Oxidative stress and autophagy: the clash between damage and metabolic needs

G Filomeni; D De Zio; F Cecconi

doi:10.1038/cdd.2014.150

Oxidative stress and autophagy: the clash between damage and metabolic needs

Filomeni, G; De Zio, D; Cecconi, F 2014-09-26 00:00:00 Cell Death and Differentiation (2015) 22, 377–388 OPEN & 2015 Macmillan Publishers Limited All rights reserved 1350-9047/15 www.nature.com/cdd Review Oxidative stress and autophagy: the clash between damage and metabolic needs ,1,2 1,2 ,1,2 G Filomeni* , D De Zio and F Cecconi* Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli, with reactive oxygen species (ROS) and reactive nitrogen species (RNS) being among the main intracellular signal transducers sustaining autophagy. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth. Cell Death and Differentiation (2015) 22, 377–388; doi:10.1038/cdd.2014.150; published online 26 September 2014 Facts Open Questions How do ROS and oxidative stress affect autophagy? Reactive oxygen species (ROS) production and thiol redox Which are the main ROS able to signal autophagy being state imbalance are induced immediately upon nutrient activated and going on? deprivation and represent important mediators of Does nitric oxide act as a real inhibitor of autophagy? autophagy. How does autophagy sense DNA damage? ROS and reactive nitrogen species (RNS) irreversibly How can autophagy contribute to DNA damage repair? oxidize DNA and cellular biomolecules, thereby represent- ing the primary source of damage in biological systems. 1,2 In the 1950s, Christian de Duve, contextually with the Autophagy contributes to clearing the cells of all irreversibly discovery of glucagon, clarified the intracellular localization of oxidized biomolecules (proteins, DNA and lipids), this is all the more reason why it could be included in the antioxidant several enzymes by setting up centrifugation-based tissue and DNA damage repair systems. fractionation of rat liver homogenates. During his work, he 1 2 Cell Stress and Survival Unit, Danish Cancer Society Research Center, Copenhagen, Denmark and IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy *Corresponding author: F Cecconi, Cell Stress and Survival Unit, Danish Cancer Society Research Center, Strandboulevarden 49, Copenhagen DK-2100, Denmark. Tel: +45 35257401; Fax: +45 35271811; E-mail: [email protected] or Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, 00133 Rome, Italy. Tel: +39 06 72594230; Fax:+39 06 72594311; E-mail: [email protected] or G Filomeni, Cell Stress and Survival Unit, Danish Cancer Society Research Center, Strandboulevarden 49, Copenhagen DK-2100, Denmark. Tel: +45 35257402; Fax: +45 35271811; E-mail: [email protected] or Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, 00133 Rome, Italy. Tel: +39 06 72594243; Fax: +39 06 72594311; E-mail: [email protected] Abbreviations: 4E-BP1, EIF4E-binding protein 1; 53BP1, p53 binding protein 1; 8-OHdG, 8-hydroxydeoxyguanosine; 8-OHG, 8-hydroxyguanine; Ambra1, activating molecule in Beclin1-regulated autophagy; AMPK, AMP-dependent protein kinase; ARE, antioxidant-responsive element; Atg4, autophagy related gene 4; Atg5, autophagy related gene 5; Atg7, autophagy related gene 7; Atg13, autophagy related gene 13; ATM, Ataxia telangiectasia mutated; Bnip3, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3; Cvt, cytoplasm-to-vacuole targeting; DDR, DNA damage response; DMPK, myotonic dystrophy protein kinase; DSB, double-strand break; eEF, eukariotic elongation factor; eIF, eukaryotic initiation factor; Esp1, extra spindle pole bodies homologue 1; FIP200, FAK-family interacting protein of 200 kDa; GABARAP, GABA receptor-associated protein; GSH-Px or GPx, glutathione peroxidise; Grx, glutaredoxin; GSH, reduced glutathione; GSNOR, S-nitrosoglutathione reductase; GSSG, disulphide glutathione; HKII, hexokinase II; IKKβ,IκB kinase β; JNK1, c-Jun-N-terminal kinase 1; Keap1, Kelch-like ECH- associated protein 1; LC3, light chain 3; LKB1, liver kinase B1; MRP1, multidrug resistance protein 1; mtDNA, mitochondrial DNA; mTORC1, mammalian target of rapamycin complex 1; NO, nitric oxide; NOX, NADPH oxidase; Nrf2, nuclear factor erythroid 2-related factor 2; OGG1, 8-oxoguanine glycosylase; p70S6K, p70 ribosomal protein S6 kinase; PAR, polyADP-ribose; PARP1, polyADP-ribose polymerase 1; PHD, prolyl hydroxylase; PI3K, phosphoinositide 3-kinase; PINK1, PTEN-induced putative kinase 1; PMN, piecemeal microautophagy; Psd1, pleckstrin and Sec7 domain containing 1; PTEN, phosphatase and tensin homologue; PTP, permeability transition pore; RAG, RAS-related GTP-binding protein; RHEB, RAS homologue enriched in brain; RNS, reactive nitrogen species; ROS, reactive oxygen species; Sae2, sporulation in the absence of SPO11 protein 2; SOD, superoxide dismutase; SQSTM1, sequestosome 1; SSB, single-strand break; TCA, tricarboxylic acid; Trx, thioredoxin; TSC2, tuberous sclerosis 2; ULK1, upstream kinase UNC51-like kinase 1; UVRAG, UV irradiation resistance-associated gene; v-ATPase, vacuolar H -ATPase; VPS34, vacuolar protein sorting 34 Received 29.6.14; revised 19.8.14; accepted 21.8.14; Edited by G Kroemer; published online 26.9.14 Oxidative stress and autophagy interplay G Filomeni et al discovered and coined the names of many organelles, whose his findings could be basically interconnected by a finely purification, characterization and distribution contributed to organized signalling system, where primary/primitive stimuli earning him the Nobel Prize for Physiology and Medicine in (e.g., nutrient availability and oxidative insults) differently 1974. In his studies on carbohydrate metabolism and insulin impinge on the maintenance of biomolecule integrity and cell action, he described for the first time the lysosomes as the viability through the intermediate activity of homeostatic intracellular granules containing the enzymes glucose-6- processes (mainly based on repair and degradation), the phosphatase and acid phosphatase, in addition to a set of most complex and versatile of which was the very same hydrolases that were deputed to digest, recycle and remove autophagy he discovered 10 years before. intracellular material, such as worn-out or damaged orga- nelles, and engulfed pathogens, by means of a process that he Autophagy: Converging Point of Different Stimuli named autophagy. More than 10 years later, in 1966, he also defined the There are three main types of autophagy culminating to structure and composition of microbodies: the cellular lysosome-mediated degradation: (1) macroautophagy (here- districts in which hydrogen peroxide is endogenously pro- after referred to as autophagy) that involves the formation of a duced to a high extent as a side effect of the reactions double-membrane vesicle (autophagosome) deputed to catalyzed by many oxidases involved in amino acid, purines sequester damaged organelles and biomolecules; (2) micro- and fatty acid metabolism, and for this reason named autophagy, by which the cytosolic material is directly engulfed peroxisomes. Although the toxicity of hydrogen peroxide had by the lysosome; and (3) chaperone-mediated autophagy. It is been reported many years before, only in the late 1950s its now well established that autophagy is a very sensitive real implications in biology were coming up. Progress in the process underlying cell response induced by almost every field of metallobiology and the fine characterization of stressful condition affecting cellular homeostasis. Through metalloenzyme-mediated catalysis provided compelling evi- autophagy, cells coordinate energy and building blocks dence for an endogenous and physiological production of demanded for vital processes (e.g., growth and proliferation) partially reduced oxygen species (nowadays usually referred with the extracellular stimuli and carbon source availability, to as reactive oxygen species (ROS)), such as superoxide such as amino acids and glucose. If they are not sufficient to anion (O ), hydrogen peroxide (H O ) and hydroxyl radical maintain the rate of protein synthesis, or to provide the 2 2 2 ( OH). Their being highly reactive towards lipids, proteins and required amount of ATP needed to sustain metabolic 7–9 DNA, and severely harmful for cell survival when present at reactions, then cells activate autophagy in order to rapidly very high concentrations, both led to the concept of oxidative degrade the old or burned-out components and reuse the stress as detrimental condition occurring in all living systems generated pool of biomolecules. and arising from the imbalance between oxidants species and Both glucose and amino acids signals converge on a unique antioxidant defence. It is not a coincidence that in the same molecular transducer of cellular needs, the mammalian target 10 12 years, Denham Harman postulated the ‘free radical theory of of rapamycin complex 1 (mTORC1) (Figure 1). Active ageing’ in which he stated that free radicals were the primary mTORC1 controls the activity of translation eukaryotic cause of massive damage to DNA and all cellular macro- initiation factors (eIFs) and eukariotic elongation factors molecules, culminating in cancer and in a diffuse cell (eEFs), namely eIF2, -3 and -4 and eEF2, by direct dysfunction distinctive of ageing. phosphorylation of two key protein targets, EIF4E-binding When de Duve characterized the peroxisomes and found protein 1 (4E-BP1) and protein S6 kinase (p70S6K). Both out that they were the organelles in which the antioxidant are required for a correct and efficient protein synthesis, as enzyme catalase resides, he probably did not realize that all they regulate the interactions between the mRNA 5′ cap, the Figure 1 Main molecular pathways activated in the presence or absence of nutrients. (a) The synergic import of leucine (Leu) and glutamine (Gln) (top left) results in mTORC1 recruitment to the lysosomal membrane and its subsequent activation by at least two distinct pathways. The first one proposes that cytosolic amino acids enter the lysosome and signal their presence to RAG-A and RAG-C (or RAG-B and RAG-D, not shown in the figure) through the lysosome-located proton pump v-ATPase and the multimolecular complex called Ragulator. The second pathway provides for the double deamination of Gln catalysed by the enzymes glutaminase (GLN) and glutamate dehydrogenase (GDH). This sequence of reactions subsequently generates glutamate (Glu) and α-ketoglutarate (αKG) that, by acting as co-substrate for prolyl hydroxylases (PHD), finally leads to RAG activation. GTP-bound RAG-A (or B) and GDP-bound RAG-C (or D) can then recruit mTORC1 to the lysosome membrane where it is activated by RHEB (middle left). Once activated, mTORC1 activates protein synthesis by phosphorylating 4EBP1 and p70S6K, and concomitantly inhibits autophagy by phosphorylating ULK1 complex at the level of ULK1 and Atg13 (bottom left). Glucose is taken up through specific transporters (GLUTs) and phosphorylated to glucose-6-phosphate (G6P) by hexokinase (the only mitochondrial isoform II, HKII, is shown in the top right side of the figure). G6P is then isomerized to fructose-6-phosphate (F6P), oxidized through the glycolytic pathway to generate pyruvate (Pyr) and acetyl-CoA that fuels the mitochondrial TCA cycle and the respiratory chain for the production of ATP (middle right) through the oxidative phosphorylation (OXPHOS). G6P can also undergo oxidation via the glucose-6-phosphate dehydrogenase (G6PDH)-mediated catalysis along the pentose phosphate pathway (middle right). In this way, electrons required for NADP -to-NADPH reduction, and sugars needed for DNA de novo synthesis (e.g., ribulose-5-phosphate, R5P) are also provided (bottom right). (b) Upon amino acid deprivation, RAGs exchange nucleotides located in their binding sites (GTP with GDP or vice versa), thus leading to mTORC1 release from the lysosome membrane (top left). These 2 events are associated with the inhibitory binding of mTORC1 to HKII that takes place upon glucose deficiency and G6P level decrease (top right). This condition leads to a decrease of NADPH and ATP levels that finally result in a reduced antioxidant capacity of the cell (especially in regenerating the reduced thiol pool) (middle right) and in energetic stress that the cell attempts to counteract by the adenylate kinase (AK)-mediated conversion of ADP into ATP and AMP (centre). AMP increase induces to the activation of AMPK that inhibits protein synthesis by phosphorylating TSC2 and mTORC1 and activates autophagy by phospho-activating ULK1. Once activated, ULK1 phosphorylates its interactors (Atg13 and FIP200) and recruits microtubule-associated PI3K complex by means of an AMBRA1-mediated process to initiate the nucleation phase of autophagic vesicles from the endoplasmic reticulum (or mitochondria, not shown). Many other factors, such as Atg proteins coming from Golgi apparatus (e.g., Atg9) contribute to phagophore elongation and autophagosome formation (bottom) Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al poly(A)-tail and the 40S and 60S ribosomal subunits leucine also, as it binds to and activates glutamate dehydro- (Figure 1). Concomitantly, active mTORC1 prevents autoph- genase, the enzyme catalysing the last deamination step agy by phospho-inhibiting the UNC51-like kinase 1 (ULK1) at leading to α-ketoglutarate production. In line with these Ser and its interacting partner, the autophagy related gene pieces of evidence, compartmentalization of mTORC1 at the 13 (Atg13) that, together with the FAK-family interacting level of the lysosomes could provide some explanations protein of 200 kDa (FIP200), form the so-called ULK1 complex regarding mTOR negative regulation on autophagy. 