Redox homeostasis, oxidative stress and mitophagy

Journal Pre-proofs Redox Homeostasis, Oxidative Stress and Mitophagy Carla Garza-Lombó, Aglaia Pappa, Mihalis I. Panayiotidis, Rodrigo Franco PII: DOI: Reference:

S1567-7249(19)30230-2 https://doi.org/10.1016/j.mito.2020.01.002 MITOCH 1438

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Mitochondrion

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31 August 2019 21 December 2019 3 January 2020

Please cite this article as: Garza-Lombó, C., Pappa, A., Panayiotidis, M.I., Franco, R., Redox Homeostasis, Oxidative Stress and Mitophagy, Mitochondrion (2020), doi: https://doi.org/10.1016/j.mito.2020.01.002

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Redox Homeostasis, Oxidative Stress and Mitophagy Carla Garza-Lombó 1, Aglaia Pappa 2, Mihalis I. Panayiotidis 3 and Rodrigo Franco 1, 

1 Redox

Biology Center and School of Veterinary Medicine and Biomedical Sciences. University

of Nebraska-Lincoln, Lincoln, NE 68583. [email protected]. 2

3

Department of Applied Sciences, Northumbria University, Newcastle Upon Tyne, NE1 8ST, UK

: Rodrigo Franco. Redox Biology Center and School of Veterinary Medicine and Biomedical Sciences. 114 VBS 0905. University of Nebraska-Lincoln, Lincoln, NE 68583. Tel: 402-472-8547. Fax: 402-472-9690. Email: [email protected]. Running head: Mitophagy and redox homeostasis Keywords: autophagy, mitochondrial dynamics, fission, fusion

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Abstract Autophagy is a ubiquitous homeostatic mechanism for the degradation or turnover of cellular components. Degradation of mitochondria via autophagy (mitophagy) is involved in a number of physiological processes including cellular homeostasis, differentiation and aging. Upon stress or injury, mitophagy prevents the accumulation of damaged mitochondria and the increased steady state levels of reactive oxygen species leading to oxidative stress and cell death. A number of human diseases, particularly neurodegenerative disorders, have been linked to the dysregulation of mitophagy. In this mini-review, we aimed to review the molecular mechanisms involved in the regulation of mitophagy and their relationship with redox signaling and oxidative stress.

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1. Introduction Autophagy is a homeostatic process in which double–membraned autophagosomes engulf cellular components to be subsequently degraded upon fusion with lysosomes (Figure 1). Autophagosome cargo degradation preserves cellular homeostasis and viability via the turnover of damaged organelles and biomolecules whose prevalence or accumulation within cells can lead to deleterious effects. Autophagy is a persistent homeostatic mechanism; almost all types of cells have basal levels of autophagy. Alterations in the autophagic cycle rate (flux), which begins with the formation of the phagophore and ends with the degradation of autophagosome cargo after its fusion with lysosome (Figure 1), are commonly observed in response to stress (Galluzzi et al., 2017; Mizushima, 2018). In most cases, induction of autophagy in response to stress acts as a pro-survival mechanism, but several examples have been reported where autophagy mediates cell death (Doherty and Baehrecke, 2018). There are three major types of autophagy, macroautophagy, microautophagy, and chaperonemediated autophagy (CMA). In microautophagy, the targeted cargo is directly sequestered and subsequently engulfed by lysosomes. The formation of the lysosomal wrapping process starts with the direct engulfment of cytoplasmic components by pre-existing primary or secondary lysosomes followed by the opposition of the extension’s ends leading to the sealing of the sequestered materials. Lysosomal enzymes are proposed to directly access the cargo by degeneration of the inner membrane (Galluzzi et al., 2017; Mijaljica et al., 2011; Mizushima, 2018). CMA specializes in breaking down cytosolic proteins identified by a chaperone that delivers them to the surface of the lysosomes, where substrate proteins unfold and cross the lysosomal membrane. CMA is mediated by the presence of an intrinsic CMA target sequence but motifs can also be generated through post-translational modifications. About 30% of soluble cytosolic proteins contain a putative CMA-targeting motif. These motifs are recognized specifically by the cytosolic protein Hsc70 (heat shock cognate protein of 70 kDa). Chaperone-

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targeted proteins bind to the lysosomal membrane via interaction with the lysosome-associated membrane protein type 2A (LAMP-2A) (Kaushik and Cuervo, 2018). Macroautophagy, herein referred as autophagy, is the most understood form of autophagy. It is responsible for the breakdown of proteins and organelles in the cell, and it is considered necessary for cell survival. Autophagy starts with the formation of a phagophore, generated de novo from pre-existing intracellular precursor molecules or multiple sources, which matures into a double-membrane autophagosome (Figure 1). The autophagosome then fuses with a lysosome, forming an autolysosome where cellular cargo is degraded by lysosomal hydrolases (Galluzzi et al., 2017; Mizushima, 2018). Non-selective autophagy is involved in bulk degradation upon stress. On the other hand, several forms of selective autophagy have been identified to participate in organelle turnover such as endoplasmic reticulum (ER)-phagy, pexophagy (peroxisomes), mitophagy (mitochondria), and ribophagy (ribosomes). Selective autophagy is not limited to degradation of organelles. Lipid (lipophagy), glycogen and pathogen (xenophagy) degradation via autophagy has been described as well (Farre and Subramani, 2016; Gatica et al., 2018; Khaminets et al., 2016). A search for mitophagy research in regard to any brain-related study in NCBI/PubMed will give >500 manuscripts published within the last 12 years, which highlights the increase interest in understanding the physio-pathological importance of this homeostatic mechanism in brainrelated biomedical research. Excellent recent reviews have been written in regards to the generalities of mitophagy (Palikaras et al., 2018), and its role in neurodegenerative diseases (Evans and Holzbaur, 2019; Kerr et al., 2017; Ryan et al., 2015) and neuronal injury (Guan et al., 2018). Alterations in redox homeostasis are central to the etiology of brain diseases. In this work, we aim to provide an update in regards to the relationship between redox balance and mitophagy.

