53-dependent manner [105].

Alterations of TRIM and CULLIN proteins are associated with human Concluding Remarks and Future Perspectives pathologies including cancer, muscular A major challenge in the autophagy field is understanding how the autophagy machinery is dystrophy, and infections. Is dysfunc- promptly activated in response to stress. Transducing stress signals to the autophagy process tional autophagy responsible for the onset and the progression of these appears as an intricate puzzle in which the activity of autophagy protein complexes is mainly diseases? regulated through a combination of post-translational modifications that control the process in a dynamic manner [6].Atfirst, these modifications allow autophagy complexes to be rapidly Mechanisms governing autophagy ter- activated and to assemble at the site of autophagosome formation [12]. Active complexes are mination have been well characterized, mainly under nutrient-deprivation con- eventually inhibited to self-limit the response, mainly by mechanisms involving proteasomal ditions. Do these mechanisms control degradation [89]. Although essential modifications of autophagy proteins, mainly represented by the duration of autophagy in response phosphorylation, ubiquitination, and more recently acetylation (Box 3), have been individually to other stresses? Do chronic stress characterized, how they are integrated and influence each other remains largely unknown. The conditions cause degenerative pathologies through defective autophagy recent structural characterization of ULK and VPS34 complexes by electron cryo-microscopy termination? (cryo-EM) may represent a powerful tool for elucidating how multiple signals impact on this process [90].

Available information on the ‘On–Off’ mechanisms of autophagy has been mainly obtained in nutrient- and energy-restricted conditions. The recent characterization of several types of selective autophagy [19], in addition to providing insights on how specific damaged components are recognized, opens up new questions on how specific stresses may signal to the autophagy machinery (see Outstanding Questions). The large families of TRIM and CULLIN E3 ubiquitin ligases are highly promising candidates for satisfying the requirements of multiple target recognition and signal transduction activation that are essential to regulate autophagy in response to specific types of damage and stimuli.

Acknowledgments This work was supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (IG2015 grant 17404 to G.M.F. and IG2014 15244 to M.P.), the Italian Ministry of University and Research (FIRB Accordi di Programma 2011 and PRIN2015), the Italian Ministry of Health (Ricerca Corrente and Ricerca Finalizzata RF2010 2305199), Fondazione Fibrosi Cistica (Progetto FFC#8/2015), FRIAS COFUND Fellowship Programme (University of Freiburg, Germany), and the Marie Curie Actions of the European Commission 7th Framework Programme (FP/2007–2013) under grant agreement 609305 to M.A.