12,15 (Figure 1). Upon autophagic stimuli, mTORC1 is inhibited, thus leading to the activation of ULK1. Once activated, ULK1 is Glucose signal. Glucose is the primary carbon source that, able to phosphorylate Atg13 and FIP200 inducing to the upon sequential oxidation steps taking place during glyco- following activation of the class III phosphoinositide 3-kinase lysis and TCA cycle, provides the electrons (energy) coming (PI3K) complex via the activating molecule in Beclin1- from the breakdown of its chemical bonds, required for ATP 15,16 regulated autophagy 1 (Ambra1). The formation of PI3K production. The maintenance of endergonic processes complex is required to initiate phagophore nucleation that strictly depends on the maintenance of ATP levels, and for represents the first step leading to autophagosome formation this reason, cells (1) actively synthesize ATP and (2) have 12,15,16 (Figure 1). evolved sophisticated mechanisms to face up energetic stress. Amino acid signal. Among the amino acids that are able to AMP-dependent protein kinase (AMPK) is the genuine signal their presence to the cell, and then let autophagy be sensor of the energetic state of the cell, and directly responds induced in case of any deficiency, leucine and glutamine play to the so-called adenylate energy charge as the enzyme is the most important roles because of their essentiality and activated by very low increases of AMP levels (and, to certain 28–30 their tight interdependence in the mechanism regulating their extent, of ADP), and deactivated by ATP. In order to 17,18 uptake. Leucine is an essential amino acid indispensible restore the correct adenylate energy charge, phospho-active for cell survival as it is nonsynthesizable de novo through AMPK concertedly stimulates catabolic pathways (e.g., alternative pathways, such as by transformation of other glycolysis and fatty acid oxidation), inhibits the rate of anabolic amino acids or by transamination of carboxylates coming up reactions (protein and fatty acid synthesis) and activates from glycolysis and the tricarboxylic acid (TCA) cycle. autophagy. Glutamine, instead, though nonessential, represents the At the molecular level, active AMPK stimulates autophagy most abundant amino acid in the human body and one of by means at least three distinct mechanisms. These include the main substrates of anaplerotic reactions fuelling the TCA (1) phosphorylation of the mTORC1 inhibitor, tuberous 1387 31 cycle. It has been estimated that hypercatabolism or other sclerosis 2 (TSC2) at Ser , which induces RHEB GTPase stressful conditions (e.g., infection and severe injuries) are activity; (2) phosphorylation of the mTORC1 component 722 792 32 accompanied by a significant decrease of glutamine in Raptor at Ser and Ser , which is preparatory for its 19 317 skeletal muscle cells, thus arguing for it being a reliable binding to 14–3–3; and (3) phosphorylation of ULK1 at Ser 777 33 marker of both amino acid and energetic status. and Ser (Figure 1). From a mechanistic point of view, the At the molecular level, the presence of an adequate amount first two phosphorylations catalysed by AMPK inhibit of amino acids induces members of the RAS-related GTP- mTORC1 and reduce its inhibitory effects on ULK1. ULK1 is binding protein (RAG) family of small GTPases (i.e., RAG then free to interact with and to be phosphoactivated by AMPK A–D) to bind guanine nucleotides (GTP or GDP in depen- (Figure 1). dence of the member), leading to their activation and, Fascinatingly, it has very recently been reported that subsequently, to the recruitment of mTORC1 on the lysosome glucose sensing by the cells does not only depend on AMPK, 20,21 membrane (Figure 1). Here, mTORC1 can be targeted by as indirect transducer of the intracellular energy state, but also RAS homologue enriched in brain (RHEB) that, upon binding relies upon more direct mechanisms, thereby making the to GTP, acts as positive regulator of mTORC1. In agreement system redundant and controlled at multiple levels. Roberts with this model, RAGs are the primary sensors that, by means et al. demonstrated that hexokinase II (HKII), the of at least two distinct mechanisms, signal amino acid mitochondria-located enzyme responsible for the first step of availability to mTORC1 and modulate its activation state. glycolysis, binds to and inhibits mTORC1, and that this One mechanism suggests that RAGs sense the amino acid interaction is enhanced by glucose deprivation, namely by a pool (mainly leucine) present within the lumen of the lysosome decrease in glucose-6-phosphate levels (Figure 1). As by the vacuolar H -ATPase (v-ATPase) and a molecular proposed by the authors, this new mechanism could 24,25 complex called Ragulator (Figure 1). This mechanism contribute to the modulation of cell metabolism in circum- could underlie the negative feedback acting on autophagy stances of glucose deficiency, but could also have deep (because of mTOR reactivation) once amino acid levels have implications in redox homeostasis. Indeed, besides many been successfully restored. Alternatively, it has been pro- indications arguing for a direct role of HKII in preventing ROS 35–37 posed that RAGs are activated by glutamine, specifically by generation from mitochondria, it should be also taken into α-ketoglutarate generated upon double deamination occurring account that glucose-6-phosphate, via the pentose phosphate 26 + in the glutaminolytic pathway. Although mTOR activation pathway, is also the primary source of electrons for NADP - through this mechanism needs to be still clarified, it seems to to-NADPH reduction (Figure 1). NADPH directly participates require prolyl hydroxylase (PHD) activity that, in fact, is in bioreductive synthesis and provides the electrons required positively regulated by α-ketoglutarate (Figure 1). As above for thiol redox homeostasis (Figure 1). In particular, NADPH reported, this mechanism has been shown to be responsive to acts as a co-substrate of glutathione reductase – the enzyme Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al responsible for the reduction of the disulphide (GSSG) to the endogenously produced by NO synthases (NOS1-3), a family sulphhydryl (GSH) form of glutathione – as well as of many of constitutive or inducible enzymes with different tissue other reductases deeply implicated in sulphhydryl regenera- distribution and that use arginine and NADPH as substrates tion and, in turn, in the defence against oxidative stress. for reaction. As already described for ROS, NO-derived oxidant species contribute to establishing oxidative conditions as well, resulting in irreversible damage to biomolecules when Oxidative Stress produced at an extent high enough to overcome the Living cells are always subjected to the hazardous effects of antioxidant response. exogenously or endogenously produced highly reactive oxidizing molecules. These can be radicals and nonradicals Redox signal. In the late 1990s, a new ‘radical free’ concept (e.g., H O ), but have in common the ability to easily take 2 2 for free radicals began to take root, and a new signalling electrons from (oxidize) molecules with which they remain in role for ROS and RNS emerged. Many lines of evidence were contact, such as all cellular biomolecules, generating chain accumulating, indicating that ROS and RNS were able to reactions and ultimately leading to cell structure damage. modify proteins in a reversible manner at the level of the Among these classes of molecules, those deriving from ROS sulphur-containing residues, cysteine and methionine, thus and reactive nitrogen species (RNS) have the main biological providing evidence for the existence of a redox-based 40,49 impact because they are endogenously produced at the signal. In particular, reactive cysteine thiol groups (SH) highest concentration, and for this reason the concept of of a growing number of proteins were revealed to be able to oxidative stress can be widened so as to nitro-oxidative stress. rapidly undergo reactions with H O and NO in biological 2 2 systems, thus forming the S-hydroxylated (S-OH) and Oxidative damage. It is commonly accepted that the S-nitrosylated (S-NO) derivatives, respectively. Upon reaction principal source of ROS in the cell is the mitochondrial with other cysteines (e.g., those belonging to GSH or other respiratory chain. Indeed, mitochondrial complexes (mainly protein thiols), both these adducts usually covert to dis- complexes I and III) can leak electrons, leading to the partial ulphide (S-S), and are finally reduced back to sulphhydryl at reduction of oxygen to O that spontaneously, or by the the expense of the reducing equivalents provided by NADPH superoxide dismutase (SOD)-mediated catalysis, very rapidly through the Trx/Trx reductase (or Grx/Grx reductase) 40,41 disproportionates into H O . It has been estimated that ROS 2 2 system. Oxidative modifications of reactive cysteines produced by mitochondria are ∼ 1–2% of the total rate of cause changes in protein structure and function; they affect oxygen consumption. This at first glance could appear very localization and physical interactions, as well as the capability low; yet, if one considers that the average rate of oxygen to undergo further posttranslational modifications (e.g., -18 utilization in each single cell of human body is ∼ 2.5 × 10 phosphorylation). This is the reason that reactive cysteines 10 39 mol/s (that means 2.2 × 10 molecules everyday), the are deemed to be the primary molecular switches that are amount of ROS daily generated intracellularly reaches ∼ 1 able to transduce a redox signal. billion molecules. Multiplying this value for the number of cells in human body (∼50 trillion) gives an idea of the intensity of Autophagy and Oxidative Stress ROS flux to which we are exposed physiologically. Moreover, ROS have been copiously reported as early inducers of considering that in some circumstances, the electron flux autophagy upon nutrient deprivation. However, to date, it is through the mitochondrial respiratory chain is intensified still unclear as to which species exactly drives the process. A (e.g., upon increased energetic demand), or that mitochon- 51 .- detailed work from Chen et al. proposes that O is the drial efficiency might decrease (e.g., during ageing), along primary ROS involved in autophagy induced by glucose, with the fact that other exogenous (e.g., UV radiation) and glutamine, pyruvate or serum deprivation. Further lines of endogenous sources of ROS (e.g., oxidases and oxyge- evidence indicate, instead, that H O is the molecule nases) can operate as well, it becomes evident that a highly 2 2 52,53 produced immediately after starvation, whereas many efficient antioxidant response has probably been selected by others just hypothesize that ROS are crucial for autophagy evolution to protect and preserve, as far as possible, the execution as treatment with antioxidants partially or comple- cellular components. The antioxidant enzymes SOD, catalase and glutathione tely reverts the process. peroxidases (GSH-Px or GPx) are those responsible for removal of O ,H O and peroxides in general. They are Mitochondria as main source of ROS in autophagy 2 2 2 present in all cellular districts and act in concert with other signalling. Although the question is still far from being proteins, such as peroxiredoxins, thioredoxins (Trx) and solved, there are at least other two issues that deserve to be glutaredoxins (Grx), as well as low-molecular-weight anti- considered. The first is ‘where ROS are so rapidly produced’. oxidants (e.g., GSH, tocopherols and ascorbate) to fully It would be actually more logical that a stimulus coming from scavenge ROS and restore the reduced protein and lipid the outside of the cell is transduced by a ROS-producing 41–43 pool. The efficiency of the antioxidant defence is also system located at, or nearby, the plasma membrane, such as important to modulate the levels of RNS, a class of molecules the NADPH oxidase (NOX) complexes. Nevertheless, deriving from peroxynitrite (ONOO ), a very dangerous although attractive, this hypothesis has been verified only in .