Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis

Upon encountering oxidative stress, proteins are oxidized extensively by highly reactive and toxic reactive oxidative species, and these damaged, oxidized proteins need to be degraded rapidly and effectively. There are two major proteolytic systems for bulk degradation in eukaryotes, the proteasome and vacuolar autophagy. In mammalian cells, the 20S proteasome and a specific type of vacuolar autophagy, chaperone-mediated autophagy, are involved in the degradation of oxidized proteins in mild oxidative stress. However, little is known about how cells remove oxidized proteins when under severe oxidative stress. Using two macroautophagy markers, monodansylcadaverine and green fluorescent protein-AtATG8e, we here show that application of hydrogen peroxide or the reactive oxidative species inducer methyl viologen can induce macroautophagy in Arabidopsis (Arabidopsis thaliana) plants. Macroautophagy-defective RNAi-AtATG18a transgenic plants are more sensitive to methyl viologen treatment than wild-type plants and accumulate a higher level of oxidized proteins due to a lower degradation rate. In the presence of a vacuolar H(+)-ATPase inhibitor, concanamycin A, oxidized proteins were detected in the vacuole of wild-type root cells but not RNAi-AtATG18a root cells. Together, our results indicate that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis.     

Disruption of mitochondrial redox circuitry in oxidative stress

The present review and commentary considers oxidative stress as a disruption of mitochondrial redox circuitry rather than an imbalance of oxidants and antioxidants. Mitochondria contain two types of redox circuits, high-flux pathways that are central to mechanisms for ATP production and low-flux pathways that utilize sulfur switches of proteins for metabolic regulation and cell signaling. The superoxide anion radical (hereafter termed "superoxide", O2*-), a well known free radical product of the high-flux mitochondrial electron transfer chain, provides a link between the high-flux and low-flux pathways. Disruption of electron flow and increased superoxide production occurs due to inhibition of electron transfer in the high-flux pathway, and this creates aberrant "short-circuit" pathways between otherwise non-interacting components. A hypothesis is presented that superoxide is not merely a byproduct of electron transfer but rather is generated by the mitochondrial respiratory apparatus to serve as a positive signal to coordinate energy metabolism. Electron mediators such as free Fe(3+) and redox-cycling agents, or potentially free radical scavenging agents, could therefore cause oxidative stress by disrupting this normal superoxide signal. Methods to map the regulatory redox circuitry involving sulfur switches (e.g., redox-western blotting of thioredoxin-2, redox proteomics) are briefly presented. Use of these approaches to identify sites of disruption in the mitochondrial redox circuitry can be expected to generate new strategies to prevent toxicity and, in particular, promote efforts to re-establish proper electron flow as a means to counteract pathologic effects of oxidative stress.     

Disruption of neuronal autophagy by infected microglia results in neurodegeneration

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.     

Denervation-induced oxidative stress and autophagy signaling in muscle

Alterations in contractile activity influence the intracellular homeostasis of muscle which results in adaptations in the performance and the phenotype of this tissue. Denervation is an effective disuse model which functions to change the intracellular environment of muscle leading to a rapid loss in mass, a decrease in mitochondrial content, and an elevation in both pro-apoptotic protein expression and myonuclear apoptosis. Recent investigations have shown that alternative degradation pathways such as autophagy are activated in conjunction with apoptosis during chronic muscle disuse. We have previously shown that 7 days of muscle disuse increases the expression of Beclin 1. Furthermore, we have also detected a significant increase in the expression of LC3-II, a known component of autophagy. In addition to its upregulation, denervation appears to induce the translocation of LC3-II to mitochondrial membranes. Collectively, these increases in protein expression suggest that autophagy signaling is upregulated in response to denervation, and that these pathways may preferentially target mitochondria for degradation in skeletal muscle.     

AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells

In Arabidopsis root tips cultured in medium containing sufficient nutrients and the membrane-permeable protease inhibitor E-64d, parts of the cytoplasm accumulated in the vacuoles of the cells from the meristematic zone to the elongation zone. Also in barley root tips treated with E-64, parts of the cytoplasm accumulated in autolysosomes and pre-existing central vacuoles. These results suggest that vacuolar and/or lysosomal autophagy occurs constitutively in these regions of cells. 3-Methyladenine, an inhibitor of autophagy, inhibited the accumulation of such inclusions in Arabidopsis root tip cells. Such inclusions were also not observed in root tips prepared from Arabidopsis T-DNA mutants in which AtATG2 or AtATG5, an Arabidopsis homolog of yeast ATG genes essential for autophagy, is disrupted. In contrast, an atatg9 mutant, in which another homolog of ATG is disrupted, accumulated a significant number of vacuolar inclusions in the presence of E-64d. These results suggest that both AtAtg2 and AtAtg5 proteins are essential for autophagy whereas AtAtg9 protein contributes to, but is not essential for, autophagy in Arabidopsis root tip cells. Autophagy that is sensitive to 3-methyladenine and dependent on Atg proteins constitutively occurs in the root tip cells of Arabidopsis.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H 2O 2) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

Apoptosis and autophagy in photoreceptors exposed to oxidative stress

Studies on human and animal models of retinal dystrophy have suggested that apoptosis may be the common pathway of photoreceptor cell death. Autophagy, the major cellular degradation process in animal cells, is important in normal development and tissue remodeling, as well as under pathological conditions. Previously we provided evidence that genes, whose products are involved in apoptosis and autophagy, may be coexpressed in photoreceptors undergoing degeneration. Here, we investigated autophagy in oxidative stress-mediated cell death in photoreceptors, analyzing the light-damage mouse model and 661W photoreceptor cells challenged with H(2)O(2). In the in vivo model, we demonstrated a time-dependent increase in the number of TUNEL-positive cells, concomitant with the formation of autophagosomes. In vitro, oxidative stress increased mRNA levels of apoptotic and autophagic marker genes. H(2)O(2) treatment resulted in the accumulation of TUNEL-positive cells, the majority of which contain autophagosomes. To determine whether autophagy and apoptosis might precede each other or co-occur, we performed inhibitor studies. The autophagy inhibitor 3-methyladenine (3-MA), silencing RNA (siRNA) against two genes whose products are required for autophagy (autophagy-related (ATG) gene 5 and beclin 1), as well as the pan-caspase-3 inhibitor, Zvad-fmk, were both found to partially block cell death. Blocking autophagy also significantly decreased caspase-3 activity, whereas blocking apoptosis increased the formation of autophagosomes. The survival effects of 3?MA and zVAD-fmk were not additive; rather treatment with both inhibitors lead to increased cell death by necrosis. In summary, the study first suggests that autophagy participates in photoreceptor cell death possibly by initiating apoptosis. Second, it confirms that cells that normally die by apoptosis will execute cell death by necrosis if the normal pathway is blocked. And third, these results argue that the up-stream regulators of autophagy need to be identified as potential therapeutic targets in photoreceptor degeneration.     

HMGB1 as an autophagy sensor in oxidative stress

High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, actively released following cytokine stimulation as well as passively during cell injury and death. Autophagy is a tightly regulated cellular stress pathway involving the lysosomal degradation of cytoplasmic organelles or proteins. Organisms respond to oxidative injury by orchestrating stress responses such as autophagy to prevent further damage. Recently, we reported that HMGB1 is an autophagy sensor in the presence of oxidative stress. Hydrogen peroxide (H₂O₂) and loss of superoxide dismutase 1 (SOD1)-mediated oxidative stress promotes cytosolic HMGB1 expression and extracellular release. Inhibition of HMGB1 release or loss of HMGB1 decreases the number of autolysosomes and autophagic flux in human and mouse cell lines under conditions of oxidative stress. These findings provide insight into how HMGB1, a damage associated molecular pattern (DAMP), triggers autophagy as defense mechanism under conditions of cellular stress.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

The impact of oxidative stress on Arabidopsis mitochondria

Treatment of Arabidopsis cell culture for 16 h with H2O2, menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H2O2/menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H2O2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O2 consumption by mitochondria was significantly decreased by H2O2 treatment.     