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References 1. Mizushima, N. and Komatsu, M. (2011) Autophagy: renovation of 29. Egan, D.F. et al. (2015) Small molecule inhibition of the autophagy cells and tissues. Cell 147, 728–741 kinase ULK1 and identification of ULK1 substrates. Mol. Cell 59, – 2. Choi, A.M. et al. (2013) Autophagy in human health and disease. 285 297 N. Engl. J. Med. 368, 1845–1846 30. Park, J.M. et al. (2016) The ULK1 complex mediates MTORC1 3. Galluzzi, L. et al. (2014) Metabolic control of autophagy. Cell 159, signaling to the autophagy initiation machinery via binding and – 1263–1276 phosphorylating ATG14. Autophagy 12, 547 564 4. Deretic, V. et al. (2013) Autophagy in infection, inflammation and 31. Di Bartolomeo, S. et al. (2010) The dynamic interaction of immunity. Nat. Rev. Immunol. 13, 722–737 AMBRA1 with the dynein motor complex regulates mammalian autophagy. J. Cell Biol. 191, 155–168 5. Sheng, R. and Qin, Z.H. (2015) The divergent roles of autophagy in ischemia and preconditioning. Acta Pharmacol. Sin. 36, 32. Antonioli, M. et al. (2014) AMBRA1 interplay with cullin E3 ubiquitin – 411–420 ligases regulates autophagy dynamics. Dev. Cell. 31, 734 746 6. Xie, Y. et al. (2015) Posttranslational modification of autophagy- 33. Nazio, F. et al. (2013) mTOR inhibits autophagy by controlling related proteins in macroautophagy. Autophagy 11, 28–45 ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol. 15, 406–416 7. Lamb, C.A. et al. (2013) The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 34. Wang, R.C. et al. (2012) Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation. Science 8. Lin, M.G. and Hurley, J.H. (2016) Structure and function of the 338, 956–959 ULK1 complex in autophagy. Curr. Opin. Cell Biol. 39, 61–68 35. Wei, Y. et al. (2013) EGFR-mediated Beclin 1 phosphorylation in 9. Mizushima, N. (2010) The role of the Atg1/ULK1 complex in autophagy suppression, tumor progression, and tumor chemo- autophagy regulation. Curr. Opin. Cell Biol. 22, 132–139 resistance. Cell 154, 1269–1284 10. Levine, B. et al. (2015) Beclin orthologs: integrative hubs of cell 36. Tan, X. et al. (2015) A kinase-independent role for EGF receptor in signaling, membrane trafficking, and physiology. Trends Cell Biol. autophagy initiation. Cell 160, 145–160 25, 533–544 37. Hardie, D.G. (2014) AMPK–sensing energy while talking to other 11. Baskaran, S. et al. (2014) Architecture and dynamics of the signaling pathways. Cell. Metab. 20, 939–952 autophagic phosphatidylinositol 3-kinase complex. Elife 3, e05115 38. Egan, D.F. et al. (2011) Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to 12. Ktistakis, N.T. and Tooze, S.A. (2016) Digesting the expanding mitophagy. Science 331, 456–461 mechanisms of autophagy. Trends Cell Biol. 26, 624–635 39. Kim, J. et al. (2011) AMPK and mTOR regulate autophagy 13. Hamasaki, M. et al. (2013) Autophagosomes form at ER–mito- through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, chondria contact sites. Nature 495, 389–393 132–141 14. Ge, L. et al. (2014) Phosphatidylinositol 3-kinase and COPII 40. Zhao, Y. et al. (2015) RACK1 promotes autophagy by enhancing generate LC3 lipidation vesicles from the ER–Golgi intermediate the Atg14L–Beclin 1–Vps34–Vps15 complex formation upon compartment. Elife 3, e04135 phosphorylation by AMPK. Cell. Rep. 13, 1407–1417 15. Yamamoto, H. et al. (2012) Atg9 vesicles are an important 41. Kim, J. et al. (2013) Differential regulation of distinct Vps34 membrane source during early steps of autophagosome forma- complexes by AMPK in nutrient stress and autophagy. Cell tion. J. Cell Biol. 198, 219–233 152, 290–303 16. Mizushima, N. et al. (2011) The role of Atg proteins in autopha- 42. Moller, A.B. et al. (2015) Physical exercise increases autophagic gosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132 signaling through ULK1 in human skeletal muscle. J. Appl. Phys- 17. Dooley, H.C. et al. (2014) WIPI2 links LC3 conjugation with PI3P, iol. 118, 971–979 autophagosome formation, and pathogen clearance by recruit- 43. Hoyer-Hansen, M. et al. (2007) Control of macroautophagy by ing Atg12-5-16L1. Mol. Cell 55, 238–252 calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. 