The Effect of CYP2E1-Dependent Oxidant Stress on Activity of Proteasomes in HepG2 Cells

Abstract

A reduction in proteasome activity and accumulation of oxidized proteins may play a role in alcoholic liver disease. The current study assessed proteasome peptidase activities and oxidative modifications of proteasomes during oxidative stress generated by CYP2E1. The model of toxicity by arachidonic acid (AA) and iron [ferric-nitrilotriacetate (Fe-NTA)] in HepG2 cells overexpressing CYP2E1 (E47 cells) and control C34 cells was used. AA/Fe-NTA treatment decreased trypsin-like (T-L) activity of the proteasome in E47 cells but not in C34 cells. This inhibition was abolished by antioxidants. Chymotrypsin-like activity of the proteasome was increased in E47 cells, and activity was not altered by AA/Fe-NTA treatment. There were no changes in content of subunits of 20S proteasomes or 19S regulator ATPase subunits S4 and p42 by AA/Fe-NTA treatment. An increased content of the PA28␣ subunit of the 11S regulator of proteasomes was detected in E47 cells. In proteasome pellets, the decline of T-L activity was accompanied by increased content of carbonyl adducts, suggesting oxidative modification of proteasomes. Higher levels of ubiquitinated, 3-nitrotyrosine-and 4-hydroxynonenal-modified proteins and lower levels of free ubiquitin were detected in untreated E47 cells in comparison with C34 cells. Accumulation of protein cross-linked, detergent-insoluble aggregates was increased with AA/Fe-NTA treatment in E47 cells. Thus, reactive oxygen species generated upon CYP2E1-dependent oxidative stress mediated a decline in T-L proteasome function, increased carbonyl adducts in proteasomes, and promoted protein aggregate formation; this may alter the balance among protein oxidation, ubiquitination, and degradation.

A Boronic-Chalcone Derivative Exhibits Potent Anticancer Activity through Inhibition of the Proteasome

“…Previous studies showed that inhibition of the proteasome activity is a major mechanism that causes increased accumulation of ubiquitinated proteins, which are normally degraded by the cellular proteasome machinery (Chung et al, 2005;Kessova and Cederbaum, 2005;Marteijn et al, 2005;Zhang et al, 2005). We therefore tested whether AM114 might inhibit proteasome activity, leading to the accumulation of ubiquitinated proteins in the cells.…”

Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal

“…These included the 26 S proteasome system, which is responsible for the regulated degradation of many intracellular proteins and is essential for cell survival. Previous work indicates that proteasome function can be inhibited by oxidants and oxidative stress (57,58). Two recent reports indicate that HNE adduction to proteasome subunits inhibits specific catalytic functions and impairs the degradation of protein substrates (59,60).…”

Ethanol metabolism alters major histocompatibility complex class I-restricted antigen presentation in liver cells

“…Enhanced activity of CYP2E1 is a common feature of numerous pathologic events induced by ethanol-elicited oxidative stress in liver cells, both in the cytosolic and mitochondrial compartments (12, 13). Loss of proteasome function due to oxidative stress appears to occur from formation of adducts with carbonyls, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine derived from peroxynitrite (8, 14, 15). The 20S proteasome removes oxidized proteins even after 26S proteasome has been inhibited by oxidants, indicating differential resistance to oxidative insult (10, 16).…”