We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation of L-ascorbate and identified it as S100b protein, an EF-hand calcium-binding protein, by sequencing its proteolytic peptides. Copper binding studies showed that this protein has four copper-binding sites per dimeric protein molecule with a dissociation constant of 0.46 M and that in the presence of L-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes induced by CuCl 2 . We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be expressed as a fusion protein with glutathione S-transferase. The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl 2 plus H 2 O 2 as compared with the control cells expressing glutathione S-transferase alone. These results indicate that S100b protein does suppress oxidative cell damage by sequestering copper ions.

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Certain proto-oncogenes transfer growth regulatory signals from the cell surface to the nucleus. These genes often show activation soon after cells are exposed to mitogenic stimulation but can also be activated as a nonmitogenic stress response. Cadmium (Cd) is a carcinogenic metal in humans and rodents and, though its mechanism of action is unknown, it could involve activation of such proto-oncogenes. Metallothionein (MT), a metal-inducible protein that binds Cd, can protect against many aspects of Cd toxicity, including genotoxicity and possibly carcinogenesis. Thus, the effects of Cd on expression of c-myc and c-jun in rat L6 myoblasts, and the effect of preactivation of the MT gene by Zn treatment on such oncogene expression, were studied. MT protein levels were determined by the Cd-heme assay, and MT, c-myc, and c-jun mRNA levels were measured using oligonucleotide hybridization and standardized to beta-actin levels. Cd (5 microM CdCl2, 0-30 h) stimulated both c-myc and c-jun mRNA expression. An initial peak of activation of c-myc expression occurred 2 h after initiation of Cd exposure, and levels remained elevated throughout the assessment period. Zn pretreatment markedly reduced the activation of c-myc expression by Cd compared to cells not receiving Zn pretreatment. Cd treatment increased c-jun mRNA levels by up to 3.5-fold. Again, Zn pretreatment markedly reduced Cd-induced activation of c-jun expression as minimal increases occurred with Cd exposures of < or = 1 h, but otherwise the Zn pretreatment prevented activation of c-jun. The Zn pretreatment elevated MT protein levels > 5-fold over control at the point of Cd exposure, but Cd exposure did not further elevate these Zn-induced MT levels. Similarly, Zn pretreatment did not result in increased relative MT mRNA levels above Cd exposure alone at various time points after Cd exposure. Therefore, Zn pretreatment, possibly by providing elevated MT protein levels at the point of Cd exposure, inhibited the Cd-induced c-myc and c-jun proto-oncogene expression. The extent of Cd-induced proto-oncogene activation thus may be limited by the presence of cellular MT.

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.