18. Nakatogawa, H. et al. (2007) Atg8, a ubiquitin-like protein Mol. Cell 25, 193–205 required for autophagosome formation, mediates membrane 44. Levine, B. et al. (2008) Bcl-2 family members: dual regulators of tethering and hemifusion. Cell 130, 165–178 apoptosis and autophagy. Autophagy 4, 600–606 19. Rogov, V. et al. (2014) Interactions between autophagy receptors 45. Luo, S. et al. (2012) Bim inhibits autophagy by recruiting Beclin 1 and ubiquitin-like proteins form the molecular basis for selective to microtubules. Mol. Cell 47, 359–370 autophagy. Mol. Cell 53, 167–178 46. Wei, Y. et al. (2008) JNK1-mediated phosphorylation of Bcl-2 20. Amaya, C. et al. (2015) Autophagy and proteins involved in regulates starvation-induced autophagy. Mol. Cell 30, 678–688 vesicular trafficking. FEBS Lett. 589, 3343–3353 47. Geeraert, C. et al. (2010) Starvation-induced hyperacetylation of 21. McEwan, D.G. et al. (2015) PLEKHM1 regulates Salmonella- tubulin is required for the stimulation of autophagy by nutrient containing vacuole biogenesis and infection. Cell. Host Microbe deprivation. J. Biol. Chem. 285, 24184–24194 17, 58–71 48. Gurkar, A.U. et al. (2013) Identification of ROCK1 kinase as a 22. Itakura, E. et al. (2012) The hairpin-type tail-anchored SNARE critical regulator of Beclin1-mediated autophagy during meta- syntaxin 17 targets to autophagosomes for fusion with endo- bolic stress. Nat. Commun. 4, 2189 somes/lysosomes. Cell 151, 1256–1269 49. Rashid, H.O. et al. (2015) ER stress: autophagy induction, inhi- 23. Liang, C. et al. (2008) Beclin1-binding UVRAG targets the class C bition and selection. Autophagy 11, 1956–1977 Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat. Cell Biol. 10, 776–787 50. Cinque, L. et al. (2015) FGF signalling regulates bone growth through autophagy. Nature 528, 272–275 24. Kim, Y.M. et al. (2015) mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation. 51. Gomez-Sanchez, J.A. et al. (2015) Schwann cell autophagy, Mol. Cell 57, 207–218 myelinophagy, initiates myelin clearance from injured nerves. J. Cell Biol. 210, 153–168 25. Wong, P.M. et al. (2013) The ULK1 complex: sensing nutrient signals for autophagy activation. Autophagy 9, 124–137 52. Zalckvar, E. et al. (2009) DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 26. Puente, C. et al. (2016) Nutrient-regulated phosphorylation of from Bcl-XL and induction of autophagy. EMBO Rep. 10, 285–292 ATG13 inhibits starvation-induced autophagy. J. Biol. Chem. 291, 6026–6035 53. Eisenberg-Lerner, A. and Kimchi, A. (2012) PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of 27. Saleiro, D. et al. (2015) Central role of ULK1 in type I interferon DAPk. Cell Death Differ. 19, 788–797 signaling. Cell. Rep. 11, 605–617 54. Maejima, Y. et al. (2013) Mst1 inhibits autophagy by promoting 28. Russell, R.C. et al. (2013) ULK1 induces autophagy by phos- the interaction between Beclin1 and Bcl-2. Nat. Med. 19, 1478– phorylating Beclin-1 and activating VPS34 lipid kinase. Nat. Cell 1488 Biol. 15, 741–750

Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy 13 TIBS 1309 No. of Pages 14

