Anna J Zandy1, Steven Bassnett. 1. Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Abstract
PURPOSE: To remove light-scattering structures from the visual axis, all intracellular organelles are eliminated from cells in the center of the developing ocular lens. Organelle degradation is accompanied by an increase in VEIDase (caspase-6-like) activity, but data from caspase-null mice suggest that the lens VEIDase is not caspase-6. The goal of the present work was to identify the lens VEIDase and determine whether it plays a role in organelle breakdown. METHODS: The approximate molecular mass of the lens VEIDase was determined by size-exclusion chromatography. Three proteasome inhibitors (NLVS, MG132, and clasto-lactacystin beta-lactone) were tested for their ability to inhibit lens VEIDase activity. Lens lysates were immunodepleted of proteasomes using an antibody against the 20S proteasome. To inhibit the ubiquitin-proteasome pathway (UPP) in vivo, lactacystin was injected into the vitreous humor of the developing chicken eye. The effect of lactacystin on mitochondrial degradation was assessed by examining the disappearance of succinate-ubiquinone oxidoreductase, an integral protein of the inner mitochondrial membrane. RESULTS: The lens VEIDase eluted at approximately 700 kDa from a size-exclusion column and was inhibited by the proteasome inhibitors NLVS, MG132, and clasto-lactacystin beta-lactone. In vivo, the trypsin-like activity of the proteasome was reduced by 60% to 70% after lactacystin injection. Proteasome inhibition was associated with the accumulation of ubiquitinated proteins and reversible opacification of the lens cortex. In lactacystin-injected eyes, the programmed degradation of succinate-ubiquinone oxidoreductase was inhibited in the central lens fiber cells. CONCLUSIONS: These data suggest that lens VEIDase activity is attributable to the proteasome and that the UPP may function in the removal of organelle components during lens fiber cell differentiation.
PURPOSE: To remove light-scattering structures from the visual axis, all intracellular organelles are eliminated from cells in the center of the developing ocular lens. Organelle degradation is accompanied by an increase in VEIDase (caspase-6-like) activity, but data from caspase-null mice suggest that the lens VEIDase is not caspase-6. The goal of the present work was to identify the lens VEIDase and determine whether it plays a role in organelle breakdown. METHODS: The approximate molecular mass of the lens VEIDase was determined by size-exclusion chromatography. Three proteasome inhibitors (NLVS, MG132, and clasto-lactacystin beta-lactone) were tested for their ability to inhibit lens VEIDase activity. Lens lysates were immunodepleted of proteasomes using an antibody against the 20S proteasome. To inhibit the ubiquitin-proteasome pathway (UPP) in vivo, lactacystin was injected into the vitreous humor of the developing chicken eye. The effect of lactacystin on mitochondrial degradation was assessed by examining the disappearance of succinate-ubiquinone oxidoreductase, an integral protein of the inner mitochondrial membrane. RESULTS: The lens VEIDase eluted at approximately 700 kDa from a size-exclusion column and was inhibited by the proteasome inhibitors NLVS, MG132, and clasto-lactacystin beta-lactone. In vivo, the trypsin-like activity of the proteasome was reduced by 60% to 70% after lactacystin injection. Proteasome inhibition was associated with the accumulation of ubiquitinated proteins and reversible opacification of the lens cortex. In lactacystin-injected eyes, the programmed degradation of succinate-ubiquinone oxidoreductase was inhibited in the central lens fiber cells. CONCLUSIONS: These data suggest that lens VEIDase activity is attributable to the proteasome and that the UPP may function in the removal of organelle components during lens fiber cell differentiation.
Authors: M Joseph Costello; Marc Kantorow; Lisa A Brennan; Subharsee Basu; Daniel Chauss; Ashik Mohamed; Kurt O Gilliland; Sönke Johnsen; Sue Menko Journal: Exp Eye Res Date: 2013-09-04 Impact factor: 3.467
Authors: Andrea Caceres; Fu Shang; Eric Wawrousek; Qing Liu; Orna Avidan; Ales Cvekl; Ying Yang; Aydin Haririnia; Andrew Storaska; David Fushman; Jer Kuszak; Edward Dudek; Donald Smith; Allen Taylor Journal: PLoS One Date: 2010-10-20 Impact factor: 3.240