The roles of autophagy in development and stress responses in Arabidopsis thaliana

Autophagy is a dynamic process that involves the recycling process of the degradation of intracellular materials. Over the past decade, our molecular and physiological understanding of plant autophagy has greatly been increased. Most essential autophagic machineries are conserved from yeast to plants. The roles that autophagy-related genes (ATGs) family play in the lifecycle of the Arabidopsis are proved to be similar to that in mammal. Autophagy is activated during certain stages of development, senescence or in response to starvation, or environmental stress in Arabidopsis. In the progression of autophagy, ATGs act as central signaling regulators and could develop sophisticated mechanisms to survive when plants are suffering unfavorable environments. It will facilitate further understanding of the molecular mechanisms of autophagy in plant. In this review, we will discuss recent advances in our understanding of autophagy in Arabidopsis, areas of controversy, and highlight potential future directions in autophagy research.     

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.     

Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis

Light absorbed by colored intermediates of chlorophyll biosynthesis is not utilized in photosynthesis; instead, it is transferred to molecular oxygen, generating singlet oxygen ((1)O(2)). As there is no enzymatic detoxification mechanism available in plants to destroy (1)O(2), its generation should be minimized. We manipulated the concentration of a major chlorophyll biosynthetic intermediate i.e., protochlorophyllide in Arabidopsis by overexpressing the light-inducible protochlorophyllide oxidoreductase C (PORC) that effectively phototransforms endogenous protochlorophyllide to chlorophyllide leading to minimal accumulation of the photosensitizer protochlorophyllide in light-grown plants. In PORC overexpressing (PORCx) plants exposed to high-light, the (1)O(2) generation and consequent malonedialdehyde production was minimal and the maximum quantum efficiency of photosystem II remained unaffected demonstrating that their photosynthetic apparatus and cellular organization were intact. Further, PORCx plants treated with 5-aminolevulinicacid when exposed to light, photo-converted over-accumulated protochlorophyllide to chlorophyllide, reduced the generation of (1)O(2) and malonedialdehyde production and reduced plasma membrane damage. So PORCx plants survived and bolted whereas, the 5-aminolevulinicacid-treated wild-type plants perished. Thus, overexpression of PORC could be biotechnologically exploited in crop plants for tolerance to (1)O(2)-induced oxidative stress, paving the use of 5-aminolevulinicacid as a selective commercial light-activated biodegradable herbicide. Reduced protochlorophyllide content in PORCx plants released the protochlorophyllide-mediated feed-back inhibition of 5-aminolevulinicacid biosynthesis that resulted in higher 5-aminolevulinicacid production. Increase of 5-aminolevulinicacid synthesis upregulated the gene and protein expression of several downstream chlorophyll biosynthetic enzymes elucidating a regulatory net work of expression of genes involved in 5-aminolevulinicacid and tetrapyrrole biosynthesis.     

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert‐butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12–Atg5 conjugate, Atg7 contents decreased but LC3‐II accumulated in tBHP‐treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ‐1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions. J. Cell. Biochem. 114: 212–219, 2012. © 2012 Wiley Periodicals, Inc.     

Inactivation of genes,encoding tocopherol biosynthetic pathway enzymes,results in oxidative stress in outdoor grown Arabidopsis thaliana

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. The wild type Columbia and two mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes – tocopherol cyclase (vte1) and γ-tocopherol methyltransferase (vte4) – were analyzed after long-term outdoor growth. The concentration of total tocopherol was up to 12-fold higher in outdoor growing wild type and vte4 plant lines than in plants grown under laboratory conditions. The vte4 mutant plants had a lower concentration of chlorophylls and carotenoids, whereas the mutant plants had a higher level of total glutathione than of wild type. The activities of antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate oxidase (AO, EC 1.10.3.3) were lower in both mutants, whereas activities of catalase (EC 1.11.1.6) and ascorbate peroxidase (APx, EC 1.11.1.11) were lower only in vte1 mutant plants in comparison to wild type plants. However, the activity of guaiacol peroxidase (GuPx, EC 1.11.1.7) was higher in vte1 and vte4 mutants than that in wild type. Additionally, both mutant plant lines had higher concentration of protein carbonyl groups and oxidized glutathione compared to the wild type, indicating the development of oxidative stress. These results demonstrate in plants that tocopherols play a crucial role for growth of plants under outdoor conditions by preventing oxidation of cellular components.