Activity of Ubiquitin-dependent Pathway in Response to Oxidative Stress

Abstract

Relations between the ubiquitin pathway and cellular stress have been noted, but data regarding responses of the ubiquitin pathway to oxidative stress are scanty. This paper documents the response of this pathway to oxidative stress in lens cells. A brief exposure of lens epithelial cells to physiologically relevant levels of H 2 O 2 induces a transient increase in activity of the ubiquitindependent pathway. Ubiquitin conjugation activity was maximal and increased 3.5-9.2-fold over the activity noted in untreated cells by 4 h after removal of H 2 O 2 . By 24 h after removal of H 2 O 2 , ubiquitin conjugation activity returned to the level noted in untreated cells. In parallel to the changes in ubiquitin conjugation activity, the activity of ubiquitin-activating enzyme (E1), as determined by thiol ester formation, increased 2-6.7-fold during recovery from oxidation. Addition of exogenous E1 resulted in an increase in ubiquitin conjugation activity and in the levels of ubiquitin carrier protein (E2)-ubiquitin thiol esters in both the untreated cells and the H 2 O 2 -treated cells. These data suggest that E1 is the rate-limiting enzyme in the ubiquitin conjugation process and that the increases in ubiquitin conjugation activity which are induced upon recovery from oxidation are primarily due to increased E1 activity. The oxidation-and recovery-induced up-regulation of E1 activity is primarily due to post-synthetic events. Substrate availability and up-regulation of E2 activities also appear to be related to the enhancement in ubiquitinylation upon recovery from oxidative stress. The oxidationinduced increases in ubiquitin conjugation activity were associated with an increase in intracellular proteolysis, suggesting that the transient increase in ubiquitinylation noted upon recovery from oxidative stress may play a role in removal of damaged proteins from the cells.

Ubiquitination capabilities in response to neocarzinostatin and H2O2 stress in cell lines from patients with ataxia-telangiectasia

“…Recently, we demonstrated that the ubiquitin pathway is also regulated by cellular redox status (JahngenHodge et al, 1997;Obin et al, 1999;Shang et al, 1997b) and that it is the major cellular pathway for removal of oxidized proteins Shang et al, 1997a). Taken together with data which suggest elevated oxidant sensitivity in A-T cells, it would appear that sequelae due to the absence of a functional ATM protein might involve the ubiquitin pathway as well.…”

“…We used H 2 O 2 in further experiments because (1) this would allow us to compare these results with prior information regarding the e ects of H 2 O 2 on ubiquitination capabilities in other types of cells (from both fast and slowly metabolizing tissues), (2) H 2 O 2 is encountered in many physiological conditions, and (3) because there was little data which related NCS treatment to alterations in redox status (Adamo et al, 1999;Jahngen-Hodge et al, 1997;Obin et al, 1998;Ramanathan et al, 1999;Shang et al, 1997b). The experiments were done at three cell densities: 0.3610 6 , 0.6610 6 and 1.0610 6 cells/mL in order to assure that cell density alone did not cause changes in ubiquitin conjugates.…”

“…(a) Wild type cells. (b) Cells from an A-T patient Shang et al, 1997b). Thus, we sought to (1) further characterize and compare the ubiquitination response in A-T and wt cells, and (2) determine if changes in the ubiquitination response were associated with changes in redox status using a more frequently employed and physiologically relevant oxidant.…”

“…Milder physiological oxidation regimes (Shang et al, 1997b) also indicate elevations in levels of ubiquitin conjugates but show either unchanged or slightly increased activity of the ubiquitin conjugating machinery. In this study we compared four lymphoblastoid cell lines from healthy individuals and three A-T cell lines for indicators of oxidative stress associated with the ubiquitin system: levels of endogenous ubiquitin conjugates, ability to form ubiquitin conjugates de novo, thiol-ester forming ability of E1 and E2s, and levels of GSH and GSSG, with and without treatment with the NCS or the physiological oxidant H 2 O 2 .…”

“…Experiments have implicated ubiquitination or ubiquitin dependent protein degradation in virtually every facet of cellular regulation, including: cell cycle progression, di erentiation, stress responses, DNA repair, signal transduction, gene regulation, control of proto-oncogenes, antigen presentation, and removal of damaged proteins (Haas and Siepmann, 1997a,b;Jahngen-Hodge et al, 1997;Johnston et al, 1998;Kornitzer and Ciechanover, 2000;Obin et al, 1999;Shang et al, 1997a; for reviews see Ciechanover, 1994;Ciechanover et al, 1985;Hochstrasser, 1996;Huang et al, 1995;Jentsch and Schlenker, 1995;Shang et al, 1997b;Shang and Taylor, 1995;Varshavsky, 1997). Most, but not all, of these functions are accomplished by degrading or preventing the degradation of relevant regulatory proteins.…”