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2. Autophagy machinery and signaling Autophagy initiation starts with activation of the autophagy-related protein (Atg) 1/unc-51-like kinase-1 (ULK1) complex, which includes the Atg scaffolds Atg13, Atg101, and the retinoblastoma-associated protein (RB1)-inducible coiled-coil protein 1 (RB1CC1 or focal adhesion kinase family kinase-interacting protein of 200 kDa, FIP200) (Corona Velazquez and Jackson, 2018) (Figure 1.1). While there are 4 isoforms of ULK in humans, ULK1 and ULK2 are the most important ones for autophagy initiation (Lin and Hurley, 2016). ULK1 is activated by autophosphorylation and phosphorylates further ULK1 complex subunits. Growth factors and amino acids stimulate the mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) via the class I phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) pathway and Rag guanosine triphosphatases (GTPases), which negatively regulate the ULK1 complex via phosphorylation of ULK1 and Atg13. Thus, withdrawal of growth factors or amino acids induce autophagy by inhibition of mTORC1 (Hurley and Young, 2017; Kundu, 2011; Mizushima, 2010) (Figure 1.1). In addition, ATP depletion upregulates autophagy by activation of the adenosine monophosphate (AMP)–activated protein kinase (AMPK), which phosphorylates ULK1 at multiple sites antagonizing autophagy inhibition by mTORC1 (Figure 1.1). Importantly, ULK1 and/or ULK2 have been reported to be dispensable for autophagy under certain conditions (Cheong et al., 2011; Corona Velazquez et al., 2018; Feng et al., 2019; Gammoh et al., 2013). Nucleation is mediated by the class III PI3K (PIK3C3 or vacuolar protein sorting 34, Vps34) complex 1 (PIK3C3–C1) that includes the B-cell lymphoma 2 (Bcl-2)-interacting myosin/moesinlike coiled-coil protein 1 (Beclin 1) and Vps15 (also known as phosphoinositide 3-kinase regulatory subunit 4, PI3KR4, or p150). PIK3C3–C1 also includes the Atg14-like protein (Atg14L, or Beclin 1-associated autophagy-related key regulator Barkor), the autophagy and beclin 1 regulator 1 (Ambra1) and the nuclear receptor binding factor 2 (NRBF2) (Figure 1.2), whereas PIK3C3-C2 contains the UV radiation resistance-associated gene protein (UVRAG)

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(Baskaran et al., 2014; Dou et al., 2010; Liang et al., 2007; Morris et al., 2015). PIK3C3-C1 is essential for autophagy. In contrast, PIK3C3-C2 is involved in multiple cellular processes including autophagy, endocytic trafficking, cytokinesis and Golgi-ER retrograde transport. ULK1 phosphorylates the PIK3C3-C1 complex activating PIK3C3 that in turn generates phosphatidylinositol-3-phosphate (PIP3) (Hurley and Young, 2017; Stjepanovic et al., 2017). PIP3 recruits effectors such as the double Fab 1-YOTB-Vac 1-early endosome antigen 1 (EEA1) (FYVE) domain-containing protein 1 (DFCP1 or ZFYVE1) and tryptophan (W)-aspartic acid (D)repeat protein interacting with phosphoinositides (WIPI) family proteins to mediate the initial stages of autophagosome formation including the recruitment of Atg16L and consequently, the Atg5-Atg12 conjugation system to a specific subcellular location termed the phagophore assembly site (PAS) (Proikas-Cezanne et al., 2015). In addition, Atg9-containing vesicles generated by the secretory pathway are recruited to the PAS for the delivery of additional lipids and proteins contributing to membrane expansion (Noda, 2017). The binding of the anti-apoptotic proteins Bcl-2, Bcl-extra large (Bcl-xl) or myeloid cell leukemia sequence-1 (Mcl-1) to Beclin 1 inhibit PIK3C3 activity and autophagy, and this is negatively regulated by death-associated protein kinase (DAPK)-dependent Beclin 1 phosphorylation. Beclin 1 has also been reported to be phosphorylated by mitogen-activated protein kinaseactivated protein kinase (MAPKAPK) 2/3 and AMPK resulting in autophagy induction (Hurley and Young, 2017). In contrast, phosphorylation of Beclin 1 by the epidermal growth factor receptor (EGFR) and Akt antagonize autophagy (Menon and Dhamija, 2018). PIK3C3–C1 is also negatively regulated by mTORC1 via Atg14L phosphorylation (Hurley and Young, 2017). Non-canonical, Beclin 1-independent autophagy has been reported as well (Chu et al., 2007; Codogno et al., 2012). Elongation of the autophagosome requires the ubiquitin (Ub)-like conjugation systems Atg5– Atg12 and the Atg8 family proteins, which include the microtubule-associated light chain 3 (LC3)

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protein subfamily (LC3A, LC3B, LC3C formed by different post-translational modifications), and the γ-aminobutyric acid receptor-associated protein (GABARAP) subfamily (GABARAP, GABARAPL1, GATE-16/GABARAPL2) (Schaaf et al., 2016) (Figure 1.3). The covalent conjugation of Atg12 to Atg5 occurs via the E1-like enzyme Atg7 and the E2-like enzyme Atg10 and is organized into a complex by a non-covalent association with Atg16 (Figure 1.3). This complex is essential for the elongation of the pre-autophagosomal membrane but dissociates from fully formed autophagosomes. The Atg12-Atg5-Atg16 complex can function as the E3 ligase for LC3 (He et al., 2003). The conjugation of phosphatidylethanolamine (PE) to soluble LC3 (LC3-I) is mediated by the sequential action of the protease Atg4, the E1-like enzyme Atg7, and the E2-like enzyme Atg3. The autophagic vesicle-associated form or lipidated form of LC3 (LC3-II) is specifically targeted to the elongating autophagosome and remains on autophagosomes until their fusion with lysosomes (Xie et al., 2015) (Figure 1.3). LC3-II localized at the cytoplasmic face of autolysosomes is delipidated by Atg4 and recycled, while LC3-II found on the internal surface of autophagosomes is degraded in the autolysosomes (Kroemer et al., 2010). From the four distinct Atg4 isoforms, Atg4B recognizes all LC3 proteins, while Atg4A is more specific to GABARAPs (Skytte Rasmussen et al., 2017). Mouse cells lacking Atg5 or Atg7 can still form autophagosomes/autolysosomes, which do not correlate with LC3-II accumulation, instead, autophagy occurs in a Rab9-dependent manner by fusion of phagophores with trans-Golgi derived vesicles and late endosomes (Arakawa et al., 2017; Nishida et al., 2009). Interestingly, the metabolic switch from oxidative phosphorylation to glycolysis required for stem cell reprogramming is mediated by an Atg5-independent mitophagy process (Ma et al., 2015). Autophagosome maturation involves the sealing of the double membrane vesicle which is catalyzed by the endosomal sorting complexes required for transport (ESCRT) (Lefebvre et al., 2018). Autophagosomes move along microtubules, which require the function of dynein motor

