Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitrated proteins.
Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 micro m) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated alpha-tubulin, SOD-1, alpha-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3-5, T240R and Q311'X). The nitric oxide synthase inhibitor, l-NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it.
Hyun
Dong-Hoon
DH
Wolfson Centre for Age-Related Diseases, GKT School of Biomedical Sciences, King's College, London, UK.
Lee
MoonHee
M
Halliwell
Barry
B
Jenner
Peter
P
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018014:Gene Transfer Techniques; D006801:Humans; D008025:Ligases / Q000096:biosynthesis / Q000235:genetics; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D016900:Neurofilament Proteins / Q000096:biosynthesis; D009566:Nitrates / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D051843:Synucleins; D014404:Tubulin / Q000096:biosynthesis; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D051844:alpha-Synuclein
D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D009566:Nitrates; D011506:Proteins; C497604:SNCA protein, human; C000606290:SOD1 protein, human; D051843:Synucleins; D014404:Tubulin; D025801:Ubiquitin; D051844:alpha-Synuclein; C066858:neurofilament protein NF 68; C067713:lactacystin; D019001:Nitric Oxide Synthase; C470429:SOD1 G37R protein, mouse; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D000111:Acetylcysteine