BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

Structures of the cIAP2 RING domain reveal conformational changes associated with ubiquitin-conjugating enzyme (E2) recruitment

Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo.     

Role of proneural genes in the formation of the larval olfactory organ of <Emphasis Type="Italic">Drosophila</Emphasis>

In this paper, we address the role of proneural genes in the formation of the dorsal organ in the Drosophila larva. This organ is an intricate compound comprising the multineuronal dome—the exclusive larval olfactory organ—and a number of mostly gustatory sensilla. We first determine the numbers of neurons and of the different types of accessory cells in the dorsal organ. From these data, we conclude that the dorsal organ derives from 14 sensory organ precursor cells. Seven of them appear to give rise to the dome, which therefore may be composed of seven fused sensilla, whereas the other precursors produce the remaining sensilla of the dorsal organ. By a loss-of-function approach, we then analyze the role of atonal, amos, and the achaete-scute complex (AS-C), which in the adult are the exclusive proneural genes required for chemosensory organ specification. We show that atonal and amos are necessary and sufficient in a complementary way for four and three of the sensory organ precursors of the dome, respectively. AS-C, on the other hand, is implicated in specifying the non-olfactory sensilla, partially in cooperation with atonal and/or amos. Similar links for these proneural genes with olfactory and gustatory function have been established in the adult fly. However, such conserved gene function is not trivial, given that adult and larval chemosensory organs are anatomically very different and that the development of adult olfactory sensilla involves cell recruitment, which is unlikely to play a role in dome formation. N. Grillenzoni and V. de Vaux contributed equally to this work.     

Anti-apoptotic potential of insect cellular and viral IAPs in mammalian cells

IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.     

A novel Apaf-1-independent putative caspase-2 activation complex

Caspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome.     

Characterizing neutral and adaptive genomic differentiation in a changing climate: The most northerly freshwater fish as a model

Arctic freshwater ecosystems have been profoundly affected by climate change. Given that the Arctic charr (Salvelinus alpinus) is often the only fish species inhabiting these ecosystems, it represents a valuable model for studying the impacts of climate change on species life‐history diversity and adaptability. Using a genotyping‐by‐sequencing approach, we identified 5,976 neutral single nucleotide polymorphisms and found evidence for reduced gene flow between allopatric morphs from two high Arctic lakes, Linne'vatn (Anadromous, Normal, and Dwarf) and Ellasjøen (Littoral and Pelagic). Within each lake, the degree of genetic differentiation ranged from low (Pelagic vs. Littoral) to moderate (Anadromous and Normal vs. Dwarf). We identified 17 highly diagnostic, putatively adaptive SNPs that differentiated the allopatric morphs. Although we found no evidence for adaptive differences between morphs within Ellasjøen, we found evidence for moderate (Anadromous vs. Normal) to high genetic differentiation (Anadromous and Normal vs. Dwarf) among morphs within Linne'vatn based on two adaptive loci. As these freshwater ecosystems become more productive, the frequency of sympatric morphs in Ellasjøen will likely shift based on foraging opportunities, whereas the propensity to migrate may decrease in Linne'vatn, increasing the frequency of the Normal morph. The Dwarf charr was the most genetically distinct group. Identifying the biological basis for small body size should elucidate the potential for increased growth and subsequent interbreeding with sympatric morphs. Overall, neutral and adaptive genomic differentiation between allopatric and some sympatric morphs suggests that the response of Arctic charr to climate change will be variable across freshwater ecosystems.     

Seasonal dynamics and habitat specificity of mosquitoes in an English wetland: implications for UK wetland management and restoration

We engaged in field studies of native mosquitoes in a Cambridgeshire Fen, investigating a) the habitat specificity and seasonal dynamics of our native fauna in an intensively managed wetland, b) the impact of water‐level and ditch management, and c) their colonization of an arable reversion to flooded grassland wetland expansion project. Studies from April to October, 2010 collected 14,000 adult mosquitoes (15 species) over 292 trap‐nights and ～4,000 pre‐imaginal mosquitoes (11 species). Open floodwater species (Aedes caspius and Aedes cinereus, 43.3%) and wet woodland species (Aedes cantans/annulipes and Aedes rusticus, 32.4%) dominated, highlighting the major impact of seasonal water‐level management on mosquito populations in an intensively managed wetland. In permanent habitats, managing marginal ditch vegetation and ditch drying significantly affect densities of pre‐imaginal anophelines and culicines, respectively. This study presents the first UK field evidence of the implications of wetland expansion through arable reversion on mosquito colonization. Understanding the heterogeneity of mosquito diversity, phenology, and abundance in intensively managed UK wetlands will be crucial to mitigating nuisance and vector species through habitat management and biocidal control.     

The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation,but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.