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proteins (Figure 1.4). Depolymerization of microtubules or inhibition of dynein-dependent transport result in inhibition of autophagy. LC3-II localized on the cytoplasmic face of autophagosomes links them to kinesin adaptors such as the FYVE and coiled-coil domaincontaining protein (FYCO1) (Cheng et al., 2016). Fusion of an autophagosome with a lysosome forms an autolysosome where engulfed cargo is digested by acidic hydrolases (Figure 1.4). Phosphorylation of the transcription factor EB (TFEB) is another mechanism for the regulation of lysosomal biogenesis and autophagy by mTORC1. Phosphorylated TFEB is retained in the cytoplasm, whereas dephosphorylated TFEB translocates to the nucleus to induce gene transcription (Napolitano and Ballabio, 2016). The autophagosome can also fuse with endosomes generating an intermediate vesicle called amphisome, which then fuses with a lysosome (Hyttinen et al., 2013). The fusion of autophagosomes with lysosomes is regulated by: 1) rat sarcoma viral oncogene homolog (Ras)associated binding proteins (small Rab GTPases Rab7 and Rab11); 2) soluble Nethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the autophagosome (syntaxin-17 [STX17], synaptosomal-associated protein 29 [SNAP-29]) and in the lysosome (vesicle-associated membrane protein 8 [VAMP8]); and 3) the homotypic fusion and vacuole protein sorting (HOPS) complex (Vps16, Vps33A, and Vps39) that is recruited to autophagosomes via the multivalent adaptor pleckstrin homology and RUN domain containing M1 (PLEKHM1) protein to mediate membrane tethering to allow SNARE-mediated fusion (Amaya et al., 2015; Marwaha et al., 2017; Rawet-Slobodkin and Elazar, 2015). 3. Mitochondria dynamics and mitophagy Mitochondria are dynamic organelles that undergo continuous events of biogenesis, remodeling and turnover. Fusion and fission are opposing processes working in concert to maintain the shape, size, number of mitochondria and their physiological function (Figure 2). Fusion enables

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content to be mixed between neighboring mitochondria and has been proposed to rescue “moderately” dysfunctional mitochondria (Dorn, 2019; Whitley et al., 2019). Fusion is mediated by oligomerization of dynamin GTPases mitofusins 1 (Mfn1) and 2 (Mfn2) at the outer membrane to tether adjacent mitochondria together (Schrepfer and Scorrano, 2016), and the subsequent fusion of the inner membranes by the optic atrophy GTPase (Opa1) (Song et al., 2009) (Figure 2.1). Fission represents a quality control mechanism to transform damaged elongated mitochondria into a form suitable for engulfment by mitophagy (Dorn, 2019; Whitley et al., 2019) (Figure 2.2). Fission requires recruitment of the dynamin-related protein 1 GTPase (Drp1) (Bleazard et al., 1999) via mitochondrial surface receptors (mitochondrial fission 1 protein [Fis1], mitochondrial fission factor [Mff] and mitochondrial dynamics proteins 49 and 51 [MiD49, MiD51]) for assembly of the fission machinery required for membrane scission (Loson et al., 2013) (Figure 2.2). AMPK phosphorylates Mff and activates mitochondrial fission in response to energy stress (Toyama et al., 2016) (Figure 2.1). Interestingly, Fis1 has been reported to promote mitochondrial fragmentation independent from Drp1, via impairment of the fusion machinery (Yu et al., 2019). Both Ub-dependent and independent pathways exist for the recognition and deliver of mitochondria to the autophagosome. Mitophagy is regulated by the phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) and the E3-Ub ligase Parkin (PARK2) whose mutations are associated with Parkinson’s disease (PD) (Harper et al., 2018). PINK1 is constitutively transported into the inner mitochondrial membrane (IMM), where it is cleaved by proteases and degraded by the Ub-proteasome system (Liu et al., 2017b; Yamano and Youle, 2013). Upon loss of mitochondrial membrane potential (ΔΨm), PINK1 is stabilized and autophosphorylated on the outer mitochondrial membrane (OMM) (Okatsu et al., 2012) (Figure 2.3). PINK1 phosphorylates a number of targets that facilitate mitophagy (Palikaras et al., 2018; Pickrell and Youle, 2015) (Figure 2.4). Parkin-phosphorylation by PINK1 promotes its

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translocation to the surface of mitochondria where it becomes activated and ubiquitinates mitochondrial substrates including Mfn1 and Mfn2 which are also phosphorylated by PINK1, abolishing mitochondrial fusion (Chen and Dorn, 2013). PINK1 and Parkin also phosphorylate and ubiquitinate the OMM mitochondrial Rho–GTPase (Miro) disrupting its interaction with the adapter protein Milton and the kinesin motor protein KIF5a. The loss of Miro and Milton/Kinesin Family Member 5A (KIF5a) interaction leads to the detachment of mitochondria from microtubules (Kazlauskaite and Muqit, 2015; Liu et al., 2012b; Wang et al., 2011) (Figure 2.6). Phosphorylation of Ub by PINK1 also facilitates Parkin activation (Ordureau et al., 2014; Sauve et al., 2015) (Figure 2.4), which seems to prevent the deubiquitination of mitochondria by Ubspecific proteases (USP) (Bingol et al., 2014; Cornelissen et al., 2014; Cunningham et al., 2015; Durcan et al., 2014). Other targets for Parkin-mediated ubiquitination include the voltagedependent anion channel-1 (VDAC1) (Novak, 2012) and the vacuole membrane protein 1 (VMP1) (Kroemer et al., 2010). Importantly, recent reports have demonstrated that basal mitophagy in neurons is independent of PINK1/Parkin (Lee et al., 2018; McWilliams et al., 2018) and that only prolonged (chronic) mitochondrial depolarization induces Parkin translocation or mitophagy in neurons (Lee et al., 2015; Van Laar et al., 2011). In contrast, a recent report demonstrated that in Drosophila the age-dependent increase in mitophagy in both muscle and dopaminergic neurons is dependent on PINK1/Parkin, and the knockdown of USP15 and USP30 rescues mitophagy in Parkin deficient organisms (Cornelissen et al., 2018). However, mitophagy induced by dysfunction in mitochondrial respiration is independent from Parkin in dopaminergic neurons (Sterky et al., 2011). PINK1/Parkin-dependent mitophagy protects against neuronal cell death-induced by experimental conditions linked to neurodegeneration or injury (Fang et al., 2019; Khalil et al., 2015; Shen et al., 2017), but contradictory findings exist in regards to the sensitization of PINK1/Parkin knockout models to experimental PD insults (Aguiar et al., 2013; Haque et al., 2012).

