Morimitsu Nishikimi

Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly.

An Rpn9-disrupted yeast strain, Delta rpn9, whose growth is temperature sensitive with defective assembly of the 26 S proteasome complex, was studied. This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type. Nondenaturing gel electrophoresis followed by activity staining revealed that Delta rpn9 yeast cells had a higher activity of 20 S proteasome than wild type and that in both Delta rpn9 and wild-type cells treated with hydrogen peroxide, 20 S proteasome activity was increased with a concomitant decrease in 26 S proteasome activity. Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells. Taken together, these results suggest that the 20 S proteasome degrades oxidized proteins without ubiquitination of target proteins.

Fragmentation and dimerization of copper-loaded prion protein by copper-catalysed oxidation

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.

Impaired reductive regeneration of ascorbic acid in the Goto-Kakizaki diabetic rat.

Ascorbic acid (AA) is a naturally occurring major antioxidant that is essential for the scavenging of toxic free radicals in both plasma and tissues. AA levels in plasma and tissues have been reported to be significantly lower than normal in diabetic animals and humans, and might contribute to the complications found at the late stages of diabetes. In this study, plasma and hepatic AA levels and AA regeneration were studied in the Goto-Kakizaki diabetic rat (GK rat) to elucidate the mechanism of decreasing plasma and hepatic AA levels in diabetes. AA concentrations in the plasma and liver were significantly lower in GK than in control rats. AA levels in primary cultured hepatocytes derived from GK rats were lower than those derived from control Wistar rats with or without dehydroascorbic acid (DHA) in the medium. Among various enzyme activities that reduce DHA to AA, the NADPH-dependent regeneration of AA in the liver was significantly suppressed in GK rats. Northern blot analysis revealed that only the expression of 3-alpha-hydroxysteroid dehydrogenase (AKR) was significantly suppressed in these rats. These results suggest that decreased AA-regenerating activity, probably through decreased expression of AKR, contributes to the decreased AA levels and increased oxidative stress in GK rats.

Carbonyl formation on a copper-bound prion protein fragment, PrP23-98, associated with its dopamine oxidase activity

The amino‐terminal part of prion protein (PrP), containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ, has been implicated in the binding of copper ion. This region possesses amino acid residues susceptible to oxidation, such as histidine, lysine, arginine and proline. In this study, we have investigated copper‐catalyzed oxidation of an N‐terminal part of human PrP, PrP23–98, that was prepared by the recombinant DNA technique. Carbonyl formations on copper‐bound PrP23–98 induced by dopamine and L‐ascorbate were analyzed kinetically, and it was found that the redox cycling of PrP23–98‐bound copper, especially induced by dopamine, was coupled to the formation of carbonyls on the protein.

Occurrence of two missense mutations in Cu‐ATPase of the macular mouse, a menkes disease model

We have investigated the genetic defect of the Cu‐ATPase gene (Atp7a) in the macular mouse, a genetic model of classical Menkes disease. Northern blot analysis showed that its placenta and kidney possess a normal amount of the Cu‐ATPase mRNA of the normal size; sequencing analysis revealed two missense mutations, His674Arg and Ser1381Pro, in a PCR‐amplified cDNA for mutant Cu‐ATPase. The latter mutation was suspected to affect the function of the ATPase, because it lies in the transmembrane segment that is thought to form a channel for the transportation of copper ions.