- compound generated by reaction between O and nitric oxide macrophages upon bacterial infection, where ROS generated 44,45 (NO). Nitric oxide is a highly reactive gaseous radical, by NOX2 are indispensable for the recruitment of the 45,46 soluble in water and diffusible across cell membranes. It is microtubule-associated protein light chain 3 (LC3) on Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al phagosomes that, thus modified, are degraded by autophagy mechanism is activated for the selective dismissal of impaired to prevent pathogen escape. A large amount of data, or dysfunctional mitochondria. It is responsive to mitochondrial instead, converge to state that the mitochondria represent depolarization and is regulated by the PTEN-induced putative the principal source of ROS required for autophagy kinase 1 (PINK1) and Parkin, a ubiquitin E3 ligase whose 56,57 induction, although they are not in close proximity to mutations have been associated with familial form of Parkin- the plasma membrane. A possible explanation for this son’s disease. PINK1 is a Ser/Thr kinase that translocates on unexpected evidence is that nutrient deprivation suddenly the outer mitochondrial membrane where it is stabilized by low results in energetic stress that, in turn, increases ATP mitochondrial transmembrane potential, thereby acting as real demand and causes mitochondrial overburden to face up sensor of mitochondrial depolarization. Here, PINK1 recruits adverse conditions. As a consequence, electron leakage and Parkin that ubiquitylates a number of outer mitochondrial ROS production also increase. Another hypothesis is that a membrane-located proteins, for example, the voltage- still uncharacterized factor could act as transducer, linking the dependent anion channel 1 (VDAC1). Once ubiquitylated, upstream autophagic signal with mitochondria. A good these proteins are recognized by p62/sequestosome 1 (p62/ 69,70 candidate could be HKII that, by sequestering mTORC1, SQSTM1, or simply p62), a ubiquitin-binding protein acting could loosen its inhibition on permeability transition pore as a scaffold for several protein aggregates and triggering their 34,35 (PTP) and its ability to decrease ROS. In support of this degradation through the proteasome, or the lysosome pathway 71,72 hypothesis, it has been reported that the two protein kinases via autophagy. p62 contains an LC3 interacting region positively regulating HKII activity, Akt and myotonic dystrophy (LIR) that has been indicated being fundamental for p62 to protein kinase (DMPK), are negative modulators of mediating mitophagy. Indeed, by means of this motif, p62 can 58,59 autophagy. bridge autophagy-targeted mitochondria to LC3 located on the autophagosomes surface, thereby driving their degradation. ROS and mitophagy. As principal sites of ROS production, Interestingly, our group has recently identified a role for mitochondria are the organelles that are able to turn on and Ambra1 in mitophagy, driven by its selective interaction with tune autophagy. However, upon chronic impairment of LC3 and independent from Parkin and p62. mitochondrial function, ROS can be generated at high extent, Redox signalling in autophagy. Another issue to be thus shifting their role from bulk autophagy inducers into a self-removal signal for mitochondria through a selective considered is ‘how oxidative stress can crosstalk with process called mitophagy. This represents a fine mechanism autophagic machinery’. As previously mentioned, antioxidant of negative feedback regulation by which autophagy elim- treatment prevents autophagy, suggesting that redox imbal- inates the source of oxidative stress and protects the cell from ance has a pivotal role in driving the process. The very fast oxidative damage (see below). induction of autophagy upon ROS production from mitochon- Although necessary, mitophagy represents an ‘extreme dria argues for a rapid (ON/OFF) response mediated by decision’ for a cell subjected to nutrient deprivation because of redox-sensitive proteins, among which AMPK could be a at least two main reasons. The first reason is that mitochondria good candidate. Indeed, AMPK has been proposed as being underpin ATP production that is fundamental upon carbon activated upon H O exposure, particularly through 2 2 source limitation. The second reason lies in the fact that S-glutathionylation (formation of a mixed disulphide with mitochondria are relatively large organelles that require being GSH) of reactive cysteines located at the α- (Cys and 304 30,75 beforehand fragmented in order to be properly recognized and Cys ) and β-subunits (still not identified) (Figure 2). engulfed within the autophagosomes. Both these issues Although the role of redox regulation in AMPK activation is contribute to explain why mitochondria are in general still a matter of debate, these results are in line with the recent refractory to undergo mitophagy, unless they are severely observations indicating that, once deprived of nutrients, cells damaged. Recently, it has been proposed that under nutrient actively extrude GSH by the drug efflux pump, multidrug deprivation, mitochondria attempt to protect themselves from resistance protein 1 (MRP1) in order to shift intracellular autophagic removal by promoting fusion and inhibiting fission redox environment towards more oxidizing conditions and 61,62 events. The combination of these two inputs results in prime redox-sensitive proteins to be oxidatively modified mitochondrial elongation that further impedes organelle (Figure 2). The evidence that the sole chemically induced engulfment within the autophagosomes and, concomitantly, oxidation of GSH is able to induce autophagy even in the 62 76 allows to maximize ATP production. Only upon prolonged absence of any autophagic stimulus underlines the starvation, mitochondria depolarize and become fragmented importance of thiol redox homeostasis in autophagy commit- in order to assist their removal by mitophagy. ment. This assumption is in line with the evidence indicating So far, at least two different molecular mechanisms under- that a number of proteins involved in both induction and lying mitophagy have been described and characterized. The execution of autophagy act by means of Cys residues. first one is mediated by NIX/Bnip3L (Bcl-2/adenovirus E1B Among them, the two ubiquitin-like systems Atg7-Atg3 and 63,64 19-kDa-interacting protein 3, long form), an atypical BH3 Atg7-Atg10, some members of Rab GTPase (e.g., Rab33b), protein that is able to directly recognize the autophagosome- and the phosphatase and tensin homologue deleted (PTEN) sited GABA receptor-associated protein (GABARAP), a func- are included. Along this line, it is worthwhile to note that p62 tional homologue of LC3 and, in turn, allow mitochondria to be contains a zinc-finger motif (ZZ) rich in cysteine residues that removed. This is a ‘programmed’ event, independent on any are necessary for metal binding and that could be redox damage response that is required, for example, in mitochondrial regulated. Although no evidence has been provided yet about 65,66 elimination during erythrocyte differentiation. The second a possible redox sensitivity of p62, it could be conceived that, Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al Conflicting role of NO and nitrosative stress in autoph- agy. Results emerging in the past 5 years suggest that NO, by means of S-nitrosylation mechanisms, has also a role in modulating autophagy. However, rather than a positive effector of the process, it seems that it could act as an inhibitory molecule. This assumption is completely in contrast with that described above for ROS, and contributes to making the functional relationship between oxidative stress and autop- hagy even more complex. Sarkar et al., indeed, demon- strated that treatment with NO donors or enhancement of NOS activity in HeLa cells results in autophagy prevention because of S-nitrosylation, and subsequent inhibition of the c-Jun-N- terminal kinase 1 (JNK1) and IκB kinase β (IKKβ) that regulate Beclin1 detachment from Bcl2 and AMPK activation, respec- tively. This is in line with results reporting that S-nitrosylation of TSC2 prevents its inhibitory activity on mTOR, thereby preventing autophagy and inducing proliferation of melanoma cells. However, these data are in contrast with the well- documented role of NO and nitrosative stress in the activation of AMPK–TSC2 pathway via Ataxia telangiectasia mutated (ATM) in response to DNA damage (see below). This discrepancy is likely because of the fact that the biological effects of NO range from prosurvival to apoptotic depending on the real concentrations used (reason why NO has been identified as Janus-faced molecule) that frequently are completely unknown (e.g., upon treatment with NO donors). In support to this, very recently it has been reported Figure 2 Crosstalk between autophagy and oxidative stress. Superoxide (O ) and H O are the main ROS produced by mitochondria upon nutrient that genetic ablation of the main denitrosylating enzyme, 2 2 2 deprivation. They positively regulate autophagy by means of at least three different S-nitrosoglutathione reductase (GSNOR) – which is the elective mechanisms, including: (1) S-glutathionylation (SH → S-SG) of cysteines located in experimental approach to study the effects of S-nitrosylation in the α and β subunits of AMPK (top right); (2) oxidation of Cys81 (SH → Sox) of Atg4 biological contexts – does not absolutely affect bulk autophagy that in turn leads to the inactivation of its ‘delipidating’ activity on LC3 and to the (Figure 3, unpublished results from Filomeni’s laboratory), but accumulation of the pro-autophagic LC3-II isoform (top centre); and (3) wide exclusively results in an impairment of the sole mitochondrial alteration of thiol redox state (e.g., decrease of GSH/GSSG ratio and general increase of oxidized thiols, Sox) that is facilitated by the release of reduced autophagy (mitophagy) in skeletal muscle models. glutathione (GSH) to the extracellular milieu through the multidrug resistance protein Altogether, these observations indicate that a straightfor- 1 (MRP1) (top left). In a redox-independent manner, it has also been demonstrated ward idea about how oxidative stress functions in autophagy is that p62, when bound to ubiquitylated protein aggregates, can undergo still lacking, despite abundant evidence corroborating its phosphorylation on Ser351, thereby sequestering Keap1 and leading to its implication in each phase of this process. detachment from Nrf2 (bottom left). Consequently, Nrf2 is no longer degraded by the ubiquitin-3 proteasome system, but translocates in the nucleus, binds to antioxidant-responsive elements (AREs) located in the promoter regions of p62/Keap1/Nrf2 system: how autophagy couples with antioxidant genes and activates their transcription (bottom right) redox response. In the past few years, autophagy and oxidative stress have been shown to be interconnected in a more intimate and coordinated maner than by a simple ON/ OFF signal. Particularly, in 2010, it was discovered that p62 activates the antioxidant transcription factor Nrf2 (nuclear similar to other ZZ-containing proteins, p62 could also factor erythroid 2-related factor 2) by a ‘non-canonical’ undergo oxidation and structural alterations that are able to pathway (Figure 2). The underlying mechanism is completely modify/regulate its role in autophagy. redox independent, and involves the recruitment of Kelch-like Notwithstanding the large amount of data supporting the ECH- associated protein 1 (Keap1) that functions as an hypothesis of a redox regulation of autophagic signalling, so adapter protein of the Cul3-ubiquitin E3 ligase complex far the only redox-based mechanism demonstrated to be able 82,83 responsible for degrading Nrf2. In agreement with this to regulate an autophagic protein goes back to 2007, when model, p62 binds to aggregates of ubiquitylated proteins and Scherz-Shouval et al. proved that H O -mediated oxidation 2 2 increases its affinity for Keap1 when phosphorylated at 351 84 of Atg4 at Cys was required for inactivating its hydrolyzing Ser (Figure 2). This event induces Keap1 degradation (delipidating) activity on LC3, thus allowing autophagosome to via autophagy and leaves Nrf2 free to accumulate and be correctly elongated (Figure 2). No further protein has translocate in the nucleus. Here, Nrf2 binds to the been added to the list since then, suggesting that redox antioxidant-responsive elements (ARE) in the promoter modifications of proteins – although reasonably proposed as regions of antioxidant and detoxifying genes, as well as likely modulators of autophagic signal transduction – are not genes involved in DNA damage response, such as the main mechanism linking ROS and autophagy. 8-oxoguanine glycosylase (OGG1) and p53 binding protein Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al Figure 3 Autophagy is not affected by S-nitrosylation. (a) Western blot analyses of S-nitrosothiols in total homogenates of skeletal muscles obtained from GSNOR-KO (KO) and wild-type (WT) mice, subjected to biotin-switch assay and revealed by HRP-conjugated streptavidin. Lactate dehydrogenase (LDH) was selected as loading control. Results show that even in the absence of any treatment with NO-delivering drugs, GSNOR ablation induces a significant increase of S-nitrosylated proteins. (b) Representative fluorescence microscopy images of satellite cell-derived myotubes isolated from KO and WT mice expressing LC3-conjugated green fluorescent protein (GFP-LC3) in heterozygosis. Cells were then subjected to two different autophagic stimuli: (1) they were treated for 6 h with 5 μM of the proteasome inhibitor MG132 or, alternatively, (2) allowed to grow for 6 h in a nutrient-deprived cell medium (stv). Images are representative of three independent experiments that gave similar results. Both genotypes displayed a significant and similar increase of fluorescent dots, plausibly representing autophagosomes, thereby indicating that autophagy is not impaired by S-nitrosylation 88 94,95 1 (53BP1), inducing their transcription (Figure 2). It has also damaged DNA. In particular, this phenomenon has been been suggested that Nrf2 activation by this pathway is observed in nuclear envelopathies where the presence of sustained by Sestrins, a highly conserved family of small perinuclear autophagosomes or autophagolysosomes con- ‘antioxidant-like’ proteins transcriptionally induced by p53 taining DNA clearly represented an operative autophagy. It upon stressful conditions, which are involved in autophagy has been reported that micronuclei containing chromosomes, since function as AMPK activators and mTORC1 inhibi- or parts of them, that are not properly incorporated in the 90,91 tors. daughter nuclei during cell division can be removed by autophagy as well, thus providing this process with a direct role in cleaning up damaged content in the nucleus and in Antioxidant Role of Autophagy: Focus on Nucleus and maintaining genomic stability. DNA Damage However, besides PMN, a number of observations indicate On the basis of what has been reported so far, antioxidant that autophagy is deeply involved in DNA damage repair, response and autophagy are mechanisms simultaneously although without any direct degradative activity on DNA. This induced by oxidative stress conditions in order to concomi- phenomenon, mainly