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Parkin and PINK1 have also been demonstrated to mediate a mitophagy-independent mechanism of mitochondria turnover via the formation of mitochondria-derived vesicles for the degradation of oxidized and damaged proteins via the lysosome (McLelland et al., 2014). It is important to consider that in addition to Parkin, other E3-Ub ligases have been reported to regulate mitophagy including the SMAD specific E3-Ub protein ligase 1 (SMURF1) (Kuang et al., 2013), the seven in absentia homolog 1 (SIAH1) (Szargel et al., 2016), the mitochondrial E3Ub protein ligase 1 MUL1 and the ariadne RBR E3-Ub protein ligase 1 (ARIH1) (Villa et al., 2017). Mitochondrial receptors facilitate the binding of mitochondria to LC3‐II allowing their engulfment by autophagosomes. The recognition of ubiquitinated mitochondria is mediated by receptors containing a Ub-binding domain and the LC3-interacting region motif (LIR or GABARAPinteracting motifs, GIMs), a Wxxleucine (L) tetrapeptide motif found in autophagic cargo recognition receptors. The receptors optineurin (OPTN) and the nuclear dot protein 52 (NDP52) mediate PINK1-dependent mitophagy (Padman et al., 2019) (Figure 2.4). On the other hand, p62/SQSTM1 (sequestrome 1) seems to be dispensable for the execution of mitophagy (Lazarou et al., 2015). Optineurin, NDP52 and p62 are phosphorylated by different kinases including the tumor necrosis receptor (TNF)-associated factor (TRAF) family memberassociated nuclear factor (NF)-kappa-light-chain-enhancer of activated B cells-activator (TANK)binding kinase 1 (TBK1), ULK1 and AMPK, but its exact role in mitophagy is unclear (Heo et al., 2015). The Ub-independent mitophagy pathway involves transmembrane receptors that interact with LC3 in autophagosomes via the LIR motif (Figure 2.5). During hypoxic conditions, transcriptional regulation of the Bcl-2 interacting protein 3 (Bnip3), its homolog Nip3-like protein X (Nix or Bnip3‐like[L]), or FUN14 domain containing 1 (FUNDC1) by the hypoxia-inducible factor-1 alpha

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(HIF1α) mediates mitophagy (Hanna et al., 2012; Jung et al., 2019; Liu et al., 2012a; Zhang et al., 2008). Furthermore, mitophagy-mediated by these mitochondrial receptors protects against ischemic injury in brain, kidney and heart tissues (Tang et al., 2019; Yuan et al., 2017; Zhang et al., 2016b; Zhang et al., 2017). Similarly, in cancer, Nix-dependent mitophagy protects glioblastoma cells against hypoxic conditions in the tumor microenvironment (Jung et al., 2019). Nix also promotes mitochondrial renewal when oxidative phosphorylation is stimulated (Melser et al., 2013), and during cell differentiation, Nix-dependent mitophagy promotes the metabolic switch to glycolysis (Esteban-Martinez and Boya, 2018). The Nix/Bnip3 homologs in Caenorhabditis elegans (DCT-1) and Drosophila melanogaster (Bnip3) have also been reported to regulate aging (Palikaras et al., 2015) and the turnover of dysfunctional mitochondria with mutated genomes (Lieber et al., 2019), respectively. Mitophagy is also important in maintaining overall tissue homeostasis and metabolism. Accordingly, Nix has been shown to regulate mitochondrial mass and lipid metabolism in the liver (Glick et al., 2012), while FUNDC1 and Bnip3 regulate mitochondria quality and metabolism in the muscle and adipose tissues (Fu et al., 2018; Wu et al., 2019). Nix and FUNDC1 also regulate mitochondrial remodeling during cardiac progenitor cell differentiation (Lampert et al., 2019). A crosstalk between the regulation of mitophagy by mitochondrial receptors and the PINK1Parkin-dependent pathway has also been established where Bnip3 and Nix promote PINK1 stabilization (Zhang et al., 2016a) and Parkin recruitment (Ding et al., 2010), respectively. In addition, Nix has been shown to be a target for ubiquitination by Parkin (Guo et al., 2016). FUNDC1 is ubiquitinated by the E3 ubiquitin-protein ligase membrane-associated ring finger (C3HC4) 5 (MARCH5 or Mitol) (Chen et al., 2017), but not by Parkin. FUNDC1 and Bnip3 interaction with the fusion (Opa1) and fission (Drp1) machinery couples mitochondrial dynamics with mitophagy (Chen et al., 2016; Landes et al., 2010; Wu et al., 2016).

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Phosphorylation of Ub-independent mitochondrial receptors seems to be key for the induction of mitophagy, but the exact kinases involved remain elusive (Rogov et al., 2017; Yuan et al., 2017). FUNDC1 is phosphorylated by Src and casein kinase 2 (CK2) (Chen et al., 2014; Liu et al., 2012a) and during hypoxia-induced mitophagy, FUNDC1 dephosphorylation by the phosphoglycerate mutase family member 5 phosphatase (PGAM5) (Chen et al., 2014) promotes its interaction with LC3. Conversely, phosphorylation of by ULK1 during hypoxia or mitochondrial uncoupling increases its binding affinity to LC3 (Wu et al., 2014; Zhu et al., 2013) (Figure 2.5). The Atg32/Bcl-2-like protein 13 (Bcl2L13) and FK506-binding protein 8 (FKBP8) are other mitophagy receptors involved in mitochondrial turnover in a Parkin-independent manner (Bhujabal et al., 2017; Murakawa et al., 2019). Similar to other mitochondrial receptors, CK2 also phosphorylates Atg32 in yeast (Kanki et al., 2013). A recent report demonstrated that Bcl2L13 recruits ULK1 to induce mitophagy (Murakawa et al., 2019). Interestingly, Bcl2L13 has been reported to promote adipogenesis by controlling mitochondrial quality control and oxidative phosphorylation (Fujiwara et al., 2019). Cardiolipin and prohibitins have also been suggested to mediate mitophagy but their exact mechanism of action is still unclear (Chao et al., 2019; Chu et al., 2014; Wei et al., 2017). 4. Cellular redox balance and mitophagy Reactive oxygen (ROS) and nitrogen (RNS) species are highly reactive molecules generated as by-products from cellular metabolism under both normal and pathological conditions or upon exposure to environmental or xenobiotic agents. Basal or physiological levels of ROS/RNS formation play an important homeostatic role regulating signal transduction involved in proliferation and survival (Finkel, 2011). When either ROS/RNS formation or endogenous antioxidant defenses are dysregulated, oxidative or reductive stress takes place. Oxidative

