Oxidative Modification of Proteasome:  Identification of an Oxidation-Sensitive Subunit in 26 S Proteasome

Abstract

Reactive oxygen species (ROS) have the potential to damage cellular components, such as protein, resulting in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress, J. Biol. Chem. 276, 12076-12083]. In the present study, to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we analyzed the protein carbonylation on SH-SY5Y cells when these cells were submitted to an endogenous inducer of ROS production. Upon exposure of SH-SY5Y cells to this endogenous electrophile, we observed significant accumulation of protein carbonyls within the cells. Proteomic analysis of oxidation-sensitive proteins showed that the major intracellular target of protein carbonylation was one of the regulatory subunits in 26 S proteasome, S6 ATPase. Accompanied by a dramatic increase in protein carbonyls within S6 ATPase, the electrophile-induced oxidative stress exerted a significant decrease in the S6 ATPase activities and a decreased ability of the 26 S proteasome to degrade substrates. Moreover, in vitro oxidation of 26 S proteasome with a metal-catalyzed oxidation system also confirmed that S6 ATPase represents the most oxidation-sensitive subunit in the proteasome. These and the observation that down-regulation of S6 ATPase by RNA interference resulted in the enhanced accumulation of ubiquitinated proteins suggest that S6 ATPase is a molecular target of ROS under conditions of electrophile-induced oxidative stress and that oxidative modification of this regulatory subunit of proteasome may be functionally associated with the altered recognition and degradation of proteasomal substrates in the cells.

S-Glutathionylation of the Rpn2 Regulatory Subunit Inhibits 26 S Proteasomal Function

“…Additionally, oxidation of Rpn1 and Rpn2 did not affect their association with the 20 S CP. However, in contrast to our study, diminished 26 S proteolytic activity has been found to be accompanied by a loss of 26 S proteasomal structure following exposure to metal-catalyzed oxidation as a result of carbonylation involving one of the S6 ATPase regulatory subunits (58). Strong oxidation of the 26 S proteasome under non-physiologic conditions can occur by exposure to hydroxyl radical generated in metal catalyzed reactions; in the present study, exposure of the 26 S proteasome to GSH and H 2 O 2 was performed in the presence of metal chelator diethylenetriaminepentaacetic acid, eliminating the possibility of such hydroxyl radical-associated events.…”

“…Our Although the present experiments, as well as findings from previous studies (58), demonstrate that H 2 O 2 can induce functionally relevant oxidative modifications of 26 S subunits that directly result in diminished proteasomal activity, it is possible that increased intracellular H 2 O 2 concentrations may also modulate proteasomal function through other mechanisms. For example, decreased expression of Rpn11, a 19 S RP subunit, was found to be associated with aging (59), a condition known be accompanied by increased ROS formation (60).…”

Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

“…Oxidation and glutathionylation itself may affect proteasome activity (51,52). Previous studies have shown that proteasomes are prone to oxidation (53).…”

Proteasome-dependent regulation of signal transduction in retinal pigment epithelial cells

“…S-Nitrosylation could also inactivate these enzymes (Yao et al, 2004). Reactive oxygen species and reactive lipid peroxidation products, such as 4-hydroxynonenal, also impair the function of the proteasome (Conconi et al, 1998;Okada et al, 1999;Caballero et al, 2003;Ishii et al, 2005). Since the UPP plays critical roles in almost every aspect of cellular function, abnormal regulation or inactivation of this pathway has been implicated in many age-related diseases, such as Alzheimer's disease (Hope et al, 2003), Parkinson's disease (Dawson and Dawson, 2003), and cataract (Jahngen-Hodge et al, 1992;Shang et al, 1997;Shang et al, 2001;Dudek et al, 2005).…”