Overexpression of human peroxiredoxin 5 in subcellular compartments of Chinese hamster ovary cells: effects on cytotoxicity and DNA damage caused by peroxides.

Peroxiredoxin 5 is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Peroxiredoxin 5 can be intracellularly localized to mitochondria, peroxisomes, the cytosol, and, to a lesser extent, the nucleus. This remarkably wide subcellular distribution compared with the five other mammalian peroxiredoxins prompted us to further investigate the antioxidant protective function of peroxiredoxin 5 in mammalian cells according to its subcellular localization. Chinese hamster ovary cells overexpressing human peroxiredoxin 5 in the cytosol, in mitochondria, or in the nucleus were established by stable transfection. Cells overexpressing peroxiredoxin 5 were exposed for 1 h to low or acute oxidative stress with exogenously added hydrogen peroxide or tert-butylhydroperoxide. Cell protection conferred by peroxiredoxin 5 was evaluated by clonogenicity and lactate dehydrogenase assays. Overexpressing peroxiredoxin 5 in either the cytosolic, mitochondrial, or nuclear compartment significantly reduced cell death, with more effective protection with overexpression of peroxiredoxin 5 in mitochondria, confirming that this organelle is a major target of peroxides. Moreover, we evaluated, with the comet assay, nuclear DNA damage induced by hydrogen peroxide or tert-butylhydroperoxide. Overexpression of peroxiredoxin 5 in the nucleus significantly decreased DNA damage induced by both peroxides. In conclusion, the present study suggests that multiple subcellular targeting of peroxiredoxin 5 in mammalian cells can be implicated in antioxidant protective mechanisms under nonpathological conditions but also during acute oxidative stress caused by peroxides occurring in pathophysiological situations.

TEM-1 beta-lactamase as a scaffold for protein recognition and assay.

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.