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

Mutations in Parkin (a ubiquitin protein ligase) are involved in autosomal recessive juvenile parkinsonism, but it is not known how they cause nigral cell death. We examined the effect of Parkin overexpression on cellular levels of oxidative damage, antioxidant defenses, nitric oxide production, and proteasomal enzyme activity. Increasing expression of Parkin by gene transfection in NT-2 and SK-N-MC cells led to increased proteasomal activity, decreased levels of protein carbonyls, 3-nitrotyrosine-containing proteins, and a trend to a reduction in ubiquitinated protein levels. Transfection of these cells with DNA encoding three mutant Parkins associated with autosomal recessive juvenile parkinsonism (Del 3–5, T240R, and Q311<i>X</i>) gave smaller increases in proteasomal activity and led to elevated levels of protein carbonyls and lipid peroxidation. Turnover of the mutant proteins was slower than that of the wild-type protein, and both could be blocked by the proteasome inhibitor, lactacystin. A rise in levels of nitrated proteins and increased levels of NO<mml:math><mml:mrow></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo></mml:mrow></mml:math>/NO<mml:math><mml:mrow></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo></mml:mrow></mml:math> was also observed in cells transfected with mutant Parkins, apparently because of increased levels of neuronal nitric-oxide synthase. The presence of mutant Parkin in substantia nigra in juvenile parkinsonism may increase oxidative stress and nitric oxide production, sensitizing cells to death induced by other insults.