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International audience; Cells need to sense stresses to initiate the execution of the dormant cell death program. Since the discovery of the first BH3only protein Bad, BH3-only proteins have been recognized as indispensable stress sensors that induce apoptosis. BH3-only proteins have so far not been identified in Drosophila despite their importance in other organisms. Here, we identify the first Drosophila BH3-only protein and name it sayonara. Sayonara induces apoptosis in a BH3 motif-dependent manner and interacts genetically and biochemically with the BCL-2 homologous proteins, Buffy and Debcl. There is a positive feedback loop between Sayonaramediated caspase activation and autophagy. The BH3 motif of sayonara phylogenetically appeared at the time of the ancestral gene duplication that led to the formation of Buffy and Debcl in the dipteran lineage. To our knowledge, this is the first identification of a bona fide BH3-only protein in Drosophila, thus providing a unique example of how cell death mechanisms can evolve both through time and across taxa.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is associated with unique changes in mitochondrial metabolism, including elevated respiration rates and morphological alterations. We examined electron transport chain (ETC) complex activity in fibroblasts derived from 18 children with ASD as well as mitochondrial morphology measurements in fibroblasts derived from the ASD participants and four typically developing controls. In ASD participants, symptoms severity was measured by the Social Responsiveness Scale and Aberrant Behavior Checklist. Mixed-model regression demonstrated that alterations in mitochondrial morphology were associated with both ETC Complex I+III and IV activity as well as the difference between ETC Complex I+III and IV activity. The subgroup of ASD participants with relative elevation in Complex IV activity demonstrated more typical mitochondrial morphology and milder ASD related symptoms. This study is limited by sample size given the invasive nature of obtaining fibroblasts from children. Furthermore, since mitochondrial function is heterogenous across tissues, the result may be specific to fibroblast respiration. Previous studies have separately described elevated ETC Complex IV activity and changes in mitochondrial morphology in cells derived from children with ASD but this is the first study to link these two findings in mitochondrial metabolism. The association between a difference in ETC complex I+III and IV activity and normal morphology suggests that mitochondrial in individuals with ASD may require ETC uncoupling to function optimally. Further studies should assess the molecular mechanisms behind these unique metabolic changes.Trial registration: Protocols used in this study were registered in clinicaltrials.gov as NCT02000284 and NCT02003170.

International audience; The underlying molecular basis for neurodevelopmental or neuropsychiatric disorders is not known. In contrast, mechanistic understanding of other brain disorders including neurodegeneration has advanced considerably. Yet, these do not approach the knowledge accrued for many cancers with precision therapeutics acting on well-characterized targets. Although the identification of genes responsible for neurodevelopmental and neuropsychiatric disorders remains a major obstacle, the few causally associated genes are ripe for discovery by focusing efforts to dissect their mechanisms. Here, we make a case for delving into mechanisms of the poorly characterized human KCTD gene family. Varying levels of evidence support their roles in neurocognitive disorders (KCTD3), neurodevelopmental disease (KCTD7), bipolar disorder (KCTD12), autism and schizophrenia (KCTD13), movement disorders (KCTD17), cancer (KCTD11), and obesity (KCTD15). Collective knowledge about these genes adds enhanced value, and critical insights into potential disease mechanisms have come from unexpected sources. Translation of basic research on the KCTD-related yeast protein Whi2 has revealed roles in nutrient signaling to mTORC1 (KCTD11) and an autophagy-lysosome pathway affecting mitochondria (KCTD7). Recent biochemical and structure-based studies (KCTD12, KCTD13, KCTD16) reveal mechanisms of regulating membrane channel activities through modulation of distinct GTPases. We explore how these seemingly varied functions may be disease related.

International audience; Cette année 2020 célèbre le centième anniversaire de la naissance de Saul Bass, designer graphique de génie et inventeur du générique de film. Moins connue, son approche de notre rapport à la Nature porte un discours moderne, en avance sur son temps, dans le champ bouillonnant de l'écologie, cette grande affaire du XXI e siècle.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causesthe infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformaticstools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection,understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to getinsight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for theroutine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemicand evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets anddevelopment of therapeutic strategies. For each tool, we briefly describe its use case and how it advances researchspecifically for SARS-CoV-2. Fil: Hufsky, Franziska. Friedrich Schiller University Jena; Alemania Fil: Lamkiewicz, Kevin. Friedrich Schiller University Jena; Alemania Fil: Almeida, Alexandre. the Wellcome Sanger Institute; Reino Unido Fil: Aouacheria, Abdel. Centre National de la Recherche Scientifique; Francia Fil: Arighi, Cecilia. Biocuration and Literature Access at PIR; Estados Unidos Fil: Bateman, Alex. European Bioinformatics Institute. Head of Protein Sequence Resources; Reino Unido Fil: Baumbach, Jan. Universitat Technical Zu Munich; Alemania Fil: Beerenwinkel, Niko. Universitat Technical Zu Munich; Alemania Fil: Brandt, Christian. Jena University Hospital; Alemania Fil: Cacciabue, Marco Polo Domingo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Chuguransky, Sara Rocío. European Bioinformatics Institute; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Drechsel, Oliver. Robert Koch-Institute; Alemania Fil: Finn, Robert D.. Biocurator for Pfam and InterPro databases; Reino Unido Fil: Fritz, Adrian. Helmholtz Centre for Infection Research; Alemania Fil: Fuchs, Stephan. Robert Koch-Institute; Alemania Fil: Hattab, Georges. University Marburg; Alemania Fil: Hauschild, Anne Christin. University Marburg; Alemania Fil: Heider, Dominik. University Marburg; Alemania Fil: Hoffmann, Marie. Freie Universität Berlin; Alemania Fil: Hölzer, Martin. Friedrich Schiller University Jena; Alemania Fil: Hoops, Stefan. University of Virginia; Estados Unidos Fil: Kaderali, Lars. University Medicine Greifswald; Alemania Fil: Kalvari, Ioanna. European Bioinformatics Institute; Reino Unido Fil: von Kleist, Max. Robert Koch-Institute; Alemania Fil: Kmiecinski, Renó. Robert Koch-Institute; Alemania Fil: Kühnert, Denise. Max Planck Institute for the Science of Human History; Alemania Fil: Lasso, Gorka. Albert Einstein College of Medicine; Estados Unidos Fil: Libin, Pieter. Hasselt University; Bélgica Fil: List, Markus. Universitat Technical Zu Munich; Alemania Fil: Löchel, Hannah F.. University Marburg; Alemania