occurring upon ROS and RNS-mediated tantly decrease ROS and RNS concentration (upstream damage to DNA, represents an issue that deserves discussion causes) and reduce the oxidative damage to biomolecules so as to comprehend how autophagy acts as a preventive and and organelles (downstream effect). This finely orchestrated reparative process upon genotoxic stress. Understanding the repair system perfectly fits the needs of a cell attempting to find underlying molecular mediators and the mechanisms would a new homeostatic state. By responding very rapidly to make it possible to clarify once and for all the antioxidant oxidative stress, and by decreasing the toxicity of oxidized activity of autophagy. molecules and organelles through their selective removal, autophagy can be in principle encompassed in the large family Oxidative Stress and DNA Damage of antioxidant processes. However, at variance with proteins and organelles that are present in several copies inside the cell ROS and RNS are one of the major sources of DNA damage (e.g., mitochondria and ribosomes), autophagy cannot med- as they could directly modify the DNA or indirectly generate iate nucleus degradation, even though it is severely damaged, different lesions, both affecting cell viability. Among ROS, because this could lead to the complete loss of genetic OH can directly attack the DNA backbone by generating five information. Genomic DNA cannot be destroyed, de novo classes of oxidative damage: oxidized bases, abasic sites, synthesised or entirely replaced like the other biomolecules. DNA–DNA intrastrand adducts, single-strand break (SSB), 97,98 Its integrity should be prevented and maintained, and any double-strand break (DSB) and DNA–protein crosslinks. damage accurately repaired. Among the nucleobases, guanine is the most susceptible to An exception to the rule is a highly selective and unusual ROS because of its low redox potential, and the main products nuclear DNA degradation by means of the so-called piece- of its oxidation are 8-hydroxyguanine and 8-hydro- meal microautophagy (PMN). Indeed, in a way resembling xydeoxyguanosine (8-OHG and 8-OHdG). Both are highly 92,93 nucleophagy occurring in fungi and nematodes, mamma- mutagenic and carcinogenic as they can match with lian cells can specifically remove part of nuclei containing both cytosine and adenine, thus leading to GC-to-AT Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al 99,100 transversions. The 8-OHG and 8-OHdG are the most genotoxic stress. However, how this occurs is still a matter of commonly used markers of DNA damage as they easily debate. In yeast, for example, it has been demonstrated the accumulate, thus being measured as good index of oxidative selective degradation of specific proteins, mostly through the · - 117 lesions to DNA. Nitric oxide and RNS (i.e., NO , ONOO ,N O so-called ‘cytoplasm to vacuole targeting’ (Cvt), is directly 2 2 3 and HNO ) can cause DNA damage as well, and are involved in: (1) inducing cell cycle arrest in G2/M phase by considered potentially mutagenic as they can induce degrading proteins involved in cell cycle progression (e.g., nitration, nitrosylation and deamination of DNA bases. Psd1 and Esp1); (2) optimizing dNTP production and DNA ROS and RNS are also harmful for mitochondrial DNA synthesis by targeting the subunit 1 of the ribonucleotide (mtDNA) integrity. This feature can deeply affect the transcrip- reductase complex; and (3) regulating the dynamics of tion of mtDNA-coded proteins and RNAs that underlie the homologous recombination by degrading the endonuclease synthesis of a number of subunits belonging to the complexes Sae2 once catalysed the resection of DNA ends. of the mitochondrial respiratory chain (except Complex II). A In higher eukaryotes, no Cvt has ever been identified, nor vicious cycle is then established in which mitochondria, with has any orthologue of proteins belonging to this pathway been oxidized mtDNA, become dysfunctional and produce a high revealed. One of the most accredited hypotheses explaining a rate of ROS, leading to further mitochondrial impairment. This role of autophagy in supporting the DDR is that by degrading condition can ultimately result in severe nuclear DNA damage damaged mitochondria (mitophagy) and toxic aggregates, and cell death. autophagy eliminates putative sources of ROS, reduces their levels and, if only indirectly, decreases DNA damage 114,121,122 accumulation. This assumption provides a general DNA Damage and Autophagy: A Complex Crosstalk explanation of the important role of autophagy in maintaining When the DNA is damaged by ROS and RNS, cells activate a genomic stability that is strictly related to its tumour suppressor 106,113,114 number of pathways in order to maintain genomic integrity, function. However, evidence supporting a direct role these being associated to the DNA damage response (DDR). for autophagy in the DDR, at least in mammals, is still lacking. Different classes of proteins are implicated in DDR, among which the sensors specifically recognize the lesions to DNA, Sensor proteins transduce the signal of DNA damage to whereas the mediators and the effectors transduce the signal autophagy. A number of works in recent years indicate that from the nucleus to the cytosol where several processes are once ROS and RNS damage the DNA, the event is contextually activated in order to better face up to adverse transduced in order to activate the DDR, and concomitantly 102,103 conditions. For instance, cell cycle checkpoints are is signalled to the autophagic pathway in order to orchestrate soon activated to block proliferation until lesions are repaired. the response. PolyADP-ribose polymerase 1 (PARP1) is However, if DNA is severely damaged or unrepaired, cells among the proteins directly linking the DDR and autophagy 123,124 remain quiescent or undergo cell death. Autophagy is (Figure 4). It is a nuclear enzyme that catalyses poly- considered both a pro-survival mechanism and a type of cell ribosylation of nuclear proteins by converting NAD into death. Therefore, once induced by DNA injury, it makes a polymers of polyADP-ribose (PAR), and deeply participates in 105,106 crucial contribution in regulating cell fate. For example, SSB repair, thereby regulating nuclear homeostasis. PARP1 many lines of evidence argue that autophagy can delay is hyperactivated upon ROS-induced DNA damage; this apoptotic cell death upon DNA damage by sustaining the leads to NAD consumption and ATP depletion (Figure 4). energy demand required to support DNA repair Such energetic imbalance results in the activation of 107,108 123,124 processes, a phenomenon that concurs to the develop- autophagy via AMPK pathway (Figure 4) in order to ment of chemoresistance mechanisms in cancer cells. recycle metabolic precursors for ATP and to provide energy Conversely, in cells where DNA is unrepaired and apoptosis is for the DDR. defective, DNA damage-induced autophagy has been Another DNA repair protein linking the DDR to autophagy is reported to contribute to cell death, thereby acting as a tumour ATM (Figure 4), a DNA damage sensor orchestrating the cell suppressor process. cycle with damage response checkpoints and DNA repair to It is worthwhile noting that cases where autophagy safeguard the integrity of the DNA. It has been demon- impairment results in DNA damage have been reported as strated that under ROS-induced cellular damage, cytosolic well, leading to the assumption that the interplay might be ATM, through the LKB1/AMPK pathway, can activate TSC2 broader, and suggesting that many molecular players could tumour suppressor to inhibit mTORC1 and induce autophagy exist to biunivocally link the two processes. In particular, it has (Figure 4). These new findings integrate different stress been demonstrated that the deficiency of autophagy genes, response pathways occurring in different cellular compart- such as Beclin 1, UV irradiation resistance-associated gene ments. From this perspective, ATM would be required to both (UVRAG), Atg5 and Atg7, leads to DNA damage accumulation initiate (nucleus) and mediate (cytosol) the DDR. 111–115 and promotes tumourigenesis. In line with this, the The principal regulator of DDR, however, remains p53 that, suppression of the ULK1-interacting protein FIP200 has been together with the other members of its family, p63 and p73, has reported to impair DDR, thus triggering cell death upon been demonstrated to modulate many autophagic genes 116 105,127–129 ionizing radiation-induced oxidative stress. (Figure 4). In particular, p53 is very rapidly activated when DNA damage occurs, and transcriptionally activates Direct and indirect roles of autophagy in the DDR. All this both DNA repair and cell cycle checkpoints proteins to allow evidence strongly suggests that autophagy participates, repair of DNA lesions. It has been recently demonstrated directly or indirectly, in the DDR to ROS and RNS-mediated that the strength of the stimulus and the dynamics of the Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al the signal (e.g., Bnip3 and AMPK) – are shared between autophagy and apoptosis. How can they be differently induced? Which are the signals, or how much high should be the threshold level to allow cell response being switched from stress adaptation and survival (autophagy) to cell dismantling (apoptosis)? These issues are still debated. Speculations and hypotheses based on the ‘strength’ of the signal have been reasonably done, but no direct evidence of the causative event underlying the decision by the cell to activate autophagy or apoptosis has been provided as yet. Concluding Remarks Several lines of evidence indicate that ROS and RNS are the upstream modulators of autophagy, likely acting at multiple levels in the process. In line with this assumption, ROS and RNS would act as the intracellular ‘alarm molecules’ of the extracellular availability of nutrients (primitive stimuli) by signalling their presence to the autophagic machinery. Through a negative feedback regulation, autophagy could be then induced to provide energy and building blocks in order to restore homeostasis and, concomitantly, remove oxidative damage. From this perspective, autophagy is required for Figure 4 Implication of autophagy in DNA damage repair. Endogenous the cell to simultaneously overcome starvation and oxidative (e.g., dysfunctional mitochondria, top right) or exogenous (e.g., radiations or stress conditions. Therefore, any dysfunction in this regard genotoxic stimuli, bottom left) sources of ROS and RNS induce DNA damage, whose has been found to be involved in the onset of pathological primary sensors are PARP1 and ATM. Once activated by DNA breaks, PARP1 states where a primary role for oxidative stress and/or catalyses poly-ADP ribosylation of itself, as well as of other nuclear proteins, thereby + alterations in metabolic demand (such as cancer) has also leading to a massive decrease of NAD and to a subsequent energetic stress (bottom been reported. Accordingly, genetic defects of autophagic left). Upon DNA damage, ATM can activate p53-mediated transcription of autophagic genes (bottom right). Alternatively, cytosolic pool of ATM could be directly activated by genes lead to an increased production of ROS and accumula- ROS through a still unidentified mechanism and it directly induces the activation of tion of damaged organelles and DNA that in turn promote LKB1 (centre). The issue of whether cytosolic and nuclear pool of ATM are metabolic reprogramming and induce tumourigenesis. interconnected still waits to be demonstrated. Both PARP1 and ATM signalling Although a number of possible mechanisms underlying the pathways converge on AMPK, whose activation induces the autophagic machinery to intimate interplay between oxidative stress and autophagy remove the main source of DNA damage and contribute to its repair through a have been postulated, to date only a few of them have been negative feedback loop (top) shown to have a role in tuning autophagy. Understanding the binding to DNA underlies the genes transactivated by p53. For fine molecular regulation of autophagy by ROS and RNS, as instance, in case of inefficient repair, p53 can shift transcrip- well as the tight relationship between metabolism and redox waf tion from cell cycle modulators (e.g., p21 ) to genes state, could therefore provide valuable information that could regulating cell senescence and apoptosis (e.g., PUMA and be useful in the future to improve anticancer treatment and BAX). In addition, autophagy genes are also induced and develop new selective therapies. their transcription finely orchestrated by p53 (Figure 4). Targets of p53 are, indeed, both upstream regulators of Acknowledgements. We thank M Acuña Villa and MW Bennett for editorial and autophagy (e.g,. PTEN, TSC2, β1, β2 and γ subunits of AMPK) secretarial work. We are also grateful to Costanza Montagna and Giuseppina and proteins directly involved in autophagosome formation Di Giacomo for having provided part of unpublished results obtained on GSNOR-KO (e.g., ULK1, UVRAG, ATG2, 4, 7, 10). It is worth noting that mice. The Unit of Cell Stress and Survival is supported by grants from the Danish p63 and p73 also share some autophagic genes as transcrip- Cancer Society (KBVU R72-A4408 to FC and KBVU R72-A4647 to GF); Lundbeck Foundation (nn. R167-2013-16100) and NovoNordisk (nn. 7559). 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Publisher: Springer Journals
Copyright: Copyright © 2015 by The Author(s)
Subject: Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Stem Cells; Apoptosis; Cell Cycle Analysis
ISSN: 1350-9047
eISSN: 1476-5403
DOI: 10.1038/cdd.2014.150
Publisher site: See Article on Publisher Site

Abstract

