Copper-catalyzed oxidation of the recombinant SHa(29–231) prion protein

Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29–231). Using Cu2+/ascorbate, we oxidized SHaPrP(29–231) in vitro. Oxidation was demonstrated by liquid chromatography/mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29–101 region, which includes the histidine-containing octarepeats, is particularly affected by oxidation. Oxidation was time- and copper concentration-dependent and was evident with copper concentrations as low as 1 μM. Concomitant with oxidation, SHaPrP(29–231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37°C with a 6-fold molar excess of Cu2+. These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.

Role of oxidation in the neurotoxic effects of intrastriatal dopamine injections.

We have examined the biochemical and histological effects of high concentrations of dopamine (0.05-1.0 micromol) injected into the rat striatum. Twenty-four hours after such injections, the oxidation products of dopamine and dihydroxyphenylacetic acid were detected as both free and protein-bound cysteinyl dopamine and cysteinyl dihydroxyphenylacetic acid. Protein-bound cysteinyl catechols were increased 7- to 20-fold above control tissue levels. By 7 days postinjection, the protein-bound cysteinyl catechols were still detectable, although reduced in concentration, whereas the free forms could no longer be measured. Histological examination of striatum at 7 days revealed a central core of nonspecific damage including neuronal loss and gliosis. This core was surrounded by a region containing a marked reduction in tyrosine hydroxylase immunoreactivity but no apparent loss of serotonin or synaptophysin immunoreactivity. When dopamine was injected with an equimolar concentration of either ascorbic acid or glutathione, the formation of protein-bound cysteinyl catechols was greatly reduced. Moreover, the specific loss of tyrosine hydroxylase immunoreactivity associated with dopamine injections was no longer detectable, although the nonspecific changes in cytoarchitecture were still apparent. Thus, following its oxidation, dopamine in high concentrations binds to protein in the striatum, an event that is correlated with the specific loss of dopaminergic terminals. We suggest that the selective degeneration of dopamine neurons in Parkinson's disease may be caused by an imbalance between the oxidation of dopamine and the availability of antioxidant defenses.