Following the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2002 and Middle East Respiratory Syndrome coronavirus (MERS-CoV) in 2012, the novel coronavirus SARS-CoV-2 emerged at the end of 2019 as a highly pathogenic infectious agent that rapidly spread around the world. SARS-CoV-2 shares high sequence homology with SARS-CoV and causes acute, highly lethal pneumonia coronavirus disease 2019 (COVID-19) with clinical symptoms similar to those reported for SARS-CoV. Like other betacoronaviruses, SARS-CoV-2 encode four major structural proteins: Spike (S), Membrane (M), Nucleocapsid (N) and Envelope (E). SARS-CoV E protein is abundant in infected cells and plays a crucial role in viral particle assembly. Moreover, SARS coronaviruses lacking E are attenuated in vivo, suggesting that CoV E may act as a critical virulence factor not only in SARS-CoV but also in the case of the new coronavirus SARS-CoV-2. Ectopic expression of SARS-CoV E was previously shown to trigger apoptosis (cell suicide) of T lymphocytes, lymphopenia being a common feature observed in fatal cases following viral infections. Importantly, T-cell apoptosis was shown to involve interaction between the C-terminal region of SARS-CoV E and the Bcl-2 family member Bcl-xL, which acts as a potent anti-apoptotic protein. Here we provide the first observation that the SARS-CoV E and SARS-CoV-2 E proteins share a conserved Bcl-2 Homology 3 (BH3)-like motif in their C-terminal region, a well-studied motif shown to be necessary for SARS-CoV E binding to Bcl-xL. We used available sequence data for SARS-CoV-2 and related coronaviruses, in combination with structural information, to study the structure to biological activity relationships of SARS-CoV-2 E in relation with its BH3-like motif. Our analysis of the SARS-CoV E interactome further revealed that the predicted SARS-CoV-2 network is extensively wired to the Bcl-2 apoptotic switch. Research is therefore needed to establish if SARS-CoV-2 E targets prosurvival Bcl-2 homologs to modulate cell viability, as part of a coronavirus strategy to interfere with apoptosis. The identification of small molecules (or the repurposing of existing drugs) able to disrupt SARS-CoV-2 E BH3-mediated interactions might provide a targeted therapeutic approach for COVID-19 treatment. Recombinant SARS-CoV-2 expressing an E protein with a deleted or mutated BH3-like motif might also be of interest for the design of a live, attenuated vaccine.

International audience; Nano-sized lipid formulations offer a great potential for topical delivery of active compounds to treat and prevent human skin damages. Of particular importance is the high loading of hydrophobic molecules, the long-term stability and the auspicious penetration capacity especially reached when using lipid nanocapsules (LNC). Unfortunately, their formation currently relies on a phase inversion process that only operates when using a poly(ethylene glycol) (PEG) based surfactant belonging to the controversial PEG family that was subject of clinical awareness. The present study proposes an alternative to this overused polymer in formulations by designing LNC made of harmless amphiphilic polyoxazolines (POx). Implementing a short sonication step in the process allowed well-defined spherical nanoparticles of ~30 nm to be obtained. The structure of the so called LNC POx was composed of an oily core surrounded by a rigid shell of phospholipids and POx, which ensures a high stability over time, temperature, centrifugation and freezing. Encapsulation of the natural quercetin antioxidant led to a drug loading three times higher than for LNC constituted of PEG (LNC PEG). The antioxidant activity of loaded LNC POx was tested on mice fibroblasts and human keratinocytes after exposure to free radicals from peroxides and UVB irradiation, respectively. The radical scavenging capacity of quercetin loaded in the LNC POx was preserved and even slightly enhanced compared to LNC PEG, highlighting the POx value in nanoformulations.

International audience; OBJECTIVE:Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function.METHODS:Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples.RESULTS:Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology.INTERPRETATION:Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7.

BCL-2 proteins correspond to a structurally, functionally, and phylogenetically heterogeneous group of regulators that play crucial roles in the life and death of animal cells. Some of these regulators also represent therapeutic targets in human diseases including cancer. In the omics era, there is great need for easy data retrieval and fast analysis of the molecular players involved in cell death. In this chapter, we present generic and specific computational resources (such as the reference database BCL2DB) as well as bioinformatics tools that can be used to investigate BCL-2 homologs and BH3-only proteins.