Cell Death and Differentiation (2015) 22, 377–388 OPEN & 2015 Macmillan Publishers Limited All rights reserved 1350-9047/15 www.nature.com/cdd Review Oxidative stress and autophagy: the clash between damage and metabolic needs ,1,2 1,2 ,1,2 G Filomeni* , D De Zio and F Cecconi* Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. A growing amount of evidence in recent years argues for oxidative stress acting as the converging point of these stimuli, with reactive oxygen species (ROS) and reactive nitrogen species (RNS) being among the main intracellular signal transducers sustaining autophagy. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth. Cell Death and Differentiation (2015) 22, 377–388; doi:10.1038/cdd.2014.150; published online 26 September 2014 Facts Open Questions How do ROS and oxidative stress affect autophagy? Reactive oxygen species (ROS) production and thiol redox Which are the main ROS able to signal autophagy being state imbalance are induced immediately upon nutrient activated and going on? deprivation and represent important mediators of Does nitric oxide act as a real inhibitor of autophagy? autophagy. How does autophagy sense DNA damage? ROS and reactive nitrogen species (RNS) irreversibly How can autophagy contribute to DNA damage repair? oxidize DNA and cellular biomolecules, thereby represent- ing the primary source of damage in biological systems. 1,2 In the 1950s, Christian de Duve, contextually with the Autophagy contributes to clearing the cells of all irreversibly discovery of glucagon, clarified the intracellular localization of oxidized biomolecules (proteins, DNA and lipids), this is all the more reason why it could be included in the antioxidant several enzymes by setting up centrifugation-based tissue and DNA damage repair systems. fractionation of rat liver homogenates. During his work, he 1 2 Cell Stress and Survival Unit, Danish Cancer Society Research Center, Copenhagen, Denmark and IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy *Corresponding author: F Cecconi, Cell Stress and Survival Unit, Danish Cancer Society Research Center, Strandboulevarden 49, Copenhagen DK-2100, Denmark. Tel: +45 35257401; Fax: +45 35271811; E-mail: [email protected] or Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, 00133 Rome, Italy. Tel: +39 06 72594230; Fax:+39 06 72594311; E-mail: [email protected] or G Filomeni, Cell Stress and Survival Unit, Danish Cancer Society Research Center, Strandboulevarden 49, Copenhagen DK-2100, Denmark. Tel: +45 35257402; Fax: +45 35271811; E-mail: [email protected] or Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica, 00133 Rome, Italy. Tel: +39 06 72594243; Fax: +39 06 72594311; E-mail: [email protected] Abbreviations: 4E-BP1, EIF4E-binding protein 1; 53BP1, p53 binding protein 1; 8-OHdG, 8-hydroxydeoxyguanosine; 8-OHG, 8-hydroxyguanine; Ambra1, activating molecule in Beclin1-regulated autophagy; AMPK, AMP-dependent protein kinase; ARE, antioxidant-responsive element; Atg4, autophagy related gene 4; Atg5, autophagy related gene 5; Atg7, autophagy related gene 7; Atg13, autophagy related gene 13; ATM, Ataxia telangiectasia mutated; Bnip3, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3; Cvt, cytoplasm-to-vacuole targeting; DDR, DNA damage response; DMPK, myotonic dystrophy protein kinase; DSB, double-strand break; eEF, eukariotic elongation factor; eIF, eukaryotic initiation factor; Esp1, extra spindle pole bodies homologue 1; FIP200, FAK-family interacting protein of 200 kDa; GABARAP, GABA receptor-associated protein; GSH-Px or GPx, glutathione peroxidise; Grx, glutaredoxin; GSH, reduced glutathione; GSNOR, S-nitrosoglutathione reductase; GSSG, disulphide glutathione; HKII, hexokinase II; IKKβ,IκB kinase β; JNK1, c-Jun-N-terminal kinase 1; Keap1, Kelch-like ECH- associated protein 1; LC3, light chain 3; LKB1, liver kinase B1; MRP1, multidrug resistance protein 1; mtDNA, mitochondrial DNA; mTORC1, mammalian target of rapamycin complex 1; NO, nitric oxide; NOX, NADPH oxidase; Nrf2, nuclear factor erythroid 2-related factor 2; OGG1, 8-oxoguanine glycosylase; p70S6K, p70 ribosomal protein S6 kinase; PAR, polyADP-ribose; PARP1, polyADP-ribose polymerase 1; PHD, prolyl hydroxylase; PI3K, phosphoinositide 3-kinase; PINK1, PTEN-induced putative kinase 1; PMN, piecemeal microautophagy; Psd1, pleckstrin and Sec7 domain containing 1; PTEN, phosphatase and tensin homologue; PTP, permeability transition pore; RAG, RAS-related GTP-binding protein; RHEB, RAS homologue enriched in brain; RNS, reactive nitrogen species; ROS, reactive oxygen species; Sae2, sporulation in the absence of SPO11 protein 2; SOD, superoxide dismutase; SQSTM1, sequestosome 1; SSB, single-strand break; TCA, tricarboxylic acid; Trx, thioredoxin; TSC2, tuberous sclerosis 2; ULK1, upstream kinase UNC51-like kinase 1; UVRAG, UV irradiation resistance-associated gene; v-ATPase, vacuolar H -ATPase; VPS34, vacuolar protein sorting 34 Received 29.6.14; revised 19.8.14; accepted 21.8.14; Edited by G Kroemer; published online 26.9.14 Oxidative stress and autophagy interplay G Filomeni et al discovered and coined the names of many organelles, whose his findings could be basically interconnected by a finely purification, characterization and distribution contributed to organized signalling system, where primary/primitive stimuli earning him the Nobel Prize for Physiology and Medicine in (e.g., nutrient availability and oxidative insults) differently 1974. In his studies on carbohydrate metabolism and insulin impinge on the maintenance of biomolecule integrity and cell action, he described for the first time the lysosomes as the viability through the intermediate activity of homeostatic intracellular granules containing the enzymes glucose-6- processes (mainly based on repair and degradation), the phosphatase and acid phosphatase, in addition to a set of most complex and versatile of which was the very same hydrolases that were deputed to digest, recycle and remove autophagy he discovered 10 years before. intracellular material, such as worn-out or damaged orga- nelles, and engulfed pathogens, by means of a process that he Autophagy: Converging Point of Different Stimuli named autophagy. More than 10 years later, in 1966, he also defined the There are three main types of autophagy culminating to structure and composition of microbodies: the cellular lysosome-mediated degradation: (1) macroautophagy (here- districts in which hydrogen peroxide is endogenously pro- after referred to as autophagy) that involves the formation of a duced to a high extent as a side effect of the reactions double-membrane vesicle (autophagosome) deputed to catalyzed by many oxidases involved in amino acid, purines sequester damaged organelles and biomolecules; (2) micro- and fatty acid metabolism, and for this reason named autophagy, by which the cytosolic material is directly engulfed peroxisomes. Although the toxicity of hydrogen peroxide had by the lysosome; and (3) chaperone-mediated autophagy. It is been reported many years before, only in the late 1950s its now well established that autophagy is a very sensitive real implications in biology were coming up. Progress in the process underlying cell response induced by almost every field of metallobiology and the fine characterization of stressful condition affecting cellular homeostasis. Through metalloenzyme-mediated catalysis provided compelling evi- autophagy, cells coordinate energy and building blocks dence for an endogenous and physiological production of demanded for vital processes (e.g., growth and proliferation) partially reduced oxygen species (nowadays usually referred with the extracellular stimuli and carbon source availability, to as reactive oxygen species (ROS)), such as superoxide such as amino acids and glucose. If they are not sufficient to anion (O ), hydrogen peroxide (H O ) and hydroxyl radical maintain the rate of protein synthesis, or to provide the 2 2 2 ( OH). Their being highly reactive towards lipids, proteins and required amount of ATP needed to sustain metabolic 7–9 DNA, and severely harmful for cell survival when present at reactions, then cells activate autophagy in order to rapidly very high concentrations, both led to the concept of oxidative degrade the old or burned-out components and reuse the stress as detrimental condition occurring in all living systems generated pool of biomolecules. and arising from the imbalance between oxidants species and Both glucose and amino acids signals converge on a unique antioxidant defence. It is not a coincidence that in the same molecular transducer of cellular needs, the mammalian target 10 12 years, Denham Harman postulated the ‘free radical theory of of rapamycin complex 1 (mTORC1) (Figure 1). Active ageing’ in which he stated that free radicals were the primary mTORC1 controls the activity of translation eukaryotic cause of massive damage to DNA and all cellular macro- initiation factors (eIFs) and eukariotic elongation factors molecules, culminating in cancer and in a diffuse cell (eEFs), namely eIF2, -3 and -4 and eEF2, by direct dysfunction distinctive of ageing. phosphorylation of two key protein targets, EIF4E-binding When de Duve characterized the peroxisomes and found protein 1 (4E-BP1) and protein S6 kinase (p70S6K). Both out that they were the organelles in which the antioxidant are required for a correct and efficient protein synthesis, as enzyme catalase resides, he probably did not realize that all they regulate the interactions between the mRNA 5′ cap, the Figure 1 Main molecular pathways activated in the presence or absence of nutrients. (a) The synergic import of leucine (Leu) and glutamine (Gln) (top left) results in mTORC1 recruitment to the lysosomal membrane and its subsequent activation by at least two distinct pathways. The first one proposes that cytosolic amino acids enter the lysosome and signal their presence to RAG-A and RAG-C (or RAG-B and RAG-D, not shown in the figure) through the lysosome-located proton pump v-ATPase and the multimolecular complex called Ragulator. The second pathway provides for the double deamination of Gln catalysed by the enzymes glutaminase (GLN) and glutamate dehydrogenase (GDH). This sequence of reactions subsequently generates glutamate (Glu) and α-ketoglutarate (αKG) that, by acting as co-substrate for prolyl hydroxylases (PHD), finally leads to RAG activation. GTP-bound RAG-A (or B) and GDP-bound RAG-C (or D) can then recruit mTORC1 to the lysosome membrane where it is activated by RHEB (middle left). Once activated, mTORC1 activates protein synthesis by phosphorylating 4EBP1 and p70S6K, and concomitantly inhibits autophagy by phosphorylating ULK1 complex at the level of ULK1 and Atg13 (bottom left). Glucose is taken up through specific transporters (GLUTs) and phosphorylated to glucose-6-phosphate (G6P) by hexokinase (the only mitochondrial isoform II, HKII, is shown in the top right side of the figure). G6P is then isomerized to fructose-6-phosphate (F6P), oxidized through the glycolytic pathway to generate pyruvate (Pyr) and acetyl-CoA that fuels the mitochondrial TCA cycle and the respiratory chain for the production of ATP (middle right) through the oxidative phosphorylation (OXPHOS). G6P can also undergo oxidation via the glucose-6-phosphate dehydrogenase (G6PDH)-mediated catalysis along the pentose phosphate pathway (middle right). In this way, electrons required for NADP -to-NADPH reduction, and sugars needed for DNA de novo synthesis (e.g., ribulose-5-phosphate, R5P) are also provided (bottom right). (b) Upon amino acid deprivation, RAGs exchange nucleotides located in their binding sites (GTP with GDP or vice versa), thus leading to mTORC1 release from the lysosome membrane (top left). These 2 events are associated with the inhibitory binding of mTORC1 to HKII that takes place upon glucose deficiency and G6P level decrease (top right). This condition leads to a decrease of NADPH and ATP levels that finally result in a reduced antioxidant capacity of the cell (especially in regenerating the reduced thiol pool) (middle right) and in energetic stress that the cell attempts to counteract by the adenylate kinase (AK)-mediated conversion of ADP into ATP and AMP (centre). AMP increase induces to the activation of AMPK that inhibits protein synthesis by phosphorylating TSC2 and mTORC1 and activates autophagy by phospho-activating ULK1. Once activated, ULK1 phosphorylates its interactors (Atg13 and FIP200) and recruits microtubule-associated PI3K complex by means of an AMBRA1-mediated process to initiate the nucleation phase of autophagic vesicles from the endoplasmic reticulum (or mitochondria, not shown). Many other factors, such as Atg proteins coming from Golgi apparatus (e.g., Atg9) contribute to phagophore elongation and autophagosome formation (bottom) Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al poly(A)-tail and the 40S and 60S ribosomal subunits leucine also, as it binds to and activates glutamate dehydro- (Figure 1). Concomitantly, active mTORC1 prevents autoph- genase, the enzyme catalysing the last deamination step agy by phospho-inhibiting the UNC51-like kinase 1 (ULK1) at leading to α-ketoglutarate production. In line with these Ser and its interacting partner, the autophagy related gene pieces of evidence, compartmentalization of mTORC1 at the 13 (Atg13) that, together with the FAK-family interacting level of the lysosomes could provide some explanations protein of 200 kDa (FIP200), form the so-called ULK1 complex regarding mTOR negative regulation on autophagy. 