Wnt signaling promotes oncogenic transformation by inhibiting c-Myc-induced apoptosis

Aberrant activation of the Wnt/beta-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/beta-catenin signaling suppressed apoptosis by inhibiting c-Myc-induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc-induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/beta-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis.     

ER–mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem‐like cells

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem‐like cells (GSC) being more sensitive to cytotoxic lymphocyte‐mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER–mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER–mitochondria contacts compared to GDCs. Forced ER–mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER–mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.     

The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.The S100 proteins comprise a multigene family of low-molecular-weight EF-hand calcium binding and zinc binding proteins (5, 13, 16, 24, 33). To date, 19 different S100 proteins have been assigned to this protein family, and they show different degrees of similarity, ranging from 25 to 56% identity at the amino acid level. With S100B, S100P, and S100Z being the exceptions, the majority of the S100 genes are clustered on human chromosome 1q21 (33). Most S100 proteins serve as calcium sensor proteins that, upon activation, regulate the function and/or subcellular distribution of specific target proteins (13, 33, 47), and they are characterized by common structural motifs, including two low-affinity (K_D [equilibrium dissociation constant] of ∼10 μM to 100 μM) helix-loop-helix calcium binding domains (EF hands) that are separated by a hinge region and flanked by amino- and carboxy-terminal domains. The carboxy-terminal domain is variable among S100 proteins, and it typically is the site that is responsible for the selective interaction of each individual S100 protein with specific target proteins (30). S100 proteins are often upregulated in cancers, in inflammation, and in response to cellular stress (14, 16), suggesting that they function in cell responses to stress situations. Consistent with this hypothesis, stress situations were necessary to reveal phenotypes associated with the S100 knockout in mice (11, 14, 33, 56). Moreover, recent observations revealed a new function for the S100 protein family that included their ability to assist and regulate multichaperone complex-ligand interactions (41, 50, 51).One member of the S100 protein family, S100B, has attracted much interest in the past few years because, like other proteins implicated in neurodegeneration (e.g., amyloid, superoxide dismutase, and dual-specificity tyrosine phosphorylation-regulated kinase 1A), its gene is located within a segment of chromosome 21, which is trisomic in Down''s syndrome (DS). Its expression in the brain of mammals coincides with defined periods of central nervous system (CNS) maturation and cell differentiation (43). In oligodendrocyte progenitor cells (OPC), S100B expression is associated with differentiation, and S100B contributes to OPC differentiation in response to demyelinating insult (11). To understand the contribution of S100B to OPC differentiation, we searched for high-affinity S100B target proteins in this cell type by using far-Western analysis. A major and highly specific S100B target protein was identified, the mitochondrial ATAD3A protein.ATAD3A belongs to a new family of eukaryote-specific mitochondrial AAA⁺ ATPase proteins (17). In the human genome, two genes, Atad3A and Atad3B, are located in tandem on chromosome 1p36.33. The Atad3A gene is ubiquitous among multicellular organisms but absent in yeast. The Atad3B gene is specific to the human genome (27). ATAD3A is a mitochondrial protein anchored into the mitochondrial inner membrane (IM) at contact sites with the outer membrane (OM). Thanks to its simultaneous interaction with the two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the inner and outer membranes and controls diverse cell responses ranging from mitochondrial metabolism, cell growth, and mitochondrial fission 20a, 25). The ATAD3A protein has also been identified as a mitochondrial DNA binding protein (23) and as a cell surface antigen in some human tumors (20, 21). The plasma membrane localization of ATAD3A in tumor cells is suggestive that ATAD3A mitochondrial routing can be compromised in pathological situations such as cancer. To understand the functional response resulting from the interaction between S100B and ATAD3A, we first characterized the minimal interaction domain on ATAD3A for S100B binding using thermodynamic studies of wild-type and ATAD3A variants as well as via nuclear magnetic resonance (NMR) spectroscopy techniques. These studies allowed us to further refine the consensus S100B binding motif, which is conserved in several other S100B target proteins, including the p53 protein and several newly discovered target proteins associated with the cell translational machinery. We next analyzed the cellular interaction of S100B with truncated ATAD3A mutants that harbor the S100B binding domain but that are deficient for mitochondrial import. These studies revealed that S100B could assist ATAD3A mutant proteins during cytoplasmic processing by preventing dysfunctional aggregation events. Our results are discussed in light of the possible function of S100B in assisting the cytoplasmic processing of proteins for proper folding and subcellular localization.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.)