Shlezinger et al . (Reports, 8 September 2017, p. 1037) report that the common fungus Aspergillus fumigatus , a cause of aspergillosis, undergoes caspase-dependent apoptosis-like cell death triggered by lung neutrophils. However, the technologies they used do not provide reliable evidence that fungal cells die via a protease signaling cascade thwarted by a fungal caspase inhibitor homologous to human survivin.

B cell lymphoma-2 (BCL-2)-related proteins control programmed cell death through a complex network of protein-protein interactions mediated by BCL-2 homology 3 (BH3) domains. Given their roles as dynamic linchpins, the discovery of novel BH3-containing proteins has attracted considerable attention. However, without a clearly defined BH3 signature sequence the BCL-2 family has expanded to include a nebulous group of nonhomologous BH3-only proteins, now justified by an intriguing twist. We present evidence that BH3s from both ordered and disordered proteins represent a new class of short linear motifs (SLiMs) or molecular recognition features (MoRFs) and are diverse in their evolutionary histories. The implied corollaries are that BH3s have a broad phylogenetic distribution and could potentially bind to non-BCL-2-like structural domains with distinct functions.

The concept of cell death has many links to the concept of death itself, defined as the opposite of life. Achievements obtained through research on apoptosis have apparently allowed us to transcend this Manichean view. Death is no longer outside, but rather inside living systems, as a constitutive force at work within the living matter. Whereas the death of cells can be positive and breed “creation” (e.g. during morphogenesis), its dysregulation can also cause or contribute to fatal diseases including cancer. It is tempting to apply this biological discourse to illuminate the relations between life and death, taken in general terms, but does this generalization actually hold? Is this discourse not essentially a metaphor? If cell death is considered as a vital aspect of various biological processes, then are we not faced with some vitalistic conception of death? Are there one or more meanings to the word “death”? Does the power to self-destruct act in opposition to other key features of living entities, or rather in juxtaposition to them? In this article, we first describe how the field of cell death has been developed on the basis of perceived and built dichotomies, mirroring the original opposition between life and death. We detail the limitations of the current paradigm of apoptosis regulation by BCL-2 family proteins, which nicely illustrate the problem of binary thinking in biology. Last, we try to show a way out of this dualistic matrix, by drawing on the notions of multiplicity, complexity, diversity, evolution and contingency., La mort est souvent perçue comme la négation de la vie, la vie comme l’antithèse de la mort. La biologie, par l’étude de la mort cellulaire, a permis de transcender ce manichéisme. La mort n’apparaît plus comme extérieure à la vie, mais comme un processus tangible, à l’œuvre dans le vivant. Si la mort cellulaire peut être créatrice, son dysfonctionnement peut aussi être la cause de pathologies mortelles (cancer). Ce discours biologique peut-il vraiment être étendu pour raisonner, en général, sur les relations entre la vie et la mort? N’est-il pas essentiellement métaphorique? Ne sommes-nous pas en train de jouer sur les différents sens du mot ¡mort¿, en prenant le risque de vitaliser la mort? Dans cet article, nous décrirons comment le champ disciplinaire de la mort cellulaire s’est construit autour d’une série de dichotomies, répétant l’opposition originelle entre vie et mort. Au travers de l’exemple des protéines de la famille de BCL-2, notre propos sera d’esquisser une voie pour sortir de ce dualisme, en s’appuyant sur les notions de multiplicité, de complexité, de diversité, d’histoire(s) évolutive(s) et de contingence.

International audience; GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode.

STUDY QUESTION: What is the expression status and subcellular localization of the maternally expressed Bcl-2 family member, BCL2L10, in early human embryos of diverse developmental stages and quality? SUMMARY ANSWER: The anti-apoptotic protein, BCL2L10, is expressed in human preimplantation embryos at least until the blastocyst stage and appears to be differentially distributed at the subcellular level between viable embryos and fragmented or arrested embryos. WHAT IS KNOWN ALREADY: BCL2L10 is an anti-apoptotic member of the BCL-2 family that shows abundant expression in human oocytes and limited sequence conservation to its mouse homologue. STUDY DESIGN, SIZE, DURATION: Embryos donated with informed consent by couples consulting for infertility in the Department of Reproductive Medicine (Hopital Femme Mere Enfant, Bron, France) were divided into two groups: high quality embryos (n = 18) and poor quality embryos (n = 30). Semen samples (n = 4) were obtained after informed consent from men consulting for couple infertility. Experiments involving human preimplantation embryos were performed between January and December 2009. PARTICIPANTS/MATERIALS, SETTING, METHODS: We examined BCL2L10 expression and subcellular localization in early human embryos by using immunofluorescence and confocal microscopy. The subcellular distribution of BCL2L10 was also studied in ejaculated sperm cells and in isolated mouse skeletal muscle fibres. MAIN RESULTS AND THE ROLE OF CHANCE: The BCL2L10 protein was detectable in healthy human preimplantation embryos at least until the blastocyst stage. In high-quality embryos, BCL2L10 was predominantly cytoplasmic with mitochondrial localization. In contrast, BCL2L10 exhibited extra-mitochondrial localization in abnormal embryos, and was nuclear-cytoplasmic in approximately half (17/30) of the poor-quality embryos. Morphologically fragmented embryos showed coexistence of blastomeres with BCL2L10-positive expression and blastomeres or fragments negative for BCL2L10. LIMITATIONS, REASONS FOR CAUTION: Future studies are needed to evaluate whether embryo quality is related to an exclusive mitochondrial localization of BCL2L10. Mechanisms mediating the nuclear translocation of BCL2L10 in abnormal embryos and functions of this nuclear pool of BCL2L10 are currently unknown. WIDER IMPLICATIONS OF THE FINDINGS: The nuclear localization of BCL2L10 in abnormal embryos suggests a potential role for this protein in pathological conditions resulting in embryo arrest. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for this study. There are no competing interests.

UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents.

International audience; Insertions or deletions (indels) of amino acids residues have been recognized as an important source of genetic and structural divergence between paralogous Bcl-2 family members. However, these signature sequences have not so far been extensively investigated amongst orthologous Bcl-2 family proteins. Bcl2l10 is an antiapoptotic member of the Bcl-2 family that has evolved rapidly throughout the vertebrate lineage and which shows conserved abundant expression in eggs and oocytes. In this paper, we have unraveled two major sites of divergence between human Bcl2l10 and its vertebrate homologs. The first one provides length variation at the N-terminus (before the BH4 domain) and the second one is located between the predicted α5-α6 pore-forming helices, providing an unprecedented case in the superfamily of helix-bundled pore-forming proteins. These two particular indels were studied phylogenetically and through biochemical and cell biological techniques, including truncation and site-directed mutagenesis. While deletion of the N-terminal extension had no significant functional impact in HeLa cells, our results suggest that the human Bcl2l10 protein evolved a calcium-binding motif in its α5-α6 interhelical region by acquiring critical negatively charged residues. Considering the reliance of female eggs on calcium-dependent proteins and calcium-regulated processes and the exceptional longevity of oocytes in the primate lineage, we propose that this microstructural variation may be an adaptive feature associated with high maternal expression of this Bcl-2 family member.

Resume Des signes d’apoptose, une forme de suicide cellulaire programme, ont ete decrits dans les gametes et l’embryon preimplantatoire de nombreuses especes de mammiferes y compris l’homme, a la fois in vitro et in vivo. Parce que le developpement embryonnaire serait lie a un equilibre entre proliferation et mort cellulaire, l’etude du controle genetique de l’apoptose dans les embryons preimplantatoires est d’une importance considerable. Par ailleurs, on sait que la qualite des gametes (en particulier des ovocytes) influencerait leur propre survie mais egalement le developpement embryonnaire precoce. Les regulateurs d’apoptose appartenant a la famille BCL-2 occupent une place centrale dans les voies de decision de vie ou de mort cellulaire. Dans cet article, nous presentons une revue de la litterature sur l’expression des membres de la famille Bcl-2 dans les gametes et l’embryon precoce. Les resultats indiquent que les trois sous-groupes composant la famille (membres multidomaines pro- et anti-apoptotiques et membres tueurs a BH3 seulement) presentent un profil d’expression dynamique tout au long de la differenciation des cellules germinales mâles et femelles et lors du developpement embryonnaire precoce. Si la proteine pro-apoptotique Bax joue un role cle dans l’apoptose des gametes et de l’embryon precoce, l’importance relative des differentes proteines de survie (Bcl-2, Bcl-xL, Bcl-w, Mcl-1) et des membres a BH3 seulement (Bim, Bad, Bik) est en revanche moins claire. Bien que l’analyse des patrons d’expression des differents membres de la famille Bcl-2 soit incontournable, l’etude d’autres proprietes telles que le controle transcriptionnel (par des stimuli environnementaux), la localisation subcellulaire ou les modifications post-traductionnelles semble particulierement pertinente. En plus de son interet fondamental, la decouverte du mecanisme d’action moleculaire des membres de la famille Bcl-2 dans les gametes et l’embryon preimplantatoire peut permettre d’ouvrir des pistes de recherche diagnostique et therapeutique interessantes.

Apoptosis has been reported in oocytes and human preimplantation embryos both in vitro and in vivo. BCL-2 family proteins are likely to play a pivotal role in controlling oocyte and early embryo degeneration. However, no BCL-2-related survival factors have been identified that would specifically function during oocyte maturation, after fertilization and during early embryogenesis. Here, we performed a comprehensive tissue expression pattern analysis of the BCL-2 family at the mRNA level. While expression of various members was detected in human oocytes and during early primate embryogenesis, our data indicate that BCL2L10 is the predominant maternally loaded Bcl-2 family transcript, revealing an evolutionary conserved expression profile at the egg-to-zygote transition. We provide evidence that BCL2L10 is associated with the microtubule binding protein translationally controlled tumor protein and mitochondria, with a stage-specific redistribution along the pericortical regulatory ooplasm. In dying oocytes, BCL2L10 colocalized with proapoptotic BAX and neutralization of BCL2L10 accelerated oocyte death. We propose BCL2L10 as a novel and prime candidate related to oocyte maturation, fertility, and embryo developmental competence.