12,15 (Figure 1). Upon autophagic stimuli, mTORC1 is inhibited, thus leading to the activation of ULK1. Once activated, ULK1 is Glucose signal. Glucose is the primary carbon source that, able to phosphorylate Atg13 and FIP200 inducing to the upon sequential oxidation steps taking place during glyco- following activation of the class III phosphoinositide 3-kinase lysis and TCA cycle, provides the electrons (energy) coming (PI3K) complex via the activating molecule in Beclin1- from the breakdown of its chemical bonds, required for ATP 15,16 regulated autophagy 1 (Ambra1). The formation of PI3K production. The maintenance of endergonic processes complex is required to initiate phagophore nucleation that strictly depends on the maintenance of ATP levels, and for represents the first step leading to autophagosome formation this reason, cells (1) actively synthesize ATP and (2) have 12,15,16 (Figure 1). evolved sophisticated mechanisms to face up energetic stress. Amino acid signal. Among the amino acids that are able to AMP-dependent protein kinase (AMPK) is the genuine signal their presence to the cell, and then let autophagy be sensor of the energetic state of the cell, and directly responds induced in case of any deficiency, leucine and glutamine play to the so-called adenylate energy charge as the enzyme is the most important roles because of their essentiality and activated by very low increases of AMP levels (and, to certain 28–30 their tight interdependence in the mechanism regulating their extent, of ADP), and deactivated by ATP. In order to 17,18 uptake. Leucine is an essential amino acid indispensible restore the correct adenylate energy charge, phospho-active for cell survival as it is nonsynthesizable de novo through AMPK concertedly stimulates catabolic pathways (e.g., alternative pathways, such as by transformation of other glycolysis and fatty acid oxidation), inhibits the rate of anabolic amino acids or by transamination of carboxylates coming up reactions (protein and fatty acid synthesis) and activates from glycolysis and the tricarboxylic acid (TCA) cycle. autophagy. Glutamine, instead, though nonessential, represents the At the molecular level, active AMPK stimulates autophagy most abundant amino acid in the human body and one of by means at least three distinct mechanisms. These include the main substrates of anaplerotic reactions fuelling the TCA (1) phosphorylation of the mTORC1 inhibitor, tuberous 1387 31 cycle. It has been estimated that hypercatabolism or other sclerosis 2 (TSC2) at Ser , which induces RHEB GTPase stressful conditions (e.g., infection and severe injuries) are activity; (2) phosphorylation of the mTORC1 component 722 792 32 accompanied by a significant decrease of glutamine in Raptor at Ser and Ser , which is preparatory for its 19 317 skeletal muscle cells, thus arguing for it being a reliable binding to 14–3–3; and (3) phosphorylation of ULK1 at Ser 777 33 marker of both amino acid and energetic status. and Ser (Figure 1). From a mechanistic point of view, the At the molecular level, the presence of an adequate amount first two phosphorylations catalysed by AMPK inhibit of amino acids induces members of the RAS-related GTP- mTORC1 and reduce its inhibitory effects on ULK1. ULK1 is binding protein (RAG) family of small GTPases (i.e., RAG then free to interact with and to be phosphoactivated by AMPK A–D) to bind guanine nucleotides (GTP or GDP in depen- (Figure 1). dence of the member), leading to their activation and, Fascinatingly, it has very recently been reported that subsequently, to the recruitment of mTORC1 on the lysosome glucose sensing by the cells does not only depend on AMPK, 20,21 membrane (Figure 1). Here, mTORC1 can be targeted by as indirect transducer of the intracellular energy state, but also RAS homologue enriched in brain (RHEB) that, upon binding relies upon more direct mechanisms, thereby making the to GTP, acts as positive regulator of mTORC1. In agreement system redundant and controlled at multiple levels. Roberts with this model, RAGs are the primary sensors that, by means et al. demonstrated that hexokinase II (HKII), the of at least two distinct mechanisms, signal amino acid mitochondria-located enzyme responsible for the first step of availability to mTORC1 and modulate its activation state. glycolysis, binds to and inhibits mTORC1, and that this One mechanism suggests that RAGs sense the amino acid interaction is enhanced by glucose deprivation, namely by a pool (mainly leucine) present within the lumen of the lysosome decrease in glucose-6-phosphate levels (Figure 1). As by the vacuolar H -ATPase (v-ATPase) and a molecular proposed by the authors, this new mechanism could 24,25 complex called Ragulator (Figure 1). This mechanism contribute to the modulation of cell metabolism in circum- could underlie the negative feedback acting on autophagy stances of glucose deficiency, but could also have deep (because of mTOR reactivation) once amino acid levels have implications in redox homeostasis. Indeed, besides many been successfully restored. Alternatively, it has been pro- indications arguing for a direct role of HKII in preventing ROS 35–37 posed that RAGs are activated by glutamine, specifically by generation from mitochondria, it should be also taken into α-ketoglutarate generated upon double deamination occurring account that glucose-6-phosphate, via the pentose phosphate 26 + in the glutaminolytic pathway. Although mTOR activation pathway, is also the primary source of electrons for NADP - through this mechanism needs to be still clarified, it seems to to-NADPH reduction (Figure 1). NADPH directly participates require prolyl hydroxylase (PHD) activity that, in fact, is in bioreductive synthesis and provides the electrons required positively regulated by α-ketoglutarate (Figure 1). As above for thiol redox homeostasis (Figure 1). In particular, NADPH reported, this mechanism has been shown to be responsive to acts as a co-substrate of glutathione reductase – the enzyme Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al responsible for the reduction of the disulphide (GSSG) to the endogenously produced by NO synthases (NOS1-3), a family sulphhydryl (GSH) form of glutathione – as well as of many of constitutive or inducible enzymes with different tissue other reductases deeply implicated in sulphhydryl regenera- distribution and that use arginine and NADPH as substrates tion and, in turn, in the defence against oxidative stress. for reaction. As already described for ROS, NO-derived oxidant species contribute to establishing oxidative conditions as well, resulting in irreversible damage to biomolecules when Oxidative Stress produced at an extent high enough to overcome the Living cells are always subjected to the hazardous effects of antioxidant response. exogenously or endogenously produced highly reactive oxidizing molecules. These can be radicals and nonradicals Redox signal. In the late 1990s, a new ‘radical free’ concept (e.g., H O ), but have in common the ability to easily take 2 2 for free radicals began to take root, and a new signalling electrons from (oxidize) molecules with which they remain in role for ROS and RNS emerged. Many lines of evidence were contact, such as all cellular biomolecules, generating chain accumulating, indicating that ROS and RNS were able to reactions and ultimately leading to cell structure damage. modify proteins in a reversible manner at the level of the Among these classes of molecules, those deriving from ROS sulphur-containing residues, cysteine and methionine, thus and reactive nitrogen species (RNS) have the main biological providing evidence for the existence of a redox-based 40,49 impact because they are endogenously produced at the signal. In particular, reactive cysteine thiol groups (SH) highest concentration, and for this reason the concept of of a growing number of proteins were revealed to be able to oxidative stress can be widened so as to nitro-oxidative stress. rapidly undergo reactions with H O and NO in biological 2 2 systems, thus forming the S-hydroxylated (S-OH) and Oxidative damage. It is commonly accepted that the S-nitrosylated (S-NO) derivatives, respectively. Upon reaction principal source of ROS in the cell is the mitochondrial with other cysteines (e.g., those belonging to GSH or other respiratory chain. Indeed, mitochondrial complexes (mainly protein thiols), both these adducts usually covert to dis- complexes I and III) can leak electrons, leading to the partial ulphide (S-S), and are finally reduced back to sulphhydryl at reduction of oxygen to O that spontaneously, or by the the expense of the reducing equivalents provided by NADPH superoxide dismutase (SOD)-mediated catalysis, very rapidly through the Trx/Trx reductase (or Grx/Grx reductase) 40,41 disproportionates into H O . It has been estimated that ROS 2 2 system. Oxidative modifications of reactive cysteines produced by mitochondria are ∼ 1–2% of the total rate of cause changes in protein structure and function; they affect oxygen consumption. This at first glance could appear very localization and physical interactions, as well as the capability low; yet, if one considers that the average rate of oxygen to undergo further posttranslational modifications (e.g., -18 utilization in each single cell of human body is ∼ 2.5 × 10 phosphorylation). This is the reason that reactive cysteines 10 39 mol/s (that means 2.2 × 10 molecules everyday), the are deemed to be the primary molecular switches that are amount of ROS daily generated intracellularly reaches ∼ 1 able to transduce a redox signal. billion molecules. Multiplying this value for the number of cells in human body (∼50 trillion) gives an idea of the intensity of Autophagy and Oxidative Stress ROS flux to which we are exposed physiologically. Moreover, ROS have been copiously reported as early inducers of considering that in some circumstances, the electron flux autophagy upon nutrient deprivation. However, to date, it is through the mitochondrial respiratory chain is intensified still unclear as to which species exactly drives the process. A (e.g., upon increased energetic demand), or that mitochon- 51 .- detailed work from Chen et al. proposes that O is the drial efficiency might decrease (e.g., during ageing), along primary ROS involved in autophagy induced by glucose, with the fact that other exogenous (e.g., UV radiation) and glutamine, pyruvate or serum deprivation. Further lines of endogenous sources of ROS (e.g., oxidases and oxyge- evidence indicate, instead, that H O is the molecule nases) can operate as well, it becomes evident that a highly 2 2 52,53 produced immediately after starvation, whereas many efficient antioxidant response has probably been selected by others just hypothesize that ROS are crucial for autophagy evolution to protect and preserve, as far as possible, the execution as treatment with antioxidants partially or comple- cellular components. The antioxidant enzymes SOD, catalase and glutathione tely reverts the process. peroxidases (GSH-Px or GPx) are those responsible for removal of O ,H O and peroxides in general. They are Mitochondria as main source of ROS in autophagy 2 2 2 present in all cellular districts and act in concert with other signalling. Although the question is still far from being proteins, such as peroxiredoxins, thioredoxins (Trx) and solved, there are at least other two issues that deserve to be glutaredoxins (Grx), as well as low-molecular-weight anti- considered. The first is ‘where ROS are so rapidly produced’. oxidants (e.g., GSH, tocopherols and ascorbate) to fully It would be actually more logical that a stimulus coming from scavenge ROS and restore the reduced protein and lipid the outside of the cell is transduced by a ROS-producing 41–43 pool. The efficiency of the antioxidant defence is also system located at, or nearby, the plasma membrane, such as important to modulate the levels of RNS, a class of molecules the NADPH oxidase (NOX) complexes. Nevertheless, deriving from peroxynitrite (ONOO ), a very dangerous although attractive, this hypothesis has been verified only in .