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P62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium.

ABSTRACT: As a signaling hub, p62/sequestosome plays important roles in cell signaling and degradation of misfolded proteins. p62 has been implicated as an adaptor protein to mediate autophagic clearance of insoluble protein aggregates in age-related diseases, including age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE). Our previous studies have shown that cigarette smoke (CS) induces oxidative stress and inhibits the proteasome pathway in cultured human RPE cells, suggesting that p62-mediated autophagy may become the major route to remove impaired proteins under such circumstances. In the present studies, we found that all p62 mRNA variants are abundantly expressed and upregulated by CS induced stress in cultured human RPE cells, yet isoform1 is the major translated form. We also show that p62 silencing exacerbated the CS induced accumulation of damaged proteins, both by suppressing autophagy and by inhibiting the Nrf2 antioxidant response, which in turn, increased protein oxidation. These effects of CS and p62 reduction were further confirmed in mice exposed to CS. We found that over-expression of p62 isoform1, but not its S403A mutant, which lacks affinity for ubiquitinated proteins, reduced misfolded proteins, yet simultaneously promoted an Nrf2-mediated antioxidant response. Thus, p62 provides dual, reciprocal enhancing protection to RPE cells from environmental stress induced protein misfolding and aggregation, by facilitating autophagy and the Nrf2 mediated antioxidant response, which might be a potential therapeutic target against AMD.

SUBMITTER: Wang L

PROVIDER: S-EPMC4019388 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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p62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium.

Wang Lei L Cano Marisol M Handa James T JT

Biochimica et biophysica acta 20140322 7

As a signaling hub, p62/sequestosome plays important roles in cell signaling and degradation of misfolded proteins. p62 has been implicated as an adaptor protein to mediate autophagic clearance of insoluble protein aggregates in age-related diseases, including age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE). Our previous studies have shown that cigarette smoke (CS) induces oxidative stress and inhibits the proteasome pathway i ...[more]

PMID: 24667411

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Project description:Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPKα1 (S485), acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.

Project description:BackgroundDJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress.MethodologyRetinal pigment epithelial (RPE) cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S) prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS) generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch's membrane (BM)/choroid from AMD eyes.Principal findingsDJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes.Conclusions/significanceDJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.

Dataset Information

P62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium.

Publications

p62 provides dual cytoprotection against oxidative stress in the retinal pigment epithelium.

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