Links Between Ubiquitin Biology, Calcium Regulation, Lens Organogenesis And Cataract

Abstract

Purpose: : Lenses from mice that express high levels of K6W-ubiquitin (K6W-Ub) against a wt-Ub background show cataracts at birth. The goal of this study was to determine developmental processes and the molecular targets that are affected by expressing K6W-Ub and that result in cataract.

Methods: : K6W-Ub was expressed in lens using an α-crystallin promoter (Caceres PLoS 2010). K6W and WT lenses from new born mice were collected at p1. Expression of proteins was determined using mass spectrometry, western blotting and immunohistochemistry. Ubiquitination targets with K6W-Ub or wt-Ub were investigated by in situ ubiquitination assay on arrays with 8000 proteins. Ca²⁺ concentrations were determined by injecting the calcium-sensitive dye, Fura2, into lens cells at various depths. Lens ultrastructure was examined by electron microscopy.

Results: : We observed stabilization of Cx43 (a Nedd4 substrate) by western and IHC in K6W-Ub lenses. Nedd4 is an ubiquitin conjugating enzyme. Protein array conjugation assays indicated more polyubiquitination of established Nedd4 substrates by K6W ubiquitin, consistent with observation that substrates that are polyubiquitinated by K6W-Ub resist degradation (Shang J.Biol.. Chem. 2005). Cx 43 was not on the array. K6W-Ub lenses also show abnormal intermediate filaments and F-actin. Whereas the intracellular Ca²⁺ varied from 351 nM in the primary fibers of p1 lenses to 123 nM at the surface, Ca²⁺ in lenses expressing K6W ubiquitin varied from 1693 nM at the center to 394 nM at the surface.

Conclusions: : Our data indicate 1) that a properly functioning ubiquitin proteolytic system is required for proper lens development. Expressing K6W-Ub 2) slows the developmental program, 3) results in stabilization of molecules (Cx43) that may be associated with decreased Cx46 and impaired Ca²⁺ outflow. We hypothesize that this results in the marked elevation of lens Ca²⁺ in the primary fibers in K6W-Ub lenses, as compared to lenses in which wt Ub was overexpressed. The data are consistent with enhanced calpain activity in K6W-Ub lenses and they rationalize our observations of extensively fragmented fodrin, vimentin, and filensin in these lenses.

Keywords: proteolysis • calcium • cataract