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, 9 (1), 256

Oxidation of SQSTM1/p62 Mediates the Link Between Redox State and Protein Homeostasis


Oxidation of SQSTM1/p62 Mediates the Link Between Redox State and Protein Homeostasis

Bernadette Carroll et al. Nat Commun.


Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.

Conflict of interest statement

The authors declare no competing financial interests.


Fig. 1
Fig. 1
p62 forms oligomers and aggregates in response to oxidation. a Mouse brain tissue, young (3 months) and old (24 months) analysed by immunoblotting for p62, PRDX-SO3 and actin as a loading control in reducing (2.5% β-ME) and non-reducing conditions. DLC disulphide-linked conjugates. Arrows indicate the positions of monomeric and oligomeric p62. b Representative images and quantification of p62 aggregates in old mouse Purkinje cells in the cerebellum. Green arrows and red arrowheads indicate Purkinje cells positive and negative for p62 aggregates, respectively. c Effect of autophagy inhibition (bafilomycin A1 (Baf, 400 nM, 4 h) and chloroquine (CQ, 50 μM, 4 h)) and oxidative stress (H2O2 (3 mM, 10 min) and PR-619 (5 μM, 30 min)) on p62 DLC (c) and p62 aggregation (d) in HeLa cells. d Anti-p62 staining analysed by confocal microscopy. Error bars represent s.e.m., n = 3, *P < 0.05, **P < 0.01, ***P < 0.005 (unpaired t-tests). Asterisk indicates a non-specific band. Scale bar: 20 µm
Fig. 2
Fig. 2
Two conserved cysteine residues located in a disordered region of p62 are required for formation of DLC and ubiquitylated aggregates. a Alignment showing cysteines 105 and 113 (highlighted) are conserved in vertebrates. Increasing conservation across species is shown by light-to-dark red. b p62−/− MEFs stably expressing either wild type or C105A,C113A FLAG-p62 were treated with H2O2 (3 mM, 1 min) or PR-619 (5 μM, 10 min) and immunoblotted in non-reducing conditions. c Formation of ubiquitylated, p62-positive aggregates in stable cells described in b following oxidative stress (1 mM H2O2 or 5 μM PR-619 for 30 min). Cells were immunostained for ubiquitin and p62, analysed by confocal microscopy (c) and quantified (d). Error bars represent s.e.m., n = 3, **P < 0.01, ***P < 0.005 (unpaired t-tests). Arrows indicate the positions of monomeric and oligomeric p62. Scale bar: 10 µm
Fig. 3
Fig. 3
Oxidation-sensitive p62 is required for pro-survival autophagy. a p62−/− MEFs stably expressing FLAG-tagged wild type or C105A,C113A p62 were treated with cycloheximide (CHX, 50 μg/ml) and H2O2 (1 mM), either in the absence (a) or presence (b) of bafilomycin A1 (Baf, 50 nM), lysed at the indicated time post treatment, immunoblotted for p62 and quantified. c Cells described in a plus one stably expressing K7A,D69A PB1-domain mutant of p62 were treated with H2O2 (1 mM, 5 h), lysed and immunoblotted for ubiquitin, LC3, p62 and actin (c) and quantified (d). e, f Stable p62 cell lines in control conditions were fixed and stained for p62 and LC3 (e) and the % cells with >20 autophagosomes was quantified (f). g, h Electron microscopy of stable p62 cell lines. White arrows: autophagosomes; black arrows: autolysosomes; blue arrow: endosome. Scale bar: 500 nm (g). Autophagosomes were quantified and graphs represent average number and % cells with three or more autophagosomes per field of view (h). i, j Stable p62 cell lines were treated as in c and % cell death was analysed by Ready Probes fluorescent dyes (i). j Stable p62 cell lines were treated as indicated and % cell death was analysed as in i. Error bars represent s.e.m., n = 3, *P < 0.05, **P < 0.01, ***P < 0.005 (unpaired t-tests). NS not significant. Scale bar: 20 µm (IF) and 500 nm (TEM)
Fig. 4
Fig. 4
Oxidation-sensitive p62 is important for the oxidative stress resistance of flies and is perturbed in human age-related disease. a Whole-fly lysates were analysed in reducing (2.5% β-ME) and non-reducing conditions for p62 homologue, Ref(2)P. Asterisk indicates a non-specific band. b Diagram representing the introduction of an 18 amino acid fragment of human p62 containing C105 and C113 to produce a ‘humanised’ Ref(2)P (Ref(2)Pox) in flies using CRISPR/Cas9. c Wild-type (WT) and Ref(2)Pox flies were treated with paraquat (PQ, 20 mM) for 12 h and whole-fly lysates were analysed by immunoblot for ubiquitin, Ref(2)P and tubulin and quantified. Error bars represent s.e.m., n = 3 (at least 10 flies per group per replicate); *P < 0.05 (unpaired t-test). d Combined survival data (of three repeats) of wild-type versus Ref(2)Pox flies in the presence of 20 mM PQ. Survival was assessed every 12 h. At least 60 flies per group per replicate were used and log-rank statistics applied. e p62−/− MEFs stably expressing FLAG-tagged wild-type p62, C105A,C113A p62 or ALS-associated mutation K102E p62 were treated with H2O2 (500 μM, 1 min) and PR-619 (20 μM, 10 min), analysed for p62 DLC formation in non-reducing conditions and quantified. Error bars represent s.e.m., n = 3; ***P < 0.005 (unpaired t-tests). f Diagram of the proposed role for p62 oxidation in the aggregation and degradation of autophagy substrates. Formation of p62 DLC is triggered by oxidative stress, which promotes degradation of p62 and bound substrates (e.g. ubiquitylated proteins) through autophagy. Mutations in p62 sequence (e.g. ALS-related K102E) can impair the formation of DLC

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