3-nitrotyrosine and lactacystin

Mutations in parkin are involved in some cases of autosomal recessive juvenile parkinsonism (AR-JP), but it is not known how they result in nigral cell death. We examined the effect of parkin overexpression on the response of cells to various insults. Wild-type and AR-JP-associated mutant parkins (Del3-5, T240R, and Q311X) were overexpressed in NT-2 and SK-N-MC cells. Overexpressed wild-type parkin delayed cell death induced by serum withdrawal, H(2)O(2), 1-methyl-4-phenylpyridinium (MPP(+)), or 4-hydroxy-2-trans-nonenal (HNE) but did not delay cell death caused by the proteasome inhibitor lactacystin. Increases in damage to proteins (protein carbonyls and 3-nitrotyrosine) were attenuated by wild-type parkin after serum withdrawal or exposure to H(2)O(2), MPP(+), or HNE but not after exposure to lactacystin. The mutant parkins (of all types) markedly accelerated cell death in response to all the insults, accompanied by increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation, and 3-nitrotyrosine and decreased levels of GSH. The viability loss induced by all the insults showed apoptotic features. The presence of parkin mutations in substantia nigra in Parkinson's disease may increase neuronal vulnerability to a range of toxic insults.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; Aldehydes; Apoptosis; Cell Death; Cell Line, Tumor; Drug Resistance; Enzyme Inhibitors; Genetic Predisposition to Disease; Glutamic Acid; Guanine; Humans; Hydrogen Peroxide; Mutation; Nerve Degeneration; Neurons; Neurotoxins; Oxidative Stress; Parkinsonian Disorders; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Substantia Nigra; Tyrosine; Ubiquitin-Protein Ligases

Exposure of proteins to oxidants leads to increased oxidation followed by preferential degradation by the proteasomal system. The role of the biologically occurring oxidants singlet oxygen and peroxynitrite in oxidation of proteins in living cells and enhanced degradation of these proteins was examined in this study. Subsequent to treatment of an isolated model protein, ferritin, with singlet oxygen or peroxynitrite, there was enhanced degradation by the isolated 20S proteasome. Treatment of clone 9 liver cells (normal liver epithelia) with two different singlet oxygen-generating systems or peroxynitrite leads to a concentration-dependent increase in cellular protein turnover. At high concentrations of these oxidants, the protein turnover decreases without significant loss of cell viability and proteasome activity. To compare the increase of intracellular protein turnover with that obtained with other oxidants, cells were exposed to hydrogen peroxide or xanthine/xanthine oxidase. The maximal increase in protein turnover was similar with the various oxidants. The oxidized protein moieties were removed by enhanced protein turnover. Removal of singlet oxygen- or peroxynitrite-damaged proteins is dependent on the proteasomal system, as suggested by the sensitivity to lactacystin. Our results provide evidence that the proteasomal system is able to selectively recognize and degrade proteins modified by singlet oxygen or peroxynitrite in vitro as well as in living cells.

Topics: Acetylcysteine; Animals; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Ferritins; Free Radicals; Hydrogen Peroxide; Liver; Multienzyme Complexes; Naphthols; Nitric Oxide; Oxidation-Reduction; Peroxynitrous Acid; Proteasome Endopeptidase Complex; Proteins; Rats; Singlet Oxygen; Tyrosine

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.

Topics: Acetylcysteine; Cell Physiological Phenomena; Cell Survival; Cells; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Damage; Free Radicals; Glutathione; Humans; Lipid Metabolism; Multienzyme Complexes; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitrogen; Oxidative Stress; Proteasome Endopeptidase Complex; Proteins; Superoxide Dismutase; Superoxide Dismutase-1; Tumor Cells, Cultured; Tyrosine; Ubiquitins