- compound generated by reaction between O and nitric oxide macrophages upon bacterial infection, where ROS generated 44,45 (NO). Nitric oxide is a highly reactive gaseous radical, by NOX2 are indispensable for the recruitment of the 45,46 soluble in water and diffusible across cell membranes. It is microtubule-associated protein light chain 3 (LC3) on Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al phagosomes that, thus modified, are degraded by autophagy mechanism is activated for the selective dismissal of impaired to prevent pathogen escape. A large amount of data, or dysfunctional mitochondria. It is responsive to mitochondrial instead, converge to state that the mitochondria represent depolarization and is regulated by the PTEN-induced putative the principal source of ROS required for autophagy kinase 1 (PINK1) and Parkin, a ubiquitin E3 ligase whose 56,57 induction, although they are not in close proximity to mutations have been associated with familial form of Parkin- the plasma membrane. A possible explanation for this son’s disease. PINK1 is a Ser/Thr kinase that translocates on unexpected evidence is that nutrient deprivation suddenly the outer mitochondrial membrane where it is stabilized by low results in energetic stress that, in turn, increases ATP mitochondrial transmembrane potential, thereby acting as real demand and causes mitochondrial overburden to face up sensor of mitochondrial depolarization. Here, PINK1 recruits adverse conditions. As a consequence, electron leakage and Parkin that ubiquitylates a number of outer mitochondrial ROS production also increase. Another hypothesis is that a membrane-located proteins, for example, the voltage- still uncharacterized factor could act as transducer, linking the dependent anion channel 1 (VDAC1). Once ubiquitylated, upstream autophagic signal with mitochondria. A good these proteins are recognized by p62/sequestosome 1 (p62/ 69,70 candidate could be HKII that, by sequestering mTORC1, SQSTM1, or simply p62), a ubiquitin-binding protein acting could loosen its inhibition on permeability transition pore as a scaffold for several protein aggregates and triggering their 34,35 (PTP) and its ability to decrease ROS. In support of this degradation through the proteasome, or the lysosome pathway 71,72 hypothesis, it has been reported that the two protein kinases via autophagy. p62 contains an LC3 interacting region positively regulating HKII activity, Akt and myotonic dystrophy (LIR) that has been indicated being fundamental for p62 to protein kinase (DMPK), are negative modulators of mediating mitophagy. Indeed, by means of this motif, p62 can 58,59 autophagy. bridge autophagy-targeted mitochondria to LC3 located on the autophagosomes surface, thereby driving their degradation. ROS and mitophagy. As principal sites of ROS production, Interestingly, our group has recently identified a role for mitochondria are the organelles that are able to turn on and Ambra1 in mitophagy, driven by its selective interaction with tune autophagy. However, upon chronic impairment of LC3 and independent from Parkin and p62. mitochondrial function, ROS can be generated at high extent, Redox signalling in autophagy. Another issue to be thus shifting their role from bulk autophagy inducers into a self-removal signal for mitochondria through a selective considered is ‘how oxidative stress can crosstalk with process called mitophagy. This represents a fine mechanism autophagic machinery’. As previously mentioned, antioxidant of negative feedback regulation by which autophagy elim- treatment prevents autophagy, suggesting that redox imbal- inates the source of oxidative stress and protects the cell from ance has a pivotal role in driving the process. The very fast oxidative damage (see below). induction of autophagy upon ROS production from mitochon- Although necessary, mitophagy represents an ‘extreme dria argues for a rapid (ON/OFF) response mediated by decision’ for a cell subjected to nutrient deprivation because of redox-sensitive proteins, among which AMPK could be a at least two main reasons. The first reason is that mitochondria good candidate. Indeed, AMPK has been proposed as being underpin ATP production that is fundamental upon carbon activated upon H O exposure, particularly through 2 2 source limitation. The second reason lies in the fact that S-glutathionylation (formation of a mixed disulphide with mitochondria are relatively large organelles that require being GSH) of reactive cysteines located at the α- (Cys and 304 30,75 beforehand fragmented in order to be properly recognized and Cys ) and β-subunits (still not identified) (Figure 2). engulfed within the autophagosomes. Both these issues Although the role of redox regulation in AMPK activation is contribute to explain why mitochondria are in general still a matter of debate, these results are in line with the recent refractory to undergo mitophagy, unless they are severely observations indicating that, once deprived of nutrients, cells damaged. Recently, it has been proposed that under nutrient actively extrude GSH by the drug efflux pump, multidrug deprivation, mitochondria attempt to protect themselves from resistance protein 1 (MRP1) in order to shift intracellular autophagic removal by promoting fusion and inhibiting fission redox environment towards more oxidizing conditions and 61,62 events. The combination of these two inputs results in prime redox-sensitive proteins to be oxidatively modified mitochondrial elongation that further impedes organelle (Figure 2). The evidence that the sole chemically induced engulfment within the autophagosomes and, concomitantly, oxidation of GSH is able to induce autophagy even in the 62 76 allows to maximize ATP production. Only upon prolonged absence of any autophagic stimulus underlines the starvation, mitochondria depolarize and become fragmented importance of thiol redox homeostasis in autophagy commit- in order to assist their removal by mitophagy. ment. This assumption is in line with the evidence indicating So far, at least two different molecular mechanisms under- that a number of proteins involved in both induction and lying mitophagy have been described and characterized. The execution of autophagy act by means of Cys residues. first one is mediated by NIX/Bnip3L (Bcl-2/adenovirus E1B Among them, the two ubiquitin-like systems Atg7-Atg3 and 63,64 19-kDa-interacting protein 3, long form), an atypical BH3 Atg7-Atg10, some members of Rab GTPase (e.g., Rab33b), protein that is able to directly recognize the autophagosome- and the phosphatase and tensin homologue deleted (PTEN) sited GABA receptor-associated protein (GABARAP), a func- are included. Along this line, it is worthwhile to note that p62 tional homologue of LC3 and, in turn, allow mitochondria to be contains a zinc-finger motif (ZZ) rich in cysteine residues that removed. This is a ‘programmed’ event, independent on any are necessary for metal binding and that could be redox damage response that is required, for example, in mitochondrial regulated. Although no evidence has been provided yet about 65,66 elimination during erythrocyte differentiation. The second a possible redox sensitivity of p62, it could be conceived that, Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al Conflicting role of NO and nitrosative stress in autoph- agy. Results emerging in the past 5 years suggest that NO, by means of S-nitrosylation mechanisms, has also a role in modulating autophagy. However, rather than a positive effector of the process, it seems that it could act as an inhibitory molecule. This assumption is completely in contrast with that described above for ROS, and contributes to making the functional relationship between oxidative stress and autop- hagy even more complex. Sarkar et al., indeed, demon- strated that treatment with NO donors or enhancement of NOS activity in HeLa cells results in autophagy prevention because of S-nitrosylation, and subsequent inhibition of the c-Jun-N- terminal kinase 1 (JNK1) and IκB kinase β (IKKβ) that regulate Beclin1 detachment from Bcl2 and AMPK activation, respec- tively. This is in line with results reporting that S-nitrosylation of TSC2 prevents its inhibitory activity on mTOR, thereby preventing autophagy and inducing proliferation of melanoma cells. However, these data are in contrast with the well- documented role of NO and nitrosative stress in the activation of AMPK–TSC2 pathway via Ataxia telangiectasia mutated (ATM) in response to DNA damage (see below). This discrepancy is likely because of the fact that the biological effects of NO range from prosurvival to apoptotic depending on the real concentrations used (reason why NO has been identified as Janus-faced molecule) that frequently are completely unknown (e.g., upon treatment with NO donors). In support to this, very recently it has been reported Figure 2 Crosstalk between autophagy and oxidative stress. Superoxide (O ) and H O are the main ROS produced by mitochondria upon nutrient that genetic ablation of the main denitrosylating enzyme, 2 2 2 deprivation. They positively regulate autophagy by means of at least three different S-nitrosoglutathione reductase (GSNOR) – which is the elective mechanisms, including: (1) S-glutathionylation (SH → S-SG) of cysteines located in experimental approach to study the effects of S-nitrosylation in the α and β subunits of AMPK (top right); (2) oxidation of Cys81 (SH → Sox) of Atg4 biological contexts – does not absolutely affect bulk autophagy that in turn leads to the inactivation of its ‘delipidating’ activity on LC3 and to the (Figure 3, unpublished results from Filomeni’s laboratory), but accumulation of the pro-autophagic LC3-II isoform (top centre); and (3) wide exclusively results in an impairment of the sole mitochondrial alteration of thiol redox state (e.g., decrease of GSH/GSSG ratio and general increase of oxidized thiols, Sox) that is facilitated by the release of reduced autophagy (mitophagy) in skeletal muscle models. glutathione (GSH) to the extracellular milieu through the multidrug resistance protein Altogether, these observations indicate that a straightfor- 1 (MRP1) (top left). In a redox-independent manner, it has also been demonstrated ward idea about how oxidative stress functions in autophagy is that p62, when bound to ubiquitylated protein aggregates, can undergo still lacking, despite abundant evidence corroborating its phosphorylation on Ser351, thereby sequestering Keap1 and leading to its implication in each phase of this process. detachment from Nrf2 (bottom left). Consequently, Nrf2 is no longer degraded by the ubiquitin-3 proteasome system, but translocates in the nucleus, binds to antioxidant-responsive elements (AREs) located in the promoter regions of p62/Keap1/Nrf2 system: how autophagy couples with antioxidant genes and activates their transcription (bottom right) redox response. In the past few years, autophagy and oxidative stress have been shown to be interconnected in a more intimate and coordinated maner than by a simple ON/ OFF signal. Particularly, in 2010, it was discovered that p62 activates the antioxidant transcription factor Nrf2 (nuclear similar to other ZZ-containing proteins, p62 could also factor erythroid 2-related factor 2) by a ‘non-canonical’ undergo oxidation and structural alterations that are able to pathway (Figure 2). The underlying mechanism is completely modify/regulate its role in autophagy. redox independent, and involves the recruitment of Kelch-like Notwithstanding the large amount of data supporting the ECH- associated protein 1 (Keap1) that functions as an hypothesis of a redox regulation of autophagic signalling, so adapter protein of the Cul3-ubiquitin E3 ligase complex far the only redox-based mechanism demonstrated to be able 82,83 responsible for degrading Nrf2. In agreement with this to regulate an autophagic protein goes back to 2007, when model, p62 binds to aggregates of ubiquitylated proteins and Scherz-Shouval et al. proved that H O -mediated oxidation 2 2 increases its affinity for Keap1 when phosphorylated at 351 84 of Atg4 at Cys was required for inactivating its hydrolyzing Ser (Figure 2). This event induces Keap1 degradation (delipidating) activity on LC3, thus allowing autophagosome to via autophagy and leaves Nrf2 free to accumulate and be correctly elongated (Figure 2). No further protein has translocate in the nucleus. Here, Nrf2 binds to the been added to the list since then, suggesting that redox antioxidant-responsive elements (ARE) in the promoter modifications of proteins – although reasonably proposed as regions of antioxidant and detoxifying genes, as well as likely modulators of autophagic signal transduction – are not genes involved in DNA damage response, such as the main mechanism linking ROS and autophagy. 8-oxoguanine glycosylase (OGG1) and p53 binding protein Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al Figure 3 Autophagy is not affected by S-nitrosylation. (a) Western blot analyses of S-nitrosothiols in total homogenates of skeletal muscles obtained from GSNOR-KO (KO) and wild-type (WT) mice, subjected to biotin-switch assay and revealed by HRP-conjugated streptavidin. Lactate dehydrogenase (LDH) was selected as loading control. Results show that even in the absence of any treatment with NO-delivering drugs, GSNOR ablation induces a significant increase of S-nitrosylated proteins. (b) Representative fluorescence microscopy images of satellite cell-derived myotubes isolated from KO and WT mice expressing LC3-conjugated green fluorescent protein (GFP-LC3) in heterozygosis. Cells were then subjected to two different autophagic stimuli: (1) they were treated for 6 h with 5 μM of the proteasome inhibitor MG132 or, alternatively, (2) allowed to grow for 6 h in a nutrient-deprived cell medium (stv). Images are representative of three independent experiments that gave similar results. Both genotypes displayed a significant and similar increase of fluorescent dots, plausibly representing autophagosomes, thereby indicating that autophagy is not impaired by S-nitrosylation 88 94,95 1 (53BP1), inducing their transcription (Figure 2). It has also damaged DNA. In particular, this phenomenon has been been suggested that Nrf2 activation by this pathway is observed in nuclear envelopathies where the presence of sustained by Sestrins, a highly conserved family of small perinuclear autophagosomes or autophagolysosomes con- ‘antioxidant-like’ proteins transcriptionally induced by p53 taining DNA clearly represented an operative autophagy. It upon stressful conditions, which are involved in autophagy has been reported that micronuclei containing chromosomes, since function as AMPK activators and mTORC1 inhibi- or parts of them, that are not properly incorporated in the 90,91 tors. daughter nuclei during cell division can be removed by autophagy as well, thus providing this process with a direct role in cleaning up damaged content in the nucleus and in Antioxidant Role of Autophagy: Focus on Nucleus and maintaining genomic stability. DNA Damage However, besides PMN, a number of observations indicate On the basis of what has been reported so far, antioxidant that autophagy is deeply involved in DNA damage repair, response and autophagy are mechanisms simultaneously although without any direct degradative activity on DNA. This induced by oxidative stress conditions in order to concomi- phenomenon, mainly occurring upon ROS and RNS-mediated tantly decrease ROS and RNS concentration (upstream damage to DNA, represents an issue that deserves discussion