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Epoxomicin, a Selective Proteasome Inhibitor, Activates AIM2 Inflammasome in Human Retinal Pigment Epithelium Cells

ABSTRACT: Emerging evidence suggests that the intracellular clearance system plays a vital role in maintaining homeostasis and in regulating oxidative stress and inflammation in retinal pigment epithelium (RPE) cells. Dysfunctional proteasomes and autophagy in RPE cells have been associated with the pathogenesis of age-related macular degeneration. We have previously shown that the inhibition of proteasomes using MG-132 activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome in human RPE cells. However, MG-132 is a non-selective proteasome inhibitor. In this study, we used the selective proteasome inhibitor epoxomicin to study the effect of non-functional intracellular clearance systems on inflammasome activation. Our data show that epoxomicin-induced proteasome inhibition promoted both nicotinamide adenine dinucleotide phosphate oxidase and mitochondria-mediated oxidative stress and release of mitochondrial DNA to the cytosol, which resulted in potassium efflux-dependent absence in melanoma 2 (AIM2) inflammasome activation and subsequent interleukin-1β secretion in ARPE-19 cells. The non-specific proteasome inhibitor MG-132 activated both NLRP3 and AIM2 inflammasomes and oxidative stress predominated as the activation mechanism, but modest potassium efflux was also detected. Collectively, our data suggest that a selective proteasome inhibitor is a potent inflammasome activator in human RPE cells and emphasize the role of the AIM2 inflammasome in addition to the more commonly known NLRP3 inflammasome.

SUBMITTER: Sridevi Gurubaran I

PROVIDER: S-EPMC9311580 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

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Epoxomicin, a Selective Proteasome Inhibitor, Activates AIM2 Inflammasome in Human Retinal Pigment Epithelium Cells

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