By controlling mitochondrial membrane permeability, the Bcl-2 family of proteins plays a crucial role in the regulation of cell death. Members of this family are functionally categorized as either pro- or anti-apoptotic based on their ability to promote or inhibit mitochondrial-dependent cell death signals. The critical role of Bcl-2 family proteins in human diseases has made them the focus of intense research by the pharmaceutical industry and academia. Anti-apoptotic Bcl-2 homologues have been widely explored as potential therapeutic targets for anticancer therapy through the development of nucleic-acid and peptide-based inhibitory molecules. Recently, strategies for the development of peptides or polypeptides derived from specific Bcl-2 family members have also been devised. This paper provides a brief review of the most recent patents that have been published relating to this area.

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

The mitochondrion descends from a bacterium that, about two billion years ago, became endosymbiotic. This organelle represents a Pandora’s box whose opening triggers cytochrome-c release and apoptosis of cells from multicellular animals, which evolved much later, about six hundred million years ago. BCL-2 proteins, which are critical apoptosis regulators, were recruited at a certain time point in evolution to either lock or unlock this mitochondrial Pandora’s box. Hence, particularly intriguing is the issue of when and how the “BCL-2 proteins–mitochondria–apoptosis” triptych emerged. This chapter explains what it takes from an evolutionary perspective to evolve a BCL-2-regulated apoptotic pathway, by focusing on the events occurring upstream of mitochondria.

International audience; Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia m?ri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

The anti-proliferative effect of Bcl-2 acts mainly at the level of the G0/G1 phase of the cell cycle. Deletions and point mutations in the bcl-2 gene show that the anti-proliferative activity of Bcl-2, can in some cases, be dissociated from its anti-apoptotic function. This indicates that the effect of Bcl-2 on cell cycle progression can be a direct effect and not only a consequence of its anti-apoptotic activity. Bcl-2 appears to mediate its anti-proliferative effect by acting on both signal transduction pathways (NFAT, ERK) and on specific cell cycle regulators (p27, p130).

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.

International audience; We made use of QNR cells transformed by a thermosensitive (tsNY68) strain of the Rous sarcoma virus (RSV) to compare the effect of p60(v-src) and serum in cultured nerve cells. In this system, both p60(v-src) heat inactivation and serum removal resulted in growth arrest in G1. In both cases, growth arrest was reversible since cell proliferation was rapidly re-induced following respectively p60v-src renaturation or serum re-addition. However, cells did not fully recover their ability to grow in soft agar, suggesting that, in contrast to the cell cycle machinery, the transforming capacities of these cells have been irreversibly altered. We found that p60(v-src) kinase activity prevented detachment from the substratum and cell death following serum removal. Thermal inactivation of p60(v-src) at restrictive temperature (41.5 degrees C), but not serum removal, resulted in dramatic morphological changes, which occurred 4 h after temperature shift up to 41.5 degrees C. Later on, typical features of apoptotic cells could be observed. Cell death was greatly reduced by the caspase-3 inhibitor ZVAD.FMK, but not by the caspase-1 inhibitor Ac-YVAD.CHO. Together, these results suggested that p60(v-src) and serum factors act on distinct pathways, at least in part. In an attempt to identify the signalling pathways involved in the cell response to p60(v-src) down regulation, we found that Erk and Rac were rapidly inactivated following temperature shift up to 41.5 degrees C. Thus, the combined effects of p60(v-src) and serum factors on the cytoskeleton dynamics and the apoptosis machinery are essential for full neoplastic transformation of neuroretina cells.We made use of QNR cells transformed by a thermosensitive (tsNY68) strain of the Rous sarcoma virus (RSV) to compare the effect of p60(v-src) and serum in cultured nerve cells. In this system, both p60(v-src) heat inactivation and serum removal resulted in growth arrest in G1. In both cases, growth arrest was reversible since cell proliferation was rapidly re-induced following respectively p60v-src renaturation or serum re-addition. However, cells did not fully recover their ability to grow in soft agar, suggesting that, in contrast to the cell cycle machinery, the transforming capacities of these cells have been irreversibly altered. We found that p60(v-src) kinase activity prevented detachment from the substratum and cell death following serum removal. Thermal inactivation of p60(v-src) at restrictive temperature (41.5 degrees C), but not serum removal, resulted in dramatic morphological changes, which occurred 4 h after temperature shift up to 41.5 degrees C. Later on, typical features of apoptotic cells could be observed. Cell death was greatly reduced by the caspase-3 inhibitor ZVAD.FMK, but not by the caspase-1 inhibitor Ac-YVAD.CHO. Together, these results suggested that p60(v-src) and serum factors act on distinct pathways, at least in part. In an attempt to identify the signalling pathways involved in the cell response to p60(v-src) down regulation, we found that Erk and Rac were rapidly inactivated following temperature shift up to 41.5 degrees C. Thus, the combined effects of p60(v-src) and serum factors on the cytoskeleton dynamics and the apoptosis machinery are essential for full neoplastic transformation of neuroretina cells.

BCL2DB (http://bcl2db.ibcp.fr) is a database designed to integrate data on BCL-2 family members and BH3-only proteins. These proteins control the mitochondrial apoptotic pathway and probably many other cellular processes as well. This large protein group is formed by a family of pro-apoptotic and anti-apoptotic homologs that have phylogenetic relationships with BCL-2, and by a collection of evolutionarily and structurally unrelated proteins characterized by the presence of a region of local sequence similarity with BCL-2, termed the BH3 motif. BCL2DB is monthly built, thanks to an automated procedure relying on a set of homemade profile HMMs computed from seed reference sequences representative of the various BCL-2 homologs and BH3-only proteins. The BCL2DB entries integrate data from the Ensembl, Ensembl Genomes, European Nucleotide Archive and Protein Data Bank databases and are enriched with specific information like protein classification into orthology groups and distribution of BH motifs along the sequences. The Web interface allows for easy browsing of the site and fast access to data, as well as sequence analysis with generic and specific tools. BCL2DB provides a helpful and powerful tool to both 'BCL-2-ologists' and researchers working in the various fields of physiopathology. Database URL: http://bcl2db.ibcp.fr.

