Abstract
Abstract: :
Purpose:Accumulation and precipitation of oxidatively modified proteins is causally related to cytotoxicity and age–related damage in many types of cells and tissues. It would appear that selective proteolytic activity is essential to maintain cellular function and delay age– or stress–related dysfunctions which are related to the accumulation of oxidatively damaged proteins. Because it offers opportunities for selectivity, we hypothesized that the ubiquitin–dependent proteolytic pathway plays a role in the selective removal of oxidatively modified proteins. Methods: His6–labeled variants of ubiquitin were constructed in which K6 was replaced with W. The K6W variant was expressed in human lens epithelial cells (HLEC) and in 293 cells via a replication deficient adenovirus vector. Results: High levels of expression were achieved as indicated by higher levels of monomeric ubiquitin in cell lysates. When HLEC were briefly exposed to H2O2 levels of ubiquitin conjugates increased. Levels increased more when the ubiquitin variant was expressed as compared with native ubiquitin. This is because rates of deubiquitination and proteolysis are slower when the K6W ubiquitin variant is incorporated into the conjugates. Oxidative stress also induced a transient increase in protein carbonyls, an indicator of oxidative modification. Immunoblots using anti DNP to detect carbonyls of ubiquitin conjugates which were affinity purified by Ni chromatography indicated that the ubiquitin conjugates had at least four–fold more carbonyls than unbound proteins. Cell viability was diminished when the ubiquitin variant was expressed and when proteolysis via the ubiquitin pathway was inhibitedConclusion: These data indicate that the ubiquitin pathway shows selectivity for oxidized proteins. and they establish an essential role for the ubiquitin pathway in response to oxidative stress. Supported by NIH grants EY13250 (AT), EY11717 (FS), a Core grant to the Tufts Center for Vision Research (EY13078), and the U.S. Department of Agriculture, under agreement No. 58–1950–9–001
Keywords: proteolysis • aging • cataract