Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis

Abstract

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.

Autophagy promotes cell and organismal survival by maintaining NAD(H) pools

“…Mammalian cells: Immunoblotting on cells was performed as described previously 42 . In brief, cells were lysed on ice in RIPA buffer (Sigma) supplemented with 1x Halt TM protease and phosphatase inhibitor cocktail (Thermo Scientific).…”

“…Insoluble material was removed by centrifugation and the supernatant was subjected to SDS-PAGE analysis. D. melanogaster: Fly sample preparation and immunoblotting were performed as described previously 42 . Briefly, 20 flies per group were homogenized on ice in a specialised buffer (1.5% Triton X-100 (Promega), complete mini EDTA-free proteinase inhibitor (Sigma), PBS 1X).…”

“…Immunofluorescence Immunofluorescence analysis was performed on primary human fibroblasts as described previously 42 . In brief, cells were washed once with 1x PBS (New England Biolabs) and fixed and permeabilised in 100% pre-chilled methanol (Fisher Scientific) for 5 min at -20 °C.…”

“…Densitometry analyses on immunoblots were performed by ImageJ (version 1.41; NIH) software as described previously 42 . Data of the control condition was normalised to 100% and graphical data denote the mean ± s.e.m.…”

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation

“…Autophagy is an evolutionarily conserved degradation system of defective proteins and subcellular structures, and an early event in autophagy induction is the nuclear translocation and transactivation of the basic Helix-Loop-Helix transcription factor HLH-30 39,38 . Oxidative stress is one of the various conditions that activate autophagy (reviewed in 64,65 ) and several components of the autophagy machinery are redox regulated by ROS-mediated mechanisms in different organisms 66,67,68 . Using a HyPer fluorescent biosensor 69 we found that gsr-1(m+,z-) worms have wild type levels of H2O2 (A. Miranda-Vizuete, unpublished results), suggesting that they are not under oxidative stress and consistent with the lack of HLH-30::GFP nuclear translocation or increased SQST-1:GFP foci in these animals.…”