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stress is defined as an increase in the steady-state levels of ROS/RNS that surpasses their catabolism or detoxification. When oxidative stress exceeds the capacity of the cell to repair biomolecule oxidation (nucleic acids, lipids and proteins), oxidative damage occurs (Franco and Cidlowski, 2009; Sies et al., 2017). Several organelles within the cell have the ability to produce ROS. However, mitochondria are considered the main source for ROS (Murphy, 2009). In the mitochondria, superoxide anion (O2-) is produced in the matrix by the one-electron reduction of O2 through complex I of the electron transport chain (ETC) (Grivennikova and Vinogradov, 2006), and in both the matrix and the inner membrane space (IMS) by complex III (Chen et al., 2003; Muller et al., 2004). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX or dual oxidases DUOX) (Figure 1) and nitric oxide synthases (NOS) are the most important enzymatic sources for the generation of ROS and RNS triggered by growth factor/cytokine signaling (Brewer et al., 2015; Martinez-Ruiz et al., 2011). The most important mitochondrial defenses against O2- are superoxide dismutases (SODs), which are compartmentalized in the mitochondrial matrix (manganese SOD [MnSOD or SOD2]) and the IMS (copper-zinc SOD [CuZnSOD or SOD1]). SODs generate hydrogen peroxide (H2O2) which if not detoxified can induce oxidative damage to proteins (iron-sulfur clusters and oxidative modifications to cysteine), DNA, and lipids (Fukai and Ushio-Fukai, 2011). Detoxification of H2O2 and derived peroxides in the mitochondria is performed primarily by the glutathione (GSH) peroxidase system (Gpx1 and mGpx4) and peroxiredoxins (Prx3 and Prx5), while cysteine modifications (glutathionylation and disulfide bonds) are reversed by the glutaredoxin (Grx2) and thioredoxin/thioredoxin reductase (Trx2/TrxR2) systems (Ren et al., 2017). H2O2 can also act as a second messenger due to its low reactivity, specificity for cysteine residues and ability to diffuse across membranes, including those of the mitochondria (Reczek and Chandel, 2015).

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Alterations in the autophagy flux/rate have been shown to regulate both redox balance and ROS formation under distinct circumstances. Mitophagy is induced by oxidative stress and is involved in the removal of dysfunctional mitochondria (Shefa et al., 2019). Direct generation of mitochondrial ROS using a mitochondrial-targeted photosensitizer has been reported to induce mitophagy (Wang et al., 2012). Parkin/PINK1-dependent mitophagy has been reported to require O2- production (Xiao et al., 2017). Interestingly, Prx6 has been shown to translocate to depolarized mitochondria regulating redox homeostasis and PINK1-dependent mitophagy (Ma et al., 2016). In contrast, in cytochrome C- and mitochondrial mtDNA-deficient ρ0 cells, STSinduced autophagy was not correlated with ROS formation and remained unaffected by antioxidant enzymes, suggesting that mitochondrial ROS are not required for mitophagy (Jiang et al., 2011). Mild oxidative stress selectively triggers mitophagy in the absence of autophagy, which is dependent on Drp1 (Frank et al., 2012). Interestingly, both non-selective autophagy (atg1Δ yeast) and ubiquitin-independent mitophagy (atg32Δ or atg11Δ yeast)-deficient cells are characterized by an enhanced accumulation of ROS upon starvation (Kurihara et al., 2012; Suzuki et al., 2011). Importantly, the mechanisms mediating ROS accumulation are different. In non-selective autophagy-deficient cells, ROS accumulation upon starvation is associated with a reduction in the cellular amino acid pool and a reduction in the expression levels of mitochondrial respiratory and scavenger proteins (Suzuki et al., 2011). In contrast, in mitophagy-deficient cells excess mitochondria are not degraded, spontaneously age, and produce ROS in excess (Kurihara et al., 2012). Although autophagy has a clear role in regulating mitochondrial homeostasis, signaling cascades involved in autophagy can indirectly regulate mitochondrial function. For example, mTOR inhibition with rapamycin decreases the expression of the peroxisome-proliferator-activated receptor coactivator (PGC)-1α whose transcriptional activity regulates mitochondrial gene expression and biogenesis and consequently ROS formation (Cunningham et al., 2007).

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During viral infection, ROS triggers Bnip3- and Nix-dependent mitophagy in natural killer (NK) cells for the turnover of dysfunctional mitochondria that also contributes to the generation of NK cell memory (O'Sullivan et al., 2015). Defects in Bnip3-dependent mitophagy promotes mammary tumor progression and metastasis by the upregulation of ROS, HIF1α signaling, glycolysis and angiogenesis (Chourasia et al., 2015). In cancer, Nix-dependent mitophagy also protects against hypoxia-induced mitochondrial ROS in glioblastoma cells (Jung et al., 2019). In response to oxidative stress, the transcription of antioxidant defenses is mediated by the nuclear factor (erythroid-derived 2)-like 2 transcription factor (Nrf2) through the recognition of cis-acting sequences known as antioxidant response elements (ARE). Nrf2 is sequestered in the cytoplasm by the kelch-like ECH-associated protein 1 (Keap1)-Cul3 complex and degraded in an ubiquitin-proteasome dependent manner. Oxidant- or electrophile-induced modification of Keap1 reactive cysteine residues inhibits Nrf2 ubiquitination allowing its translocation to the nucleus (Brigelius-Flohe and Flohe, 2011; Dinkova-Kostova and Abramov, 2015). p62dependent autophagic degradation of Keap1 also activates Nrf2 and protects against oxidative stress (Komatsu et al., 2010; Taguchi et al., 2012). Transcriptional upregulation of p62 by Nrf2 subsequently creates a positive feedback loop (Jain et al., 2010). Nrf2 also regulates PINK1 expression upon oxidative stress (Murata et al., 2015), corroborating a link between Nrf2 and mitophagy. Accordingly, Keap1 inhibitors trigger mitophagy (Georgakopoulos et al., 2017). Because Nrf2 also regulates mitochondrial biogenesis (Gureev et al., 2019), Nrf2 can be considered a central regulator of mitochondrial homeostasis. Interestingly, a recent report described a novel pathway for mitophagy where p62 translocates Keap1 to the mitochondria to ubiquitinate this organelle and trigger mitophagy (Yamada et al., 2018) (Figure 2.4). FUNDC1 phosphatase PGAM5 is also a substrate for Keap1 (Lo and Hannink, 2006), suggesting a link between PGAM5 ubiquitination and degradation and the dephosphorylation of FUNDC1 and the final induction of FUNDC1-dependent mitophagy.