International audience; Bcl-2 proteins are acknowledged as key regulators of programmed cell death. However, increasing data suggest additional roles, including regulation of the cell cycle, metabolism and cytoskeletal dynamics. Here we report the discovery and characterization of a new Bcl-2-related multidomain apoptosis accelerator, Bcl-wav, found in fish and frogs. Genetic and molecular studies in zebrafish indicate that Bcl-wav and the recently identified mitochondrial calcium uniporter (MCU) contribute to the formation of the notochord axis by controlling blastomere convergence and extension movements during gastrulation. Furthermore, we found that Bcl-wav controls intracellular Ca(2+) trafficking by acting on the mitochondrial voltage-dependent anion channel, and possibly on MCU, with direct consequences on actin microfilament dynamics and blastomere migration guidance. Thus, from an evolutionary point of view, the original function of Bcl-2 proteins might have been to contribute in controlling the global positioning system of blastomeres during gastrulation, a critical step in metazoan development.

Bcl-2 family proteins regulate apoptosis in animals. This protein family includes several homologous proteins and a collection of other proteins lacking sequence similarity except for a Bcl-2 homology (BH)3 motif. Thus, membership in the Bcl-2 family requires only one of the four BH motifs. On this basis, a growing number of diverse BH3-only proteins are being reported. Although compelling cell biological and biophysical evidence validates many BH3-only proteins, claims of significant BH3 sequence similarity are often unfounded. Computational and phylogenetic analyses suggest that only some BH3 motifs arose by divergent evolution from a common ancestor (homology), whereas others arose by convergent evolution or random coincidence (homoplasy), challenging current assumptions about which proteins constitute the extended Bcl-2 family.

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

In women, up to 99.9% of the oocyte stockpile formed during fetal life is decimated by apoptosis. Apoptotic features are also detected in human preimplantation embryos bothin vivoandin vitro. Despite the important consequences of cell death processes to oocyte competence and early embryonic development, little is known about its genetic and molecular control. B cell lymphoma-2 (BCL2) family proteins are major regulators of cell death and survival. Here, we present a literature review on BCL2 family expression and protein distribution in human and animal oocytes and early embryos. Most of the studies focused on the expression of two antagonistic members: the founding and survival family member BCL2 and its proapoptotic homolog BAX. However, recent transcriptomic analyses have identified novel candidate genes related to oocyte and/or early embryonic viability (such asBCL2L10) or commitment to apoptosis (e.g.BIK). Interestingly, some BCL2 proteins appear to be differentially distributed at the subcellular level during oocyte maturation and early embryonic development, a process probably linked to the functional compartmentalization of the ooplasm and blastomere. Assessment of BCL2 family involvement in regulating the survival of human oocytes and embryos may be of particular value for diagnosis and assisted reproductive technology. We suggest that implications of not only aberrant gene expression but also abnormal subcellular protein redistribution should be established in pathological conditions resulting in infertility.

SUMMARYAlthough many cancer cells are primed for apoptosis, they usually develop resistance to cell death at multiple levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members like Bax, is considered as a point-of-no-return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on using minimal active version of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, able to target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived poropeptide produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins evolutionary functionalized to form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.

Dear Editor, BCL-2 family proteins, through balanced interaction of proand anti-death members, play a pivotal role in controlling cell life and death [1]. Based on multiple sequence alignments, amino acid sequence similarity in a least one of four Bcl-2 homology (BH) domains (BH1–BH4) has been used to define the family membership for more than a decade. The number of available genomic sequences is now growing fast, due to the development of massive sequencing techniques, and comparisons among them can yield further important information for functional analysis. Using genomic data observed in today’s living species, we recently reported an extensive phylogenomic analysis of BCL-2-related sequences that enabled us to identify several phylogenetic subsets within the BCL-2 family [2]. The most well-defined subset is a monophyletic group, distributed across the Metazoa, which consists of ‘multidomain’ BCL-2-like (BCL-2, BCL-xL, XR11, BCL-W, XR1, MCL-1, BCL2L10, NR-13, BFL-1, CED-9, BUFFY) and BAX-like members (BAX, BAK, BOK, dBOK) and of a mix of homologous proteins (including BID, BCL-G, BCL-RAMBO/BCL2L13, BFK, BPR, and BHP1-2), whose common denominator is to share a same ‘globular’ fold. In contrast, BNip proteins (BNip1-3) and BH3-only members [3] other than BID (BAD, BIK, BIM, BMF, NOXA, PUMA, HRK, EGL-1) appear to form a polyphyletic assemblage with distinct ancestries and probably a more complex evolutionary history. Since the function of proand anti-apoptotic BCL-2 proteins is thought to determine cell fate in a wide range of cell types and species including human, it is an important effort to collect and classify the sequence, structure and annotation data available for BCL-2 family members (especially within the ‘helix-bundled’ class), with links to internet-based resources, and to integrate all these information in a database made freely available over the World Wide Web. Despite being one of the most studied protein class in cell biology, such a resource dedicated to the BCL2 family is still lacking. Former initiatives ([4]; http://www-personal.umich.edu/;ino/List/2342.htm) are no longer up-to-date or maintained and, to our knowledge, the only published web-based apoptosis tool currently available is the database of caspase substrates [5]. Here, we present BCL2DB, a database-backed web resource on the BCL-2 family of apoptosis regulators. At present, the database covers only those phylogenetically related BCL-2 family members that share a similar ‘‘pore-forming’’ helical bundle fold. BCL2DB supplies a variety of data for download, such as PDB files or nucleotide and protein sequences grouped by name, species and homology group. This work will provide the basis for higher order sequence and phylogenetic analyses. Moreover, we feel that protein function determination and drug discovery may benefit from this approach. BCL2DB is available at http:// bcl2db.ibcp.fr. S. V. Blaineau A. Aouacheria (&) IBCP, Institut de Biologie et Chimie des Proteines, CNRS, UMR 5086, 7 passage du Vercors, 69367 Lyon cedex, France e-mail: a.aouacheria@ibcp.fr