55. Wei, Y. et al. (2015) The stress-responsive kinases MAPKAPK2/ 80. Kim, B.H. et al. (2012) IFN-inducible GTPases in host cell MAPKAPK3 activate starvation-induced autophagy through defense. Cell. Host Microbe 12, 432–444 Beclin 1 phosphorylation. Elife 4, e05289 81. Chauhan, S. et al. (2015) IRGM governs the core autophagy 56. Criollo, A. et al. (2011) Inhibition of autophagy by TAB2 and machinery to conduct antimicrobial defense. Mol. Cell 58, 507–521 – TAB3. EMBO J. 30, 4908 4920 82. Singh, S.B. et al. (2010) Human IRGM regulates autophagy and 57. Liang, Q. et al. (2014) Crosstalk between the cGAS DNA sensor cell-autonomous immunity functions through mitochondria. Nat. and Beclin-1 autophagy protein shapes innate antimicrobial Cell Biol. 12, 1154–1165 – immune responses. Cell. Host Microbe 15, 228 238 83. Efeyan, A. et al. (2013) Regulation of mTORC1 by the Rag 58. Konno, H. et al. (2013) Cyclic dinucleotides trigger ULK1 (ATG1) GTPases is necessary for neonatal autophagy and survival. phosphorylation of STING to prevent sustained innate immune Nature 493, 679–683 – signaling. Cell 155, 688 698 84. Yu, L. et al. (2010) Termination of autophagy and reformation of 59. Sakurai, H. (2012) Targeting of TAK1 in inflammatory disorders lysosomes regulated by mTOR. Nature 465, 942–946 – and cancer. Trends Pharmacol. Sci. 33, 522 530 85. Xia, P. et al. (2014) RNF2 is recruited by WASH to ubiquitinate 60. Inokuchi-Shimizu, S. et al. (2014) TAK1-mediated autophagy and AMBRA1 leading to downregulation of autophagy. Cell Res. 24, fatty acid oxidation prevent hepatosteatosis and tumorigenesis. 943–958 – J. Clin. Invest. 124, 3566 3578 86. Liu, C.C. et al. (2016) Cul3–KLHL20 ubiquitin ligase governs the 61. Barber, G.N. (2015) STING: infection, inflammation and cancer. turnover of ULK1 and VPS34 complexes to control autophagy Nat. Rev. Immunol. 15, 760–770 termination. Mol. Cell 61, 84–97 62. Grabbe, C. et al. (2011) The spatial and temporal organization of 87. Lee, Y.R. et al. (2010) The Cullin 3 substrate adaptor KLHL20 ubiquitin networks. Nat. Rev. Mol. Cell Biol. 12, 295–307 mediates DAPK ubiquitination to control interferon responses. – 63. Oeckinghaus, A. et al. (2011) Crosstalk in NF-kappaB signaling EMBO J. 29, 1748 1761 pathways. Nat. Immunol. 12, 695–708 88. Xiao, J. et al. (2015) FBXL20-mediated Vps34 ubiquitination as a 64. Shi, C.S. and Kehrl, J.H. (2010) TRAF6 and A20 regulate lysine p53 controlled checkpoint in regulating autophagy and receptor – 63-linked ubiquitination of Beclin-1 to control TLR4-induced degradation. Genes Dev. 29, 184 196 autophagy. Sci. Signal. 3, ra42 89. Feng, Y. et al. (2015) How to control self-digestion: transcrip- 65. Xia, P. et al. (2013) WASH inhibits autophagy through suppres- tional, post-transcriptional, and post-translational regulation of – sion of Beclin 1 ubiquitination. EMBO J. 32, 2685–2696 autophagy. Trends Cell Biol. 25, 354 363 66. Platta, H.W. et al. (2012) Nedd4-dependent lysine-11-linked 90. Hurley, J.H. and Schulman, B.A. (2014) Atomistic autophagy: the – polyubiquitination of the tumour suppressor Beclin 1. Biochem. structures of cellular self-digestion. Cell 157, 300 311 J. 441, 399–406 91. Kvansakul, M. and Hinds, M.G. (2013) Structural biology of 67. Xu, C. et al. (2014) Regulation of autophagy by E3 ubiquitin the Bcl-2 family and its mimicry by viral proteins. Cell. Death ligase RNF216 through BECN1 ubiquitination. Autophagy 10, Dis. 4, e909 2239–2250 92. Marino, G. et al. (2014) Self-consumption: the interplay of – 68. Zhang, T. et al. (2015) G-protein-coupled receptors regulate autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 15, 81 94 autophagy by ZBTB16-mediated ubiquitination and proteasomal 93. Strappazzon, F. et al. (2015) AMBRA1 is able to induce mitoph- degradation of Atg14L. Elife 4, e06734 agy via LC3 binding, regardless of PARKIN and p62/SQSTM1. – 69. Liu, J. et al. (2011) Beclin1 controls the levels of p53 by regulating Cell Death Differ. 