Oleosins of Brassica napus L. (oilseed rape) synthesized by in-vitro translation were found to be very efficiently targeted to microsomal membranes but only poorly translocated to oil bodies or emulsified oil. The use of other bilayer membranes as controls showed that this interaction was specific. The rate of oleosin synthesis in the presence of microsomes was enhanced about threefold, indicative of the involvement of the signal-recognition particle in the targeting process. There is no evidence for the cleavage of the protein during targeting and the protein sequence reveals no consensus cleavage site for the signal peptide. Protection experiments using Proteinase K revealed that about 6 kDa of the protein is exposed on the cytoplasmic side of the ER but the remainder is protected. Carbonate (pH 11) washing of microsomal membranes after in-vitro translation confirmed that oleosins have a domain which remains inserted in the ER rather than the protein being transported completely into the lumen of the ER. These results indicate that oleosins are transported via the ER prior to their accumulation on oil bodies.     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive ⁸⁶Rb uptake and amiloride-sensitive ²⁴Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na⁺/H⁺ and Na⁺/K⁺ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na⁺/H⁺ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na⁺/H⁺ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na⁺ pump, Na⁺/H⁺ exchange) without eliciting corresponding regulatory mechanisms (Na⁺ stat, pH stat).     

Effects of Interference Between Selected Loci on the Mutation Load,Inbreeding Depression,and Heterosis

A classical prediction from single-locus models is that inbreeding increases the efficiency of selection against partially recessive deleterious alleles (purging), thereby decreasing the mutation load and level of inbreeding depression. However, previous multilocus simulation studies found that increasing the rate of self-fertilization of individuals may not lead to purging and argued that selective interference among loci causes this effect. In this article, I derive simple analytical approximations for the mutation load and inbreeding depression, taking into account the effects of interference between pairs of loci. I consider two classical scenarios of nonrandomly mating populations: a single population undergoing partial selfing and a subdivided population with limited dispersal. In the first case, correlations in homozygosity between loci tend to reduce mean fitness and increase inbreeding depression. These effects are stronger when deleterious alleles are more recessive, but only weakly depend on the strength of selection against deleterious alleles and on recombination rates. In subdivided populations, interference increases inbreeding depression within demes, but decreases heterosis between demes. Comparisons with multilocus, individual-based simulations show that these analytical approximations are accurate as long as the effects of interference stay moderate, but fail for high deleterious mutation rates and low dominance coefficients of deleterious alleles.     

The bactericidal effect of TiO2 photocatalysis involves adsorption onto catalyst and the loss of membrane integrity

The bactericidal effect of photocatalysis with TiO2 is well recognized, although its mode of action is still poorly characterized. It may involve oxidation, as illuminated TiO2 generates reactive oxygen species. Here we analyze the bactericidal effect of illuminated TiO2 in NaCl-KCl or sodium phosphate solutions. We found that adsorption of bacteria on the catalyst occurred immediately in NaCl-KCl solution, whereas it was delayed in the sodium phosphate solution. We also show that the rate of adsorption of cells onto TiO2 is positively correlated with its bactericidal effect. Importantly, adsorption was consistently associated with a reduction or loss of bacterial membrane integrity, as revealed by flow cytometry. Our work suggests that adsorption of cells onto aggregated TiO2, followed by loss of membrane integrity, is key to the bactericidal effect of photocatalysis.     

Characterization of permissivity for hobo -mediated gonadal dysgenesis in Drosophila melanogaster

The hobo transposon is responsible for one of the three hybrid dysgenic systems that have been described in Drosophila melanogaster. Most studies on the hobo dysgenic system have been carried out using the PM system as a reference. However, these two systems differ significantly. In particular, several studies have failed to find any correlation between the molecular structures of hobo elements, the instability of the transposon and the incidence of gonadal dysgenic (GD) sterility. On the other hand, no study of the ability of females to permit hobo activity in their progeny when they are crossed with males harboring active hobo elements (permissivity) has yet been reported. In order to investigate the parameters involved in hobo permissivity, four E strains were studied with regard to the molecular nature of their hobo sequences and the GD sterility induced by a controlled source of hobo transposase. We show that hobo permissivity varies both within and between E strains. Moreover, permissivity decreases with age in E females. Our results are discussed with respect to similar phenomena that have been described in relation to the reactivity of the IR dysgenic system. Received: 17 August 1998 / Accepted: 17 December 1998