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Post-translational modification of protein cysteines (protein thiols) mediate redox signaling by regulating protein activity, localization, and/or protein-protein interactions (Fomenko et al., 2008). Redox-sensitive cysteines undergo reversible and irreversible thiol modifications in response to ROS or RNS (Winterbourn and Hampton, 2008). Almost all physiological oxidants react with thiols (Winterbourn and Hampton, 2008). O2•- and peroxides (H2O2, and ONOO-), mediate one- and two-electron oxidation of protein cysteines, leading to the formation of the reactive intermediates protein sulfenic acids and protein thiyl radicals, respectively (Trujillo et al., 2016). Reaction of sulfenic acids with another protein cysteine or GSH will generate a disulfide bond or a glutathionylated residue. Protein glutathionylation is considered a protective modification against irreversible cysteine oxidation (Gao et al., 2009). Sulfenic acids can also undergo further reaction with H2O2 to irreversibly generate protein sulfinic, and sulfonic acids (Rehder and Borges, 2010). Nitros(yl)ation refers to the reversible covalent adduction of a nitroso group to a cysteine thiol. Nitros(yl)ation can be mediated by NO•, nitros(yl)ating agents such as N2O3 or by transition metal catalyzed addition of NO, but transfer of NO between nitrosoglutathione (GSNO) and protein cysteine thiols has been suggested to be the major mechanism mediating nitros(yl)ation (transnitros[yl]ation). GSNO is formed by the reaction of •NO

with GSH, and as a minor byproduct from the oxidation of GSH by ONOO- (Foster et al.,

2009). Protein denitros(yl)ation can be regulated by Trxs and also through the metabolism of GSNO via GSH-dependent formaldehyde dehydrogenase or class III alcohol dehydrogenase, also known as GSNO reductase (GSNOR) (Benhar et al., 2009). Trx can also transnitros(yl)ate proteins by first becoming nitros(yl)ated and then relaying oxidative equivalent to protein targets (Barglow et al., 2011; Wu et al., 2011). Table 1 summarizes the redox modifications in peripheral signals that regulate autophagy (growth factors, hypoxia, energy depletion), as well direct modifications in signaling proteins

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within the core autophagy machinery, mitochondrial dynamics and mitochondrial receptors involved in mitophagy. Next, we will focus only in this last due to space restrictions. Few studies have determined the role oxidative cysteine modification in the redox regulation of autophagy. Upon starvation, increased generation of mitochondrial H2O2 oxidizes and inactivates Atg4 following the initial cleavage of LC3, ensuring the structural integrity of the mature autophagosome (Scherz-Shouval et al., 2007) (Figure 1.3). More recently, a thiol/disulfide exchange reaction between Atg4 and thioredoxin has also been reported to regulate Atg4 activation (Perez-Perez et al., 2014). A recent report also demonstrated that oxidative stress inactivates Atg3 and Atg7 via the formation of a heterodimer linked by an interdisulfide bond, which inhibits LC3 lipidation (Frudd et al., 2018) (Figure 1.3). Mitochondrial dynamics and mitophagy are also modulated by redox signaling. A redox-based mechanism has been demonstrated to regulate mitochondrial fusion where GSH disulfide (GSSG or oxidized GSH) induces the generation of Mfn oligomers via disulfide bond formation, causing a conformational change in the regions that aid in the tethering of Mfns to enhance membrane fusion (Mattie et al., 2018; Ryan and Stojanovski, 2012; Shutt et al., 2012) (Figure 2.7). Cysteine nitros(yl)ation and sulfen(yl)ation of Drp1 promote mitochondrial fragmentation (Cho et al., 2009; Kim et al., 2018b) (Figure 2.2). In human-derived neurons from sporadic PD cases, PINK1 nitros(yl)ation reduces Parkin-dependent mitophagy (Oh et al., 2017) (Figure 2.4). The effect that oxidation has on Parkin is still controversial as both inhibition (Chung et al., 2004; Meng et al., 2011; Yao et al., 2004) and stimulation of its E3-Ub ligase activity (Ozawa et al., 2013) have been reported, but this seems to depend on the cysteine residues modified. Inhibition of Parkin by nitros(yl)ation increases Drp1 levels and mitochondrial fragmentation (Zhang et al., 2016c). In contrast, increased nitrosylation of cysteine 323 activates Parkin and induces mitophagy (Ozawa et al., 2013).

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GSNOR downregulation during senescence promotes mitochondrial nitrosative stress, nitros(yl)ation of Drp1 and Parkin, and impairment of mitochondrial dynamics and mitophagy (Rizza et al., 2018) (Figure 2.2 and 2.4). During myocardial ischemia, homodimerization of the mitochondrial receptor Bnip3 via the oxidation of Cys64 triggers cell death, but whether this phenomenon is linked to mitophagy is unknown (Kubli et al., 2008). In contrast oxidative stress increases p62-dependent autophagy and protein turnover via formation of disulfide-linked conjugates (Carroll et al., 2018). Methionine is another sulfur containing amino acid susceptible to reversible oxidation. Addition of an O2 oxidizes methionine to methionine sulfoxide, and a strong oxidant oxidizes methionine sulfoxide irreversibly to methionine sulfone. Methionine sulfoxide can be reduced by methionine sulfoxide reductases (Msrs) (Kaya et al., 2015). It has been recently demonstrated that mitophagy can be regulated by oxidation of Met129 in Parkin, and mutation of this amino acid in Parkin is linked to PD (Lee et al., 2019).

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Conclusions Mitophagy is a central homeostatic mechanism involved in the turnover of damaged and dysfunctional mitochondria generated as a result of cell injury or disease-associated processes. In addition, mitophagy regulates cellular bioenergetics and redox signaling during differentiation and aging. Recent studies have revealed the complexity of mechanisms involved in the regulation of mitophagy outside the “canonical” Ub-dependent PINK1/parkin-mediated mitochondrial targeting by autophagosomes. Because mitochondria are considered the primary source of ROS, in this minireview we have aimed to highlight the interrelationship between the regulation of mitochondrial ROS formation by autophagy and the redox-dependent mechanisms by which ROS regulate mitophagy. Oxidative modifications in redox sensitive amino acids (cysteine and methionine) have been shown to regulate both mitophagy as well as mitochondrial dynamics (fusion and fission). It is expected that as the complexity of mitophagy processes get untangled, and controversies are reconciled, new redox-mechanisms involved will be uncovered.

