Cellular metabolic and autophagic pathways: traffic control by redox signaling

Abstract

It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function.

Keywords: 4-hydroxy-2-nonenal; AMP kinase; AMPK; Aging; Ambra; Atg; Autophagy; B-cell lymphoma 2; BH3; BNIP; Bcl-2; Bcl-2 homology domain 3; Bcl-2/adenovirus E18 19-kDa-interacting protein; CMA; Cardiovascular disease; Cellular bioenergetics; Diabetes; ER; ERK; ESCRT3; Free radicals; G6PD; GAPDH; GSH; GSSG; Glucose; Glutathione; Glycolysis; HIF; HNE; Hsc70; IRE; JNK; LC3; MEF; Mitochondria; Mitophagy; NADPH oxidase; NIP3-like X; NIX; NOX; Neurodegeneration; Oxidative stress; PI3K; PI3P; PINK1; PPP; PTEN-induced kinase 1; RNS; ROS; Rab5; Rac1; Ras-associated protein; Ras-related C3 botulinum toxin substrate 1; Redox signaling; SNARE; TIGAR; TP53-induced glycolysis and apoptosis regulator; TSC; ULK1 and ULK2; UV radiation resistance-associated gene protein; UVRAG; VDAC; Vacuolar protein sorting.; Vps; activating molecule in beclin 1-regulated autophagy; autophagy-related gene; c-Jun N-terminal kinase; chaperone-mediated autophagy; eNOS; endoplasmic reticulum; endosomal sorting complexes required for transport 3; endothelial nitric oxide synthase; extracellular signal-regulated kinase; glucose-6-phosphate dehydrogenase; glutathione; glyceraldehyde-3-phosphate dehydrogenase; heat shock cognate 70; hypoxia-inducible factor; inositol requiring enzyme; mTOR; mammalian target of rapamycin; microtubule-associated protein 1 light-chain subunit 3; mouse embryonic fibroblast; oxidized glutathione; pentose phosphate pathway; phosphatidylinositol 3-phosphate; phosphoinositide 3-kinase; reactive nitrogen species; reactive oxygen species; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; tuberous sclerosis complex; uncoordinated family member-51-like kinases 1 and 2; voltage-dependent anion channel.

Figure 1. The three main autophagic pathways

There are three main pathways of autophagy: 1) microautophagy, 2) chaperone-mediated autophagy, and 3) macroautophagy. Microautophagy involves direct engulfment of cargo by the lysosome. Chaperone-mediated autophagy involves targeting of specific proteins that have a consensus sequence (KFERQ) by heat shock cognate protein 70 (Hsc70) to the lysosome membrane associated protein 2A (LAMP2A), which delivers the unfolded protein into the lysosome. Macroautophagy involves the bulk sequestration of cargo in the cytoplasm into a double membrane bound autophagosome, which delivers its contents to the lysosome. Once in the lysosome, all cargo is degraded by lysosomal hydrolases. Each type of autophagy can be activated by a number of stimuli including oxidative stress, nutrient deprivation, protein aggregates, pathogen invasion, or damage to organelles.

Figure 2. Regulation of autophagy through the nutrient sensing mTOR pathway, the Beclin complex, and the LC3 lipidation processes

Nutrient deprivation can activate autophagy through mTOR mediated signaling mechanisms. There are two mTOR complexes, mTORC1 and mTORC2. mTORC1 is activated by amino acid deprivation or by AMPK sensing intracellular AMP. Insulin receptor activation, on the other hand, suppresses autophagy by activating Akt, which subsequently phosphorylates and inhibits the TSC1/2 complex, removing its inhibition of Rheb, which can interact with and activate mTORC1, and thereby inhibiting autophagy. Activation of autophagy in response to nutrient deprivation inhibits mTORC1 and activates ULK1, resulting in the initiation of autophagy via the Vps34, Vps15, Beclin-1 initiation complex, which then associates with the phagophore, and extends the double membrane structure which is rich in PI3P, a product of the class III PI3K, Vps34. ULK1 can also be activated directly by AMP kinase. Microtubule associated protein light chain 3/LC3 lipidation is a hallmark of autophagy and require a multi-step conjugation process. Atg4 cleaves pro-LC3 to LC3-I. Ubiquitin-like step-wise conjugation of LC3 with Atg7, Atg3, followed by lipidation by phosphatidylethanolamine (PE); formation of Atg5-Atg12-Atg16 and Beclin-1/Vps34/Vps15 complexes are involved in initiation and expansion of the phagophore, which is a double membrane structure with LC3-II insertion and rich in PI3P, a product of Vps34. Rapamycin has been found to inhibit mTORC1 activity and thereby activate autophagy. This autophagy activating property of rapamycin has enabled research into effects of autophagy in cell function and survival with and without rapamycin. 3-methyladenine, which is a class III PI3K inhibitor, has been used as a pharmacological agent, to inhibit autophagy in various pathological studies, as a complementary approach to molecular interventions of siRNA of Beclin, Vps34, LC3, Atg5 or Atg7. Mouse knockouts of many of these genes involved in autophagy have been used in elucidating autophagy signaling and execution in vivo.