Research demonstrated that the function of mitochondria extends well beyond that of being cell powerhouses and revealed that these organelles fulfil a dual role in both cellular life and death. In most vertebrates, execution of the mitochondrial pathway of apoptosis requires permeabilization of the mitochondrial outer membrane, an event which allows for the release of a variety of intramembrane space proteins, leading to the activation of caspases and ultimately cell demise. Bcl-2 family proteins, which include pro- and antiapoptotic members, positively or negatively regulate mitochondrial outer membrane permeabilization, i.e. a barrier to apoptosis induction. Over-expression of Bcl-2 and Bcl-x(L) is associated with tumor progression and may be responsible for drug resistance, making pro-survival Bcl-2 family members important targets for the development of anticancer agents. Pharmacological apoptosis modulation by manipulation of pro-apoptotic Bcl-2 family proteins, with the goal to treat disorders associated with uncontrolled cell death or to kill unwanted cells, is likely to represent an additional research focus in the coming years. The purpose of this review is to describe, with examples taken from recent patents, novel strategies for targeting the Bcl-2 family of apoptotic regulators through peptide-based approaches and selective delivery of functional nucleic acids.

International audience; Single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variations in numerous organisms, have emerged as important tools for the study of complex genetic traits and deciphering of genome evolution. High-throughput genome sequencing projects worldwide provide an unprecedented opportunity for whole-genome SNP analysis in a variety of species. To facilitate SNP discovery in vertebrates, we have developed a web-based, user-friendly, and fully automated application, DigiPINS, for genome-wide identification of exonic SNPs from EST data. Currently, the database can be used to the mining of exonic SNPs in six complete genomes (Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus and Danio rerio). In addition to providing information on sequence conservation, DigiPINS allows compilation of comprehensive sets of polymorphisms within cancer candidate genes or identification of novel cancer markers, making it potentially useful for cancer association studies. The DigiPINS server is available via the internet at http://pbil.univ-lyon1.fr/gem/DigiPINS/query_DigiPINS.php.Single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variations in numerous organisms, have emerged as important tools for the study of complex genetic traits and deciphering of genome evolution. High-throughput genome sequencing projects worldwide provide an unprecedented opportunity for whole-genome SNP analysis in a variety of species. To facilitate SNP discovery in vertebrates, we have developed a web-based, user-friendly, and fully automated application, DigiPINS, for genome-wide identification of exonic SNPs from EST data. Currently, the database can be used to the mining of exonic SNPs in six complete genomes (Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus and Danio rerio). In addition to providing information on sequence conservation, DigiPINS allows compilation of comprehensive sets of polymorphisms within cancer candidate genes or identification of novel cancer markers, making it potentially useful for cancer association studies. The DigiPINS server is available via the internet at http://pbil.univ-lyon1.fr/gem/DigiPINS/query_DigiPINS.php.