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Acknowledgements This work was supported by the National Institutes of Health Grant P20RR017675 and the Office of Research of the University of Nebraska-Lincoln

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FIGURE LEGENDS Figure 1. Autophagy. Autophagy requires the formation of distinct complexes during four sequential stages: (1) Induction and (2) Nucleation of the phagophore; (3) Elongation and closure of the autophagosomes; and (4) Fusion between autophagosomes and lysosomes. (1) The mechanistic target of rapamycin complex 1 (mTORC1) is regulated by stimulation of the class I phosphoinositide 3-kinase (PI3K)–protein kinase B (AKT) pathway by growth factors, and via the regulation of Rag guanosine triphosphatases (GTPases) and the adenosine monophosphate (AMP)-activated protein kinase (AMPK) by amino acid and energy depletion, respectively. mTORC1 negatively regulates the unc-51-like kinase-1 (ULK1) and class III PI3K complex I (PIK3C3-CI). Starvation or growth factor withdrawal inhibit mTORC1, leading to the dephosphorylation / activation of ULK1. (2) ULK1 activation phosphorylates and activates the PIK3C3-CI that in turn generates phosphatidylinositol-3-phosphate (PIP3), which recruit the autophagy-related protein (Atg) 5-Atg12 and Atg8 / microtubule-associated light chain 3 (LC3) conjugation systems. (3) LC3 lipidation to LC3-II is used as a platform for the recognition of cellular targets, including mitochondria via specific receptors. (4) Subsequently, autophagosomes move across microtubules to encounter lysosomes. Autophagosomelysosome fusion (autolysosome) is the final step where lysosomal hydrolases are released for the degradation of autophagosome cargo. P, highlights phosphorylation events; Ox, highlights sites for redox regulation of autophagy. Ambra1, autophagy and beclin 1 regulator 1; Beclin 1, B-cell lymphoma 2-interacting myosin/moesin-like coiled-coil protein 1; NRBF2, nuclear receptor binding factor 2; NOX, nicotinamide adenine dinucleotide phosphate oxidase; PDK1, phosphoinositide-dependent kinase 1; PIP2, phosphatidylinositol 4,5-bisphosphate; PTEN, phosphatase and tensin homolog; PTP1B, protein tyrosine phosphatase 1B; RB1CC1, retinoblastoma-associated protein-inducible coiled-coil protein 1; Rheb, Ras homolog enriched in brain; RTK, receptor tyrosine kinase; SNAREs, soluble N-ethylmaleimide-sensitive factor

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attachment protein receptors; TSC1/2, tuberous sclerosis complex proteins 1 and 2; Vps15; vacuolar protein sorting 15. Figure 2. Mitochondrial fusion, fission and mitophagy. Mitochondrial maintenance is a dynamic process undergoing continuous events of fission and fusion to preserve proper mitochondrial functions. (1) Fission requires local organization of the mitochondrial fission 1 protein (Fis1) and recruitment of the dynamin-related protein 1 (Drp1) guanosine triphosphatase (GTPase) for assembly of the fission machinery that subsequently leads to membrane scission. (2) Fusion is mediated by dynamin GTPases mitofusins 1 (Mfn1) and 2 at the outer membrane and optic atrophy protein (Opa1) at the inner membrane that tether adjacent mitochondria together. Mitochondrial fission precedes mitophagy, in order to transform elongated mitochondria into a form suitable for engulfment, or upon oxidative stress, to mediate the degradation of damaged mitochondria decreasing mitochondria-derived ROS formation. (3) Loss of mitochondrial membrane potential (ΔΨm) leads to the translocation of the phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) and the E3-ubiquitin (Ub) ligase Parkin (PARK2) to the mitochondria, where it promotes the ubiquitination of proteins in the mitochondrial membrane, which recruit the autophagy receptors (p62, optineurin [OPTN] and the nuclear dot protein 52 [NDP52]) that target these mitochondria for removal (4). (5) Ubiquitin-independent mitophagy is involved primarily in metabolic reprogramming during differentiation and cancer, as well as during mitochondria turnover in response to hypoxia, and is mediated by several mitophagy receptors including NIP3-like protein X (Nix), Bcl-2 interacting protein 3 (Bnip3) and FUN14 domain containing 1 (FUNDC1) protein. (6) Transport of mitochondria to axonal and dendritic terminations is essential to meet energy demands associated with synaptic transmission. PINK1 and Parkin mediate phosphorylation, ubiquitination and degradation of the mitochondrial Rho–GTPase (Miro) leading to the detachment of mitochondria from microtubules. (7) Oxidized glutathione (GSSG) accumulation

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has been demonstrated to mediate the oxidation of Mfn’s cysteines (Cys) by disulphide bond formation (-S-S-), causing a conformational change that aid in tethering of Mfns to enhance membrane fusion. P, highlights phosphorylation events; Ox, highlights sites for redox regulation of mitophagy. AMPK, adenosine monophosphate (AMP)-activated protein kinase; HIF1α, hypoxia-inducible factor-1α, Keap1, kelch Like ECH Associated Protein 1; KIF5a, Kinesin Family Member 5A; ULK1, unc-51-like kinase-1.

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Table 1. Redox regulation of Autophagy, Mitophagy and Peripheral Signaling Pathways (from figures 1-2). Effects are described based on the modulation of their signaling activity or the induction of changes in autophagy, mitophagy or mitochondrial dynamics Peripheral Signaling Pathways

Receptor Tyrosine Kinases (RTK)

Function

Activation PI3K/Akt via Growth Factors

Redox Modifications Sulfen(yl)ation of EGFR (C797). Reversed by glutathionylation. Nitros(yl)ation of EGFR Nitros(yl)ation of EGFR (C166 and C305)

Protein tyrosine phosphatase 1B (PTP1B)

Phosphatase and tensin homolog (PTEN)

Phosphoinositidedependent kinase 1 (PDK1)

Protein kinase B (Akt)

Inhibition of RTK signaling

Dephosphorylates PIP3 negatively regulating Akt signaling

Effects

Enhanced activation

Activation Inhibition

Sulfen(yl)ation (C215). Reversed by Trx and protected by nitros(yl)ation

Reversible inhibition

Nitros(yl)ation (C215)

Reversible inhibition

Nitros(yl)ation (C83)

Inhibition of autophagy Inhibition

Disulfide bond (C124-C71)

Phosphorylates Akt to promote its activation

Nitros(yl)ation (C128)

Phosphorylates the tuberous sclerosis complex (TSC 1/2) releasing its inhibitory effect on mTOR

Nitros(yl)ation (C224) Disulfide bond (C60-C77) at the pleckstrin homology domain Disulfide bond (C297-C311) at the kinase domain of Akt1

38

Induction of autophagy via mTOR

References (Paulsen et al., 2011; Schwartz et al., 2014; Truong and Carroll, 2012) (Switzer et al., 2012) (MurilloCarretero et al., 2009) (Chen et al., 2008; Dagnell et al., 2013; Meng et al., 2002) (Hsu et al., 2016) (Numajiri et al., 2011; Zhu et al., 2019) (Lee et al., 2002) (Kim et al., 2018a)