22, 419 432 the deubiquitination activity of USP10 and USP13. Cell 147, 94. Fimia, G.M. et al. (2013) Ambra1 at the crossroad between 223–234 autophagy and cell death. Oncogene 32, 3311–3318 70. Jin, S. et al. (2016) USP19 modulates autophagy and antiviral 95. Molejon, M.I. et al. (2013) The VMP1–Beclin 1 interaction regu- immune responses by deubiquitinating Beclin-1. EMBO J. 35, lates autophagy induction. Sci. Rep. 3, 1055 – 866 880 96. Tang, D. et al. (2010) Endogenous HMGB1 regulates autophagy. 71. Elgendy, M. et al. (2014) Beclin 1 restrains tumorigenesis through J. Cell Biol. 190, 881–892 Mcl-1 destabilization in an autophagy-independent reciprocal 97. Murthy, A. et al. (2014) A Crohn's disease variant in Atg16l1 manner. Nat. Commun. 5, 5637 enhances its degradation by caspase 3. Nature 506, 456–462 72. Wu, B. and Guo, W. (2015) The exocyst at a glance. J. Cell. Sci. 98. Strappazzon, F. et al. (2016) Prosurvival AMBRA1 turns into a – 128, 2957 2964 proapoptotic BH3-like protein during mitochondrial apoptosis. 73. Bodemann, B.O. et al. (2011) RalB and the exocyst mediate the Autophagy 12, 963–975 cellular starvation response by direct activation of autophago- 99. Zhu, X. et al. (2015) Cytosolic HMGB1 controls the cellular – some assembly. Cell 144, 253 267 autophagy/apoptosis checkpoint during inflammation. J. Clin. 74. Simicek, M. et al. (2013) The deubiquitylase USP33 discriminates Invest. 125, 1098–1110 between RALB functions in autophagy and innate immune 100. Banreti, A. et al. (2013) The emerging role of acetylation in the – response. Nat. Cell Biol. 15, 1220 1230 regulation of autophagy. Autophagy 9, 819–829 75. Joffre, C. et al. (2015) The pro-apoptotic STK38 kinase is a new 101. Lin, S.Y. et al. (2012) GSK3–TIP60–ULK1 signaling pathway links Beclin1 partner positively regulating autophagy. Curr. Biol. 25, growth factor deprivation to autophagy. Science 336, 477–481 2479–2492 102. Sun, T. et al. (2015) Acetylation of Beclin 1 inhibits autophago- 76. Mandell, M.A. et al. (2014) TRIM proteins regulate autophagy and some maturation and promotes tumour growth. Nat. Commun. can target autophagic substrates by direct recognition. Dev. Cell. 6, 7215 30, 394–409 103. Huang, R. et al. (2015) Deacetylation of nuclear LC3 drives 77. Kimura, T. et al. (2015) TRIM-mediated precision autophagy autophagy initiation under starvation. Mol. Cell 57, 456–466 targets cytoplasmic regulators of innate immunity. J. Cell Biol. 104. Chang, C. et al. (2015) AMPK-dependent phosphorylation of 210, 973–989 GAPDH triggers Sirt1 activation and is necessary for autophagy fl 78. Liu, T. et al. (2016) TRIM11 suppresses AIM2 in ammasome by upon glucose starvation. Mol. Cell 60, 930–940 degrading AIM2 via p62-dependent selective autophagy. Cell. 105. Sebti, S. et al. (2014) BAT3 modulates p300-dependent acety- Rep. 16, 1988–2002 lation of p53 and autophagy-related protein 7 (ATG7) during 79. Ra, E.A. et al. (2016) TRIM31 promotes Atg5/Atg7-independent autophagy. Proc. Natl. Acad. Sci. U.S.A. 111, 4115–4120 autophagy in intestinal cells. Nat. Commun. 7, 11726

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