The myb-related gene stonewall induces both hyperplasia and cell death in Drosophila: rescue of fly lethality by coexpression of apoptosis inducers.

We carried out gain-of-function mutagenesis screening and identified a mutant in which GAL4 induction led to both hyperplasia and apoptosis. The gene involved was identified as stonewall (stwl), a myb-related gene involved in germ cell proliferation and differentiation during oogenesis. As observed with dmyb, the ectopic expression of stwl(UY823) inhibited endoreplication in salivary glands. We also found that stwl(UY823) overexpression, like overexpression of the wild-type gene, activated G1/S transition and apoptosis. The apoptosis triggered by stwl(UY823) expression is correlated to induction of the proapoptotic gene reaper. Finally, the death of flies induced by ectopic stwl(UY823) expression is efficiently prevented in vivo by triggering cell death in stwl(UY823)-expressing cells. Our results suggest that stwl(UY823) kills flies by causing inappropriate cell cycle entry, and that triggering the death of these overproliferating cells or slowing their proliferation restores viability.

TNF-alpha activates at least two apoptotic signaling cascades.

Apoptosis, the process whereby cells activate an intrinsic death program, can be induced in HeLa cells by TNF-alpha treatment. The aims of the present study were (i) to examine the precise role and the origin of Reactive Oxygen Species (ROS) in the TNF-alpha-induced programmed cell death, (ii) to characterize and order the morphological and mitochondrial changes associated with this process and (iii) to link these events with the activation of caspases. Analyses were performed on TNF-alpha-treated cells in the presence of an anti-oxidant, or of a general caspase inhibitor. To assess the role of mitochondria in the cell death signal transduction, these studies were also realized on HeLa-variant cell lines lacking functional mitochondrial respiratory chain. We show that at least two separate signaling cascades, both mediated by Z-VAD-sensitive caspase(s), contribute to the TNF-alpha-induced apoptosis of HeLa cells. One signaling pathway involves an early mitochondria-dependent ROS production, the other being ROS-independent.

Down-regulation of actin genes precedes microfilament network disruption and actin cleavage during p53-mediated apoptosis.

Inactivation of Simian Virus 40 large T antigen, in cells immortalized with conditional mutants, leads to activation of p53 and apoptosis. We used the mRNA differential display method to identify genes differentially expressed during this process. We found that steady-state levels of mRNA for cytoplasmic actins decreased early during apoptosis. We also showed that, although the steady-state level of the corresponding proteins is not profoundly affected, they are substrates for an interleukin 1-beta converting enzyme (ICE)-like protease activated during the process. However, only a very small fraction of actin is proteolysed during the early stages of apoptosis. The microfilament network is affected and non polymerized actin accumulates in apoptotic bodies after the decrease of mRNA levels, but before a significant amount of actin is cleaved. This suggests that down-regulation of actin genes may be involved in microfilament rearrangements during p53-mediated apoptosis.

Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40.

Rodent embryo cells immortalized with temperature-sensitive mutants of simian virus 40 large tumor (T) antigen have a proliferative potential that depends on temperature. At the restrictive temperature, heat-inactivation of large T antigen causes p53 release, growth arrest, and cell death. Morphological and molecular analysis indicate that the induced cell death corresponds to apoptosis. Flow cytometric analysis using a combination of forward light scatter and side scatter allows a discrimination of cells committed to apoptosis within the whole population. These cells display a reduction in cell size and a higher cellular density, confirming the apoptotic nature of the cell death. When cells exhibiting the morphological features of apoptosis were stained with a fluorescent probe of the mitochondrial membrane potential, a decreased accumulation of the dye was recorded. Measures of cellular respiration, performed with whole-cell populations, showed that the lower mitochondrial membrane potential (delta psi m) correlates, as expected, with an uncoupling of electron transport from ATP production and is linked to the induction of apoptosis. We also show that this decrease in delta psi m is associated with a decrease in the rate of mitochondrial translation. These events are detected at early stages of the apoptotic process, when most of the cells are not irreversibly committed to death, suggesting that mitochondria could be a primary target during apoptosis.

Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase.

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.

Proteolytic cleavage of Hansenula anomala flavocytochrome b2 into its two functional domains. Isolation of a highly active flavodehydrogenase and a cytochrome b2 core.

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. Our results are discussed in terms of 'gene fusion'.

How the loss of several residues, at the level of one interglobule junction, modulates the lactate dehydrogenase activity of yeast flavocytochrome b2: a study of the nicked enzymes resulting from clostripain and trypsin action.

The native chain of flavocytochrome b2 is folded into three globules linked together by two protease-sensitive bridges "a" and "cd". We show in this paper that zone "a" of H-flavocytochrome b2 is the first to be cleaved under clostripain action. The alpha c and beta c fragments thus formed are homologous to alpha T and beta'T trypsic fragments. The remaining activities of the resulting (alpha c beta c) and alpha T beta'T) forms are only 25 per cent and 4 per cent of the native flavocytochrome b2 one. The study of the catalytic properties of (alpha c beta'T) and (alpha T beta c) species resulting from the crossed reassociation of the isolated fragments show that the beta type fragment plays a critical role in the catalytic process. A dramatic activity decrease may be correlated with the loss of 6 amino acid residues at the N-terminal of beta c. Our best hypothesis is that these amino acids are involved in the active site, which may be located in the contact zone between alpha and beta. These results are in agreement with previous results obtained in this laboratory which showed the necessity of both alpha T and beta'T fragments for the correct conformation of the flavin binding site.

A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains.

Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4.

Structural studies of yeast flavocytochrome b2: cooperative roles of the alpha and beta globules in the formation of the flavin-binding sites.

The purpose of the study reported here was the localization of the heme binding sites on the two globular fragments, alpha and beta, of the 'cleaved' form of the flavocytochrome b2 chain. These fragments were partially resolved by means of molecular sieving under denaturing conditions (3 M or 6 M guanidine in the presence of 2-mercaptoethanol). They were then renatured in the presence of excesses of FMN and protoheme. The protoheme was found to be quantitatively bound to the alpha subunit, confirming previous findings. The flavin binds neither to alpha alone nor to beta alone, but only to the reassociated alphabeta protomer. the results are discussed in terms of the possible occurrence of gene fusion in the formation of the complex flavocytochrome chain of this very particular L-lactate cytochrome c reductase found specifically in yeasts.