causes) and reduce the oxidative damage to biomolecules so as to comprehend how autophagy acts as a preventive and and organelles (downstream effect). This finely orchestrated reparative process upon genotoxic stress. Understanding the repair system perfectly fits the needs of a cell attempting to find underlying molecular mediators and the mechanisms would a new homeostatic state. By responding very rapidly to make it possible to clarify once and for all the antioxidant oxidative stress, and by decreasing the toxicity of oxidized activity of autophagy. molecules and organelles through their selective removal, autophagy can be in principle encompassed in the large family Oxidative Stress and DNA Damage of antioxidant processes. However, at variance with proteins and organelles that are present in several copies inside the cell ROS and RNS are one of the major sources of DNA damage (e.g., mitochondria and ribosomes), autophagy cannot med- as they could directly modify the DNA or indirectly generate iate nucleus degradation, even though it is severely damaged, different lesions, both affecting cell viability. Among ROS, because this could lead to the complete loss of genetic OH can directly attack the DNA backbone by generating five information. Genomic DNA cannot be destroyed, de novo classes of oxidative damage: oxidized bases, abasic sites, synthesised or entirely replaced like the other biomolecules. DNA–DNA intrastrand adducts, single-strand break (SSB), 97,98 Its integrity should be prevented and maintained, and any double-strand break (DSB) and DNA–protein crosslinks. damage accurately repaired. Among the nucleobases, guanine is the most susceptible to An exception to the rule is a highly selective and unusual ROS because of its low redox potential, and the main products nuclear DNA degradation by means of the so-called piece- of its oxidation are 8-hydroxyguanine and 8-hydro- meal microautophagy (PMN). Indeed, in a way resembling xydeoxyguanosine (8-OHG and 8-OHdG). Both are highly 92,93 nucleophagy occurring in fungi and nematodes, mamma- mutagenic and carcinogenic as they can match with lian cells can specifically remove part of nuclei containing both cytosine and adenine, thus leading to GC-to-AT Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al 99,100 transversions. The 8-OHG and 8-OHdG are the most genotoxic stress. However, how this occurs is still a matter of commonly used markers of DNA damage as they easily debate. In yeast, for example, it has been demonstrated the accumulate, thus being measured as good index of oxidative selective degradation of specific proteins, mostly through the · - 117 lesions to DNA. Nitric oxide and RNS (i.e., NO , ONOO ,N O so-called ‘cytoplasm to vacuole targeting’ (Cvt), is directly 2 2 3 and HNO ) can cause DNA damage as well, and are involved in: (1) inducing cell cycle arrest in G2/M phase by considered potentially mutagenic as they can induce degrading proteins involved in cell cycle progression (e.g., nitration, nitrosylation and deamination of DNA bases. Psd1 and Esp1); (2) optimizing dNTP production and DNA ROS and RNS are also harmful for mitochondrial DNA synthesis by targeting the subunit 1 of the ribonucleotide (mtDNA) integrity. This feature can deeply affect the transcrip- reductase complex; and (3) regulating the dynamics of tion of mtDNA-coded proteins and RNAs that underlie the homologous recombination by degrading the endonuclease synthesis of a number of subunits belonging to the complexes Sae2 once catalysed the resection of DNA ends. of the mitochondrial respiratory chain (except Complex II). A In higher eukaryotes, no Cvt has ever been identified, nor vicious cycle is then established in which mitochondria, with has any orthologue of proteins belonging to this pathway been oxidized mtDNA, become dysfunctional and produce a high revealed. One of the most accredited hypotheses explaining a rate of ROS, leading to further mitochondrial impairment. This role of autophagy in supporting the DDR is that by degrading condition can ultimately result in severe nuclear DNA damage damaged mitochondria (mitophagy) and toxic aggregates, and cell death. autophagy eliminates putative sources of ROS, reduces their levels and, if only indirectly, decreases DNA damage 114,121,122 accumulation. This assumption provides a general DNA Damage and Autophagy: A Complex Crosstalk explanation of the important role of autophagy in maintaining When the DNA is damaged by ROS and RNS, cells activate a genomic stability that is strictly related to its tumour suppressor 106,113,114 number of pathways in order to maintain genomic integrity, function. However, evidence supporting a direct role these being associated to the DNA damage response (DDR). for autophagy in the DDR, at least in mammals, is still lacking. Different classes of proteins are implicated in DDR, among which the sensors specifically recognize the lesions to DNA, Sensor proteins transduce the signal of DNA damage to whereas the mediators and the effectors transduce the signal autophagy. A number of works in recent years indicate that from the nucleus to the cytosol where several processes are once ROS and RNS damage the DNA, the event is contextually activated in order to better face up to adverse transduced in order to activate the DDR, and concomitantly 102,103 conditions. For instance, cell cycle checkpoints are is signalled to the autophagic pathway in order to orchestrate soon activated to block proliferation until lesions are repaired. the response. PolyADP-ribose polymerase 1 (PARP1) is However, if DNA is severely damaged or unrepaired, cells among the proteins directly linking the DDR and autophagy 123,124 remain quiescent or undergo cell death. Autophagy is (Figure 4). It is a nuclear enzyme that catalyses poly- considered both a pro-survival mechanism and a type of cell ribosylation of nuclear proteins by converting NAD into death. Therefore, once induced by DNA injury, it makes a polymers of polyADP-ribose (PAR), and deeply participates in 105,106 crucial contribution in regulating cell fate. For example, SSB repair, thereby regulating nuclear homeostasis. PARP1 many lines of evidence argue that autophagy can delay is hyperactivated upon ROS-induced DNA damage; this apoptotic cell death upon DNA damage by sustaining the leads to NAD consumption and ATP depletion (Figure 4). energy demand required to support DNA repair Such energetic imbalance results in the activation of 107,108 123,124 processes, a phenomenon that concurs to the develop- autophagy via AMPK pathway (Figure 4) in order to ment of chemoresistance mechanisms in cancer cells. recycle metabolic precursors for ATP and to provide energy Conversely, in cells where DNA is unrepaired and apoptosis is for the DDR. defective, DNA damage-induced autophagy has been Another DNA repair protein linking the DDR to autophagy is reported to contribute to cell death, thereby acting as a tumour ATM (Figure 4), a DNA damage sensor orchestrating the cell suppressor process. cycle with damage response checkpoints and DNA repair to It is worthwhile noting that cases where autophagy safeguard the integrity of the DNA. It has been demon- impairment results in DNA damage have been reported as strated that under ROS-induced cellular damage, cytosolic well, leading to the assumption that the interplay might be ATM, through the LKB1/AMPK pathway, can activate TSC2 broader, and suggesting that many molecular players could tumour suppressor to inhibit mTORC1 and induce autophagy exist to biunivocally link the two processes. In particular, it has (Figure 4). These new findings integrate different stress been demonstrated that the deficiency of autophagy genes, response pathways occurring in different cellular compart- such as Beclin 1, UV irradiation resistance-associated gene ments. From this perspective, ATM would be required to both (UVRAG), Atg5 and Atg7, leads to DNA damage accumulation initiate (nucleus) and mediate (cytosol) the DDR. 111–115 and promotes tumourigenesis. In line with this, the The principal regulator of DDR, however, remains p53 that, suppression of the ULK1-interacting protein FIP200 has been together with the other members of its family, p63 and p73, has reported to impair DDR, thus triggering cell death upon been demonstrated to modulate many autophagic genes 116 105,127–129 ionizing radiation-induced oxidative stress. (Figure 4). In particular, p53 is very rapidly activated when DNA damage occurs, and transcriptionally activates Direct and indirect roles of autophagy in the DDR. All this both DNA repair and cell cycle checkpoints proteins to allow evidence strongly suggests that autophagy participates, repair of DNA lesions. It has been recently demonstrated directly or indirectly, in the DDR to ROS and RNS-mediated that the strength of the stimulus and the dynamics of the Cell Death and Differentiation Oxidative stress and autophagy interplay G Filomeni et al the signal (e.g., Bnip3 and AMPK) – are shared between autophagy and apoptosis. How can they be differently induced? Which are the signals, or how much high should be the threshold level to allow cell response being switched from stress adaptation and survival (autophagy) to cell dismantling (apoptosis)? These issues are still debated. Speculations and hypotheses based on the ‘strength’ of the signal have been reasonably done, but no direct evidence of the causative event underlying the decision by the cell to activate autophagy or apoptosis has been provided as yet. Concluding Remarks Several lines of evidence indicate that ROS and RNS are the upstream modulators of autophagy, likely acting at multiple levels in the process. In line with this assumption, ROS and RNS would act as the intracellular ‘alarm molecules’ of the extracellular availability of nutrients (primitive stimuli) by signalling their presence to the autophagic machinery. Through a negative feedback regulation, autophagy could be then induced to provide energy and building blocks in order to restore homeostasis and, concomitantly, remove oxidative damage. From this perspective, autophagy is required for Figure 4 Implication of autophagy in DNA damage repair. Endogenous the cell to simultaneously overcome starvation and oxidative (e.g., dysfunctional mitochondria, top right) or exogenous (e.g., radiations or stress conditions. Therefore, any dysfunction in this regard genotoxic stimuli, bottom left) sources of ROS and RNS induce DNA damage, whose has been found to be involved in the onset of pathological primary sensors are PARP1 and ATM. Once activated by DNA breaks, PARP1 states where a primary role for oxidative stress and/or catalyses poly-ADP ribosylation of itself, as well as of other nuclear proteins, thereby + alterations in metabolic demand (such as cancer) has also leading to a massive decrease of NAD and to a subsequent energetic stress (bottom been reported. Accordingly, genetic defects of autophagic left). Upon DNA damage, ATM can activate p53-mediated transcription of autophagic genes (bottom right). Alternatively, cytosolic pool of ATM could be directly activated by genes lead to an increased production of ROS and accumula- ROS through a still unidentified mechanism and it directly induces the activation of tion of damaged organelles and DNA that in turn promote LKB1 (centre). The issue of whether cytosolic and nuclear pool of ATM are metabolic reprogramming and induce tumourigenesis. interconnected still waits to be demonstrated. Both PARP1 and ATM signalling Although a number of possible mechanisms underlying the pathways converge on AMPK, whose activation induces the autophagic machinery to intimate interplay between oxidative stress and autophagy remove the main source of DNA damage and contribute to its repair through a have been postulated, to date only a few of them have been negative feedback loop (top) shown to have a role in tuning autophagy. Understanding the binding to DNA underlies the genes transactivated by p53. For fine molecular regulation of autophagy by ROS and RNS, as instance, in case of inefficient repair, p53 can shift transcrip- well as the tight relationship between metabolism and redox waf tion from cell cycle modulators (e.g., p21 ) to genes state, could therefore provide valuable information that could regulating cell senescence and apoptosis (e.g., PUMA and be useful in the future to improve anticancer treatment and BAX). In addition, autophagy genes are also induced and develop new selective therapies. their transcription finely orchestrated by p53 (Figure 4). Targets of p53 are, indeed, both upstream regulators of Acknowledgements. We thank M Acuña Villa and MW Bennett for editorial and autophagy (e.g,. PTEN, TSC2, β1, β2 and γ subunits of AMPK) secretarial work. We are also grateful to Costanza Montagna and Giuseppina and proteins directly involved in autophagosome formation Di Giacomo for having provided part of unpublished results obtained on GSNOR-KO (e.g., ULK1, UVRAG, ATG2, 4, 7, 10). It is worth noting that mice. The Unit of Cell Stress and Survival is supported by grants from the Danish p63 and p73 also share some autophagic genes as transcrip- Cancer Society (KBVU R72-A4408 to FC and KBVU R72-A4647 to GF); Lundbeck Foundation (nn. R167-2013-16100) and NovoNordisk (nn. 7559). 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Oxidative stress and autophagy: the clash between damage and metabolic needs

Oxidative stress and autophagy: the clash between damage and metabolic needs

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Oxidative stress and autophagy: the clash between damage and metabolic needs

Oxidative stress and autophagy: the clash between damage and metabolic needs

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