International audience; BACKGROUND: A promising application of the huge amounts of genetic data currently available lies in developing a better understanding of complex diseases, such as cancer. Analysis of publicly available databases can help identify potential candidates for genes or mutations specifically related to the cancer phenotype. In spite of their huge potential to affect gene function, no systematic attention has been paid so far to the changes that occur in untranslated regions of mRNA. RESULTS: In this study, we used Expressed Sequence Tag (EST) databases as a source for cancer-related sequence polymorphism discovery at the whole-genome level. Using a novel computational procedure, we focused on the identification of untranslated region (UTR)-localized non-coding Single Nucleotide Polymorphisms (UTR-SNPs) significantly associated with the tumoral state. To explore possible relationships between genetic mutation and phenotypic variation, bioinformatic tools were used to predict the potential impact of cancer-associated UTR-SNPs on mRNA secondary structure and UTR regulatory elements. We provide a comprehensive and unbiased description of cancer-associated UTR-SNPs that may be useful to define genotypic markers or to propose polymorphisms that can act to alter gene expression levels. Our results suggest that a fraction of cancer-associated UTR-SNPs may have functional consequences on mRNA stability and/or expression. CONCLUSION: We have undertaken a comprehensive effort to identify cancer-associated polymorphisms in untranslated regions of mRNA and to characterize putative functional UTR-SNPs. Alteration of translational control can change the expression of genes in tumor cells, causing an increase or decrease in the concentration of specific proteins. Through the description of testable candidates and the experimental validation of a number of UTR-SNPs discovered on the secreted protein acidic and rich in cysteine (SPARC) gene, this report illustrates the utility of a cross-talk between in silico transcriptomics and cancer genetics.BACKGROUND: A promising application of the huge amounts of genetic data currently available lies in developing a better understanding of complex diseases, such as cancer. Analysis of publicly available databases can help identify potential candidates for genes or mutations specifically related to the cancer phenotype. In spite of their huge potential to affect gene function, no systematic attention has been paid so far to the changes that occur in untranslated regions of mRNA. RESULTS: In this study, we used Expressed Sequence Tag (EST) databases as a source for cancer-related sequence polymorphism discovery at the whole-genome level. Using a novel computational procedure, we focused on the identification of untranslated region (UTR)-localized non-coding Single Nucleotide Polymorphisms (UTR-SNPs) significantly associated with the tumoral state. To explore possible relationships between genetic mutation and phenotypic variation, bioinformatic tools were used to predict the potential impact of cancer-associated UTR-SNPs on mRNA secondary structure and UTR regulatory elements. We provide a comprehensive and unbiased description of cancer-associated UTR-SNPs that may be useful to define genotypic markers or to propose polymorphisms that can act to alter gene expression levels. Our results suggest that a fraction of cancer-associated UTR-SNPs may have functional consequences on mRNA stability and/or expression. CONCLUSION: We have undertaken a comprehensive effort to identify cancer-associated polymorphisms in untranslated regions of mRNA and to characterize putative functional UTR-SNPs. Alteration of translational control can change the expression of genes in tumor cells, causing an increase or decrease in the concentration of specific proteins. Through the description of testable candidates and the experimental validation of a number of UTR-SNPs discovered on the secreted protein acidic and rich in cysteine (SPARC) gene, this report illustrates the utility of a cross-talk between in silico transcriptomics and cancer genetics.

Background Owing to the explosion of information generated by human genomics, analysis of publicly available databases can help identify potential candidate genes relevant to the cancerous phenotype. The aim of this study was to scan for such genes by whole-genome in silico subtraction using Expressed Sequence Tag (EST) data. Methods Genes differentially expressed in normal versus tumor tissues were identified using a computer-based differential display strategy. Bcl-xL, an anti-apoptotic member of the Bcl-2 family, was selected for confirmation by western blot analysis. Results Our genome-wide expression analysis identified a set of genes whose differential expression may be attributed to the genetic alterations associated with tumor formation and malignant growth. We propose complete lists of genes that may serve as targets for projects seeking novel candidates for cancer diagnosis and therapy. Our validation result showed increased protein levels of Bcl-xL in two different liver cancer specimens compared to normal liver. Notably, our EST-based data mining procedure indicated that most of the changes in gene expression observed in cancer cells corresponded to gene inactivation patterns. Chromosomes and chromosomal regions most frequently associated with aberrant expression changes in cancer libraries were also determined. Conclusion Through the description of several candidates (including genes encoding extracellular matrix and ribosomal components, cytoskeletal proteins, apoptotic regulators, and novel tissue-specific biomarkers), our study illustrates the utility of in silico transcriptomics to identify tumor cell signatures, tumor-related genes and chromosomal regions frequently associated with aberrant expression in cancer.

International audience; Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.

Last decade has led to the accumulation of large amounts of data on cancer genetics, opening an unprecedented access to the mapping of cancer genes in the human genome. Single-nucleotide polymorphisms (SNPs), the most common form of DNA variation in humans, emerge as an invaluable tool for cancer association studies. These genotypic markers can be used to assay how alleles of candidate genes correlate with the malignant phenotype, and may provide new clues into the genetic modifications that characterize cancer onset. In this cancer-oriented study, we detail an SNP mining strategy based on the analysis of expressed sequence tags among publicly available databases. Our whole-genome approach provides a comprehensive and unbiased description of nonsynonymous SNPs (nsSNPs) in tumoral versus normal tissues. To gain further insights into the possible relationships between genetic variation and altered phenotype, locations of a subset of nsSNPs were mapped onto protein domains known to be critical for protein function. Computational methods were also used to predict the potential impact of these cancer-associated nsSNPs on protein structure and function. We illustrate our approach through the detailed biochemical and structural characterization of a previously unknown cancer-associated mutation (G79C) affecting the 8 kDa dynein light chain (DNCL1).

In this report, we conducted a comprehensive survey of Bcl-2 family members, a divergent group of proteins that regulate programmed cell death by an evolutionarily conserved mechanism. Using comparative sequence analysis, we found novel sequences in mammals, nonmammalian vertebrates, and in a number of invertebrates. We then asked what conclusions could be drawn from phyletic distribution, intron/exon structures, sequence/structure relationships, and phylogenetic analyses within the updated Bcl-2 family. First, multidomain members having a sequence pattern consistent with the conservation of the Bcl-X(L)/Bax/Bid topology appear to be restricted to multicellular animals and may share a common ancestry. Next, BNip proteins, which were originally identified based on their ability to bind to E1B 19K/Bcl-2 proteins, form three independent monophyletic branches with different evolutionary history. Lastly, a set of Bcl-2 homology 3-only proteins with unrelated secondary structures seems to have evolved after the origin of Metazoa and exhibits diverse expansion after speciation during vertebrate evolution.