Figure 3. Integration of glucose metabolism and autophagy

Glucose metabolism is integrated with autophagy regulation on multiple levels, through modulating glycolysis, ER stress, cellular glutathione and reactive oxygen species levels. On the one hand, in response to glucose deprivation, autophagy can use glycogen stores to generate glucose. On the other hand, glucose levels influence glycosylation, glycolysis and redox status, all have complex relationships to the regulation of autophagy. Decreased glucose increases ER stress and thereby induces autophagy, which can be attenuated by addition of mannose, and exacerbated by thapsigargin or tunicamycin. Depletion of glucose may lead to depletion of glucose-6-phosphate, which decreases metabolites produced by the pentose phosphate pathway (PPP), resulting in increased production of reactive oxygen species (ROS), and increased autophagy. Increase of intracellular reduced glutathione, while can decrease ROS and inhibit autophagy, in certain cell types and conditions is also associated with an increase in autophagy. Low glucose-6-phosphate levels also allow GAPDH to interact with mTOR, activating autophagy. Increased glycolysis enzyme GAPDH also increases Atg12, and thereby increases autophagy. The enzyme TIGAR can be activated by p53, activates hexokinase, and shifts cells away from glycolysis to the PPP, resulting in decreased ROS production and decreased autophagy.

Figure 4. Mitophagy

A. Mitophagy can be mediated by specific factors, Atg32 in yeast, Nix and BNIP in mammals, proteins that target to mitochondria and bind to LC3. B. Mitophagy can also be stimulated by mitochondrial fission through Drp1 activation, or decrease of mitochondrial membrane potential. Decreased mitochondrial membrane potential can lead to stabilization of PINK1 that is targeted to the mitochondria, recruitment of Parkin, ubiquitination of Mfn, VDAC or other mitochondrial proteins. The modified mitochondria may be recognized by p62 or other factors and brought to the autophagosomes to be degraded. C. In addition to Nix, BNIP, PINK1 and Parkin-mediated mitophagy, DJ-1 and ERK1/2 also play a role in mitophagy signaling.

Figure 5. Oxidative and nitrative signals regulate autophagy

Oxidative and nitrative signals are derived from oxidative reactions with oxygen or nitrogen species, resulting in the generation of both radical and non-radical molecules. The radicals consist of superoxide (O₂), hydroxyl radicals (OH), peroxyl (ROO), alkoxyl radicals (RO), and nitric oxide (NO); whereas, the non-radicals consist of hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl), ozone (O₃), aldehydes (HCOR), peroxynitrite (ONOOH), and singlet oxygen (¹O₂). Superoxide (O₂) is the main reactive oxygen species produced by xanthine oxidase, the mitochondrial respiratory chain, and nitric oxide enzymes. Superoxide can interact directly with nitric oxide (NO) to form peroxynitrite (ONOO) or with membrane fatty acids to form lipid peroxidation products (LOOH). Superoxide can also be reduced to hydrogen peroxide (H₂O₂) by superoxide dismutases 1–3 (SOD1-3) or by the SOD mimetic Mn(III)-tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP). Hydrogen peroxide, which is also produced by peroxisomes, or NADPH oxidases, can interact with ferric iron (Fe²⁺) to form the highly reactive hydroxyl radical (OH). The hydroxyl radical and peroxynitrite can both interact with membrane polyunsaturated fatty acids to form lipid peroxidation products. Hydrogen peroxide can activate autophagy through a PI3K-mediated pathway, which is inhibited by catalase. The relationship between hydrogen peroxide and autophagy can be complex, in that hydrogen peroxide can also inhibit Atg4 activity, and catalase can be degraded by autophagy. Hydroxyl radicals can induce lysosomal dysfunction and inhibit autophagy. Peroxynitrite, as well as lipid peroxidation products, such as HNE, can activate autophagy, which can be inhibited by Ebselen (a glutathione peroxidase mimetic), reduced glutathione (GSH), or N-acetyl cysteine (NAC). Peroxynitrite-induced autophagy is implicated in disruption of tight junction and decreasing VEGFR2 in endothelial cells. The effects of nitric oxide and reactive nitrogen species on autophagy are also complex. Nitric oxide can inhibit autophagy by decreasing phosphorylation of Bcl-2, decreasing Beclin initiation of autophagy, and activating of mTOR. S-nitrosocysteine has also been shown to induce fission and mitochondrial depletion. Nitric oxide also activates autophagy independent of formation of peroxynitrite.