Expression of K6W‐ubiquitin in the lens perturbs calcium homeostasis and results in calpain hyperactivation and differentiation abnormality

Abstract

Ubiquitin Proteasome Systems (UPS) control multiple cellular processes but less is known about its roles in regulation of differentiation. The lens must remain clear to function. Precipitation of some lens protein, or their truncated products, result in lens opacification, or cataract. K6W‐Ub mutant enters into conjugates but such conjugates are proteolytically incompetent. There is a dose dependent relationship between expression of K6W‐Ub and the extent of lens aberrations. To initiate elucidation of effects of K6W‐Ub on lens development, we compared protein profiles of lenses from transgenic mice that express wild type ubiquitin (WT‐Ub) or K6W‐Ub. Only ~2% of the proteome was different. Among these are fragmented forms of critical lens proteins including vimentin, γ‐crystallin, filensin, fodrin (spectrin alpha 2), Caprin2 and Tdrd7. In the K6W‐Ub lens, there is a massive accumulation of Ca2+ and hyper activation of calpain. We also observed accumulation of gap junction connexin 43 and diminished connexin 46 levels, consistent with the accumulation of Ca2+ and retention of the epithelial cell type. Corroborating in vitro tests reveal for the first time an association between mutation of K6 on ubiquitin, altered UPS, Ca2+ level regulation, calpain activity, substrate cleavage, and differentiation abnormality. The data are critical to understanding the role of UPS in proper organogenesis.