Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production

Abstract

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT-2 and SK-N-MC cell lines. The selective proteasome inhibitor, lactacystin (1 µm) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3-nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO⁻) upon proteins. Higher levels (25 µm) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3-nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 µm) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO·) (measured as levels of NO₂⁻ plus NO₃⁻) by the cells; this was inhibited by N-nitro-l-arginine methyl ester (l-NAME), which also decreased cell death induced by 25 µm lactacystin and decreased levels of 3-nitrotyrosine. The NO· production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.