Oxidative stress impairs autophagy through oxidation of ATG3 and ATG7

ABSTRACT

Dysfunctional macroautophagy/autophagy has been causatively linked to aging and the pathogenesis of many diseases, which are also broadly characterized by dysregulated cellular redox. As the autophagy-related (ATG) conjugation systems that mediate autophagosome maturation are cysteine dependent, their oxidation may account for loss in this catabolic process under conditions of oxidative stress. During active autophagy, LC3 is transferred from the catalytic thiol of ATG7 to the active site thiol of ATG3, where it is conjugated to phosphatidylethanolamine. In our recent study, we show LC3 is bound to the catalytic thiols of inactive ATG3 and ATG7 through a stable thioester, which becomes transient upon autophagy stimulation. Transient interaction with LC3 exposes the catalytic thiols on ATG3 and ATG7, which under pro-oxidizing conditions undergo inhibitory oxidation. This process was found to be upregulated in aged mouse tissue and therefore may account, at least in part, for impaired autophagy observed during aging.

Lipidation of LC3 is routinely used as a measure of autophagy; however, the product is also an important component of this catabolic process. The attachment of phosphatidylethanolamine to LC3 by the E1-like and E2-like enzymes ATG7 and ATG3 contributes to the targeting of cargo, membrane extension and closure of the phagophore to generate an autophagosome, as well as autophagosome-lysosome fusion and degradation of the inner autophagosomal membrane. This process begins with newly synthesized LC3 being cleaved at its C terminus by ATG4 (to generate LC3-I), exposing a glycine residue that then binds to ATG7 through a thioester linkage, before being transferred to ATG3 where it is conjugated to phosphatidylethanolamine (to produce LC3-II). Formation of LC3-II occurs only during active autophagy as it is regulated by the membrane curvature sensing domain of ATG3, which switches on enzymatic activity at membranes characteristic of a growing phagophore.

In our recent study, we found that H₂O₂ treatment, used to mimic oxidative stress, leads to rapid impairment of amino acid starvation-induced LC3 lipidation. This ability of H₂O₂ to inhibit LC3 lipidation also prevents its translocation to the phagophore membrane, observed using fluorescence and electron microscopy. These findings are consistent with studies using a mouse model of Duchenne muscular dystrophy, where increased CYBB/Nox2-derived ROS leads to impaired autophagy.

To investigate the mechanism responsible for loss in LC3 lipidation in cells exposed to H₂O₂, autophagy was stimulated downstream of MTOR using a Tat-beclin 1 peptide. As LC3 lipidation, induced by Tat-beclin 1, is also inhibited by H₂O₂ this means the target of this oxidant is downstream of MTOR and BECN1/beclin1. As H₂O₂ leads to a rapid loss in the ability to lipidate LC3, we reasoned that the likely targets of this oxidant are the enzymes involved in its conjugation to phosphatidylethanolamine. On assessing ATG3 and ATG7 under nonreducing conditions, we discovered that when inactive these enzymes form stable thioester complexes with LC3. This was observed on non-reducing immunoblots by the formation of higher molecular mass complexes (approximately an additional 15 kDa). The identity of these complexes was substantiated by co-immunoprecipitation and siRNA knockdown experiments, as well as formation with recombinant protein. Stable thioester complexes between LC3 and both ATG3 and ATG7 become transient upon the stimulation of autophagy due to enzymatic activation, thus allowing LC3 transfer and conjugation to phosphatidylethanolamine. Loss in this stable interaction with LC3 during active autophagy sensitizes ATG3 and ATG7 to thiol oxidation under pro-oxidizing conditions. The oxidation of ATG3 and ATG7 under pro-oxidizing conditions that is potentiated upon stimulation of autophagy, leads to the formation of an intermolecular disulfide-bound complex. Using catalytic residue mutants of ATG3 (C264A) and ATG7 (C572A) we confirmed these as the sites that form the intermolecular disulfide. The formation of an oxidized covalent bound heterodimeric complex is consistent with the crystal structure of yeast Atg3 and Atg7, where catalytic thiols are in close proximity, allowing transfer of LC3 but also disulfide formation. As well as a disulfide, it is likely that under pro-oxidizing conditions S-glutathiolation of catalytic thiols on ATG3 and ATG7 could also inhibit enzymatic activity. This is consistent with experiments where oxidized glutathione inhibits ATG3 and ATG7 activity, as well as preventing their covalent interaction with LC3.

The oxidation of catalytic thiols on ATG3 and ATG7 that prevents LC3 lipidation also occurs in response to endogenous oxidant formation. In cells exposed to oxidized low-density lipoprotein (oxLDL), a disulfide ATG3–ATG7 complex can be detected as well as a loss in LC3 lipidation. This finding is consistent with the ability of oxLDL to induce reactive oxygen species formation from ALOX/lipoxygenase and mitochondrial pathways, which are implicated in the development of atherosclerotic plaque formation. Therefore, it is conceivable that loss in autophagy due to ATG3–ATG7 oxidation may contribute to the development of atherosclerosis, potentially by compromising vascular smooth muscle cell viability. As well as cells exposed to oxLDL, an increase in ATG3 and ATG7 oxidation is observed in the aorta of food-withdrawn aged mice compared to young mice. This increase in ATG3 and ATG7 oxidation in aged mice correlates with a loss of autophagy, through impaired LC3 lipidation and accumulation of SQSTM1/p62, as well as detection of a more oxidizing environment that causes PRDX/peroxiredoxin hyperoxidation and increased protein carbonylation.

Therefore, in our study we have identified a molecular mechanism for impaired autophagy under conditions of oxidative stress, which is due to the direct oxidation of ATG3 and ATG7 (Figure 1). This process is highly pertinent, as perturbations in cellular redox have been causatively linked to the pathogenesis of many diseases broadly characterized by dysregulated autophagy. Furthermore, this process may account, at least in part, for impaired autophagy during aging.

Figure 1.

When autophagy is inactive, ATG3 and ATG7 form a stable thioester with LC3 that becomes transient upon stimulation of the autophagy pathway. Transient interaction of ATG3 and ATG7 with LC3 exposes catalytic thiols, which in the presence of reactive oxygen species (ROS) undergo oxidation to form a disulfide heterodimer or a glutathione (GS) adduct. The oxidation of catalytic thiols on ATG3 and ATG7 prevents the interaction with, and lipidation of, LC3 required for functional autophagy.

Funding Statement

The British Heart Foundation (FS/14/1/30551 and FS/13/55/30643).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.