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 microM) 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 microM) 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 microM) 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 microM 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.

Effect of Wild-type or Mutant Parkin on Oxidative Damage, Nitric Oxide, Antioxidant Defenses, and the Proteasome

“…Overall, our data support the growing view that proteasomal dysfunction may be a significant contributor to neuronal cell death in the major neurodegenerative diseases (18,28,52,53,55). Our data shown that abnormal proteins such as mutated Parkins impact on the ubiquitin-proteasome system, leading to oxidative stress and excess NO production.…”

“…The mutant proteins could elevate levels of NO 2 Ϫ /NO 3 Ϫ , for which increased expression of nNOS (but not iNOS) was presumably responsible. Again, such effects have been observed in cells treated with proteasomal inhibitors (55). However, the mutant proteins did not decrease proteasomal hydrolytic activities; rather they were increased although the increase with wild-type Parkin was greater than for the mutants (as measured using substrates whose hydrolysis is not ubiquitin-dependent).…”

“…Levels of 3-nitrotyrosine, a biomarker of attack by ONOO Ϫ and other reactive nitrogen species upon protein-bound tyrosine residues (47, 48), were also significantly increased in the mutant Parkin transfectants, again independent of Parkin enzyme activity. We hypothesized that overexpression of these Parkin mutants might affect proteasomal function, since we recently found that proteasomal inhibition increases oxidative damage and protein nitration in these cell types (55). Carbonylated and nitrated proteins are degraded by the proteasome (55)(56)(57).…”

“…We hypothesized that overexpression of these Parkin mutants might affect proteasomal function, since we recently found that proteasomal inhibition increases oxidative damage and protein nitration in these cell types (55). Carbonylated and nitrated proteins are degraded by the proteasome (55)(56)(57). The mutant proteins could elevate levels of NO 2 Ϫ /NO 3 Ϫ , for which increased expression of nNOS (but not iNOS) was presumably responsible.…”

Chaperons moléculaires et repliement des protéines : L’exemple de certaines protéines de choc thermique

“…L'inhibition des activités du protéasome par des substances spécifiques (type lactacystine) induit une agrégation et une oxydation des protéines cellulaires non dégradées [13]. Ces agrégats sont constitués de protéines en fin de vie qui s'accumulent dans la cellule sous forme ubiquitinylée [14]. L'inhibition du protéasome s'accompagne également de la synthèse de Hsp70, dans un phénomène qui représente probablement une réponse Bag-1 Cochaperon déstabilisant la forme ATP du complexe Hsc70/Hsp70-substrat Tableau I. Principales protéines chaperons impliquées dans la réponse cellulaire au choc thermique.…”