We have analyzed the proteins that are oxidatively damaged when Saccharomyces cerevisiae cells are exposed to stressing conditions. Carbonyl groups generated by hydrogen peroxide or menadione on proteins of aerobically respiring cells were detected by Western blotting, purified, and identified. Mitochondrial proteins such as E2 subunits of both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, aconitase, heat-shock protein 60, and the cytosolic fatty acid synthase (alpha subunit) and glyceraldehyde-3-phosphate dehydrogenase were the major targets. In addition we also report the in vivo modification of lipoamide present in the above-mentioned E2 subunits under the stressing conditions tested and that this also occurs with the homologous enzymes present in Escherichia coli cells that were used for comparative analysis. Under fermentative conditions, the main protein targets in S. cerevisiae cells treated with hydrogen peroxide or menadione were pyruvate decarboxylase, enolase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase. Under the stress conditions tested, fermenting cells exhibit a lower viability than aerobically respiring cells and, consistently, increased peroxide generation as well as higher content of protein carbonyls and lipid peroxides. Our results strongly suggest that the oxidative stress in prokaryotic and eukaryotic cells shares common features.

In the present study, we have analyzed the role of the molecular chaperone Hsp60 in protection of Saccharomyces cerevisiae against oxidative damage. We constructed mutant strains in which the levels of Hsp60 protein, compared with wild-type cells, were four times greater, and the addition of doxycycline gradually reduces them to 20% of wild-type. Under oxidative-stress conditions, the progressive decrease in Hsp60 levels in these mutants resulted in reduced cell viability and an increase in both cell peroxide species and protein carbonyl content. Protection of Fe/S-containing enzymes from oxidative inactivation was found to be dose-dependent with respect to Hsp60 levels. As these enzymes release their iron ions under oxidative-stress conditions, the intracellular labile iron pool, monitored with calcein, was higher in cells with reduced Hsp60 levels. Consistently, the iron chelator deferoxamine protected low Hsp60-expressing cells from both oxidant-induced death and protein oxidation. These results indicate that the role of Hsp60 in oxidative-stress defense is explained by protection of several Fe/S proteins, which prevent the release of iron ions and thereby avert further damage.

The multifunctional AdhE protein of Escherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH 2 -terminal region of the AdhE protein is highly homologous to aldehyde:NAD ؉ oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe 2؉ -dependent ethanol:NAD ؉ oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol. The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA. All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 3 Thr substitution in the acetaldehyde:NAD ؉ oxidoreductase domain of AdhE. All second stage mutants grew on ethanol with a doubling time of about 90 min, and all of them produced an AdhE A267T/ E568K. Purified AdhE A267T and AdhE A267T/E568K showed highly elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhE A267T/E568K was more thermal stable than AdhE A267T . Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhE A267T .

Alcohol dehydrogenase E (AdhE) is an Fe-enzyme that, under anaerobic conditions, is involved in dissimilation of glucose. The enzyme is also present under aerobic conditions, its amount is about one-third and its activity is only one-tenth of the values observed under anaerobic conditions. Nevertheless, its function in the presence of oxygen remained ignored. The data presented in this paper led us to propose that the enzyme has a protective role against oxidative stress. Our results indicated that cells deleted in adhE gene could not grow aerobically in minimal media, were extremely sensitive to oxidative stress and showed division defects. In addition, compared with wild type, mutant cells displayed increased levels of internal peroxides (even higher than those found in a ⌬katG strain) and increased protein carbonyl content. This pleiotropic phenotype disappeared when the adhE gene was reintroduced into the defective strain. The purified enzyme was highly reactive with hydrogen peroxide (with a K i of 5 M), causing inactivation due to a metal-catalyzed oxidation reaction. It is possible to prevent this reactivity to hydrogen peroxide by zinc, which can replace the iron atom at the catalytic site of AdhE. This can also be achieved by addition of ZnSO 4 to cell cultures. In such conditions, addition of hydrogen peroxide resulted in reduced cell viability compared with that obtained without the Zn treatment. We therefore propose that AdhE acts as a H 2 O 2 scavenger in Escherichia coli cells grown under aerobic conditions.

The regulation of cell growth and proliferation is fundamental for animal development and homeostasis but the mechanisms that coordinate cell growth with cell cycle progression are poorly understood. One possibility is that "cell-size checkpoints" act to delay division until cells have achieved a minimal size or mass however, the existence of such checkpoints in mammalian cells is controversial. In this study we provide further evidence against the operation of a size checkpoint in mammalian cells. We show that primary mammalian cells proliferate at a rate that is independent of cell size or cell mass and that cell size is "set" by the balance of extracellular growth factors and mitogens. Moreover, we show that commonly used culture conditions stimulate cell growth much more than cell cycle progression resulting in cells that proliferate at sizes 300-500% larger than their in vivo counterparts. This has profound effects on cell behaviour.