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Elizabeth Whitcomb, Min-Lee Chang, Allen Taylor; The Ubiquitin Conjugating Enzyme, UbcH7, Affects Both Migration and the Epithelial to Mesenchymal Transition in Lens Epithelial Cells:A Promising Drug Target for Secondary Cataracts.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3213.
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© ARVO (1962-2015); The Authors (2016-present)
The post translational modification of proteins by ubiquitin controls many essential cellular processes by targeting substrates for degradation, facilitating interactions with other proteins, or directing relocalization within the cell. Our studies on the ubiquitin conjugating enzyme, UbcH7, elucidated an atypical regulation of the cyclin dependent kinase inhibitor p27. In contrast to expectations, expression of UbcH7 protects p27 from degradation. This protection involves formation of non-degradable ubiquitin conjugates on p27. Additionally, we determined that UbcH7 affected p27 primarily in the cytosol. Cytosolic p27 has been shown to affect both cell migration and the epithelial to mesenchymal transition (EMT) in other cell types. Given that the development of secondary cataracts is dependent on both cell migration and EMT, we asked whether the regulation of UbcH7 affected these processes in lens epithelial cells.
We developed a cell free assay using lens epithelial cell lysates and His-tagged-p27 to detect specific ubiquitination of p27. Various mutants of ubiquitin were tested to probe the composition of the ubiquitin conjugates catalyzed by UbcH7. UbcH7 was depleted from lens epithelial cells using siRNA and plasmids were used to over-express both wild-type UbcH7 as well as its active site mutant. Cell migration was determined by a scratch wound assay. EMT was assayed by treating lens epithelial cells with TGFβ and detecting changes in mesenchymal markers.
UbcH7 is able to catalyze several different types of ubiquitin chains on p27.Some of these ubiquitin chains target substrates for degradation, but others are not proteasome targets. Depletion of UbcH7 impairs cell migration in several cell types including lens epithelial cells. Over expression of the wild type UbcH7 protein promotes cell migration while over expression of the active site mutant of UbcH7 impairs cell migration. Depletion of UbcH7 inhibits EMT in lens epithelial cells.
Ubiquitination by UbcH7 catalyzes formation of degradation incompetent p27-ubiquitin conjugates, thereby protecting it from degradation. Depletion of UbcH7 impairs both migration and EMT of lens epithelial cells, suggesting it might be an attractive drug target to inhibit the development of secondary cataracts.
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