Inhibition

(Liu et al., 2017a)

Inhibition

(Yasukawa et al., 2005)

Increased recruitment to PIP3 and activation Inhibition. Reduced and protected by Grx

(Su et al., 2019) (Murata et al., 2003)

Disulfide bond (C124-C297 or C124-C311) at the kinase domain of Akt2 Disulfide bond (C2460-C2467) Intermolecular disulfide bond (C1483). Reduced by Trx

Inhibition

(Wani et al., 2011)

Increased stability

(Dames et al., 2005)

Oligomerization and inhibition

(Oka et al., 2017)

Thiol oxidants

Destabilize Raptor (mTORC1) and mTOR interaction increasing mTOR activity

(Sarbassov and Sabatini, 2005)

Rictor (mTORC2) oxidation. Reversed by Prx3

Inhibition

(Olson et al., 2017)

Nitros(yl)ation

Impairs dimerization with TSC1 leading to mTOR activation

(LopezRivera et al., 2014)

Inhibition

(Shao et al., 2014)

Activation

(Zmijewski et al., 2010)

Indirectly

Activation

(Hinchy et al., 2018)

Hydroxylation of HIF1α and its subsequent degradation

Intermolecular disulfide bond (C326) Nitros(yl)ation (C302)

Inhibition and activation of HIF1α

(Lee et al., 2016)

Inhibition

(Chowdhury et al., 2011)

Hypoxia-inducible factor-1α (HIF1α)

Transcription of Ub-independent mitochondrial receptors and induction of mitophagy

Nitros(yl)ation (C533)

Increased activity

(Li et al., 2007)

Kelch Like ECH Associated Protein 1 (Keap1)

Ubiquitination of Nrf2 for its

Electrophile reaction, Oxidation or Nitrosylation

Inactivation and release of Nrf2

(Cuadrado et al., 2019;

Mammalian-target of rapamycin (mTOR) kinase

Tuberous sclerosis complex 2 (TSC2) protein

Adenosine monophosphate (AMP)-activated protein kinase (AMPK)

Prolyl hydroxylase 2 (PHD2)

Negatively regulates autophagy via the phosphorylation of ULK1 and Atg13

When complexed with TSC1 it inactivates the Rheb GTPase, which activates mTOR Phosphorylates ULK1 antagonizing autophagy inhibition by mTORC1

Disulfide bond (C130-C174), reduced by Trx Sulfen(yl)ation and Glutathionylation (C304 and C299)

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proteasomal degradation

Autophagy

Function

Autophagy-related protein 4 (Atg4)

Atg4 initially cleaves LC3 to expose a glycine residue at the Cterminus. Then, it cleaves (delipidates) LC3PE to recycle LC3, and to promote autophagosome elongation.

Atg7-Atg3

Cytoskeleton

Mitochondrial Dynamics

(C151, C226, C257, C273, C288, C297, C434 and C613) Redox Modifications Disulfide bond (C338-C394). Reduced by Trx

Oxidation (Cys81)

Yamamoto et al., 2018)

Effects Fine-tuning of LC3 recruitment to the phagophore Inhibition of Atg4 and LC3 delipidation with concomitant stimulation of autophagy

References (PerezPerez et al., 2014)

(ScherzShouval et al., 2007)

Glutathionylation or inter molecular disulfide bond formation on (Frudd et Inhibition Catalytic Cys al., 2018) residues in Atg7 (C572) and Atg3 (C264) Cytoskeleton dynamics has essential roles in autophagy (from autophagosome biogenesis to its transport and fusion with lysosomes) through mechanisms that involve actin- and microtubule-mediated motility, cytoskeleton-membrane scaffolds and signaling proteins (Kast and Dominguez, 2017). A number of those mechanisms have been shown to be modulated by redox signaling events (Wilson and Gonzalez-Billault, 2015). Redox Function Effects References Modifications

The conjugation (lipidation) of LC3 to PE (LC3II) is mediated by the E1-like and E2-like enzymes Atg7 and Atg3, respectively

Nitros(yl)ation Dynamin-related protein 1 (Drp1)

Drp1 GTPase interacts with Fis1 and oligomerizes to form rings around dividing mitochondria

Mitofusins (Mfn)

GTPases at the OMM that oligomerize and anchor mitochondria to trigger fusion

Nitros(yl)ation or Sulfen(yl)ation (C644)

Intermolecular disulfide bond (C684) triggered by GSSG

40

None Increased dimerization and GTPase activity resulting in mitochondrial fragmentation Oligomeric mitofusins prime mitochondria for docking and fusion

(Bossy et al., 2010) (Cho et al., 2009) (Kim et al., 2018b)

(Mattie et al., 2018; Shutt et al., 2012)

Mitophagy

Parkin

PTEN-induced putative kinase 1 (PINK1)

p62

Bcl-2 interacting protein 3 (Bnip3)

Function

E3-Ub ligase that ubiquitinates mitochondrial proteins assembling monoand poly-Ub chains

A mitochondrial serine/threonineprotein kinase that phosphorylates Ub and activates Parkin A Ub-dependent receptor suggested to mediate mitophagy A Ub-independent transmembrane receptor that triggers mitophagy

Redox Modifications Nitros(yl)ation of (3 Cys or C241 and C260) the In Between Ring (IBR) domain

Effects

References

Inhibition

(Chung et al., 2004; Yao et al., 2004)

Nitros(yl)ation (C323)

Activation and induction of mitophagy

(Ozawa et al., 2013)

Sulfi(o)n(yl)ation of six Cys

Inhibition

(Meng et al., 2011)

Met oxidation (M192). Reversed by MsrB2

Reduction of oxidized M192 by MsrB2 promotes mitophagy

(Lee et al., 2019)

Nitros(yl)ation (C568)

Nhibition of Parkindependent autophagy

(Oh et al., 2017)

Intermolecular disulfide bond (C105-C113)

Oligomerization and induction of autophagy

(Carroll et al., 2014)

Homodimerization by C64 oxidation

Cell death

(Kubli et al., 2008)



The degradation of mitochondria via autophagy (mitophagy) is involved in a number of physiological processes including cellular homeostasis, differentiation and aging.



Upon stress or injury, mitophagy prevents the accumulation of damaged mitochondria and the increased steady state levels of reactive oxygen species



A number of human diseases have been linked to the dysregulation of mitophagy.



We review the molecular mechanisms involved in the regulation of mitophagy by redox signaling 41

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