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Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, β-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis. Full article

Catabolism of macromolecules is a major event in senescent cells, especially involving proteolysis of organelles and abnormally aggregated proteins, circulation of nutrients, and precise control of intracellular environmental balance. Proteasomes are distributed in the nucleus and cytoplasm; however, proteasomes in organelles are limited. In this study, multi-omics proteomic analyses of ubiquitinated proteins enriched by using antibody against “di-Gly-Lys” via a free labeling were used to investigate the global changes of protein levels and ubiquitination modification levels of upl5 mutant relative to wild-type plant; subcellular localization analysis of UPL5 was found to be located in the nucleus, cytoplasm, and plastid within the cell; and the direct lysine site patterns of UPL5 were screened by the H89R substitution in the tagged ubiquitinated assay. It suggests that UPL5 acting as a candidate of organelle E3 ligase either in the nucleus or cytoplasm or plastid modifies numerous targets related to nuclear transcription and plastid photosynthesis involving in Ca²⁺ and hormone signaling pathway in plant senescence and in response to (a)biotic stress protection. Full article