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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.

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.

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 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.