The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax,modulate functional interactions with the anti-apoptotic protein Bcl-x<Subscript>L</Subscript>

Background  

Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L.     

Bacteriophage ϕ6 nucleocapsid surface protein 8 interacts with virus-specific membrane vesicles containing major envelope protein 9

Enveloped double-stranded RNA (dsRNA) bacterial virus Pseudomonas phage ϕ6 has been developed into an advanced assembly system where purified virion proteins and genome segments self-assemble into infectious viral particles, inferring the assembly pathway. The most intriguing step is the membrane assembly occurring inside the bacterial cell. Here, we demonstrate that the middle virion shell, made of protein 8, associates with the expanded viral core particle and the virus-specific membrane vesicle.     

Thioredoxin system in obligate anaerobe Desulfovibrio desulfuricans: Identification and characterization of a novel thioredoxin 2

Metal corroding sulfate reducing bacteria have been poorly characterized at molecular level due to difficulties pertaining to isolation and handling of anaerobes. We report here for the first time the presence and characterization of thioredoxin 2 in an obligate anaerobic dissimilatory sulfate reducing bacterium Desulfovibrio desulfuricans. In silico analysis of the D. desulfuricans genome revealed the presence of thioredoxin 1 (dstrx1), thioredoxin 2 (dstrx2) and thioredoxin reductase (dstrxR) genes. These genes were found to be actively expressed by the bacteria under the anaerobic growth conditions. We have overexpressed the anaerobic thioredoxin genes in E. coli to produce functionally active recombinant proteins. Recombinant DsTrxR recognized both DsTrx1 and DsTrx2 as its substrate. Mutation studies revealed that the activity of DsTrx2 can be completely abolished with a single amino acid mutation (C69A) in the signature motif 'WCGPC'. Furthermore, the N-terminal domain of DsTrx2 containing two extra CXXC motifs was found to have a negative regulation on its biochemical activity. In conclusion, we have shown the presence of thioredoxin 2 for the first time in an obligate anaerobe which in this anaerobe may be required for its survival under either oxidative stress conditions or metal ion hemostasis.     

Social learning about egg-laying substrates in fruitflies

Social learning, defined as learning from other individuals, has had dramatic effects on some species, including humans, in whom it has generated a rich culture. As a first step in examining the evolution of and mechanisms underlying social learning in insects, we tested for social learning in fruitflies (Drosophila melanogaster). Focal females (observers) that experienced novel food together with mated females (models), who had laid eggs on that food, subsequently exhibited a stronger preference for laying eggs on that food over another novel food compared with focal females that experienced the food alone. We observed no social learning, however, when observers experienced food with potentially more ambiguous social information provided by the presence of either virgin models or aggregation pheromone. This first documentation of social learning about egg-laying substrates in fruitflies builds on recent data indicating intricate use of social information by fruitflies and opens up exciting avenues for research on the evolution and neurogenetics of social learning using biology''s major model system.     

Analysis of Multiple Ethyl Methanesulfonate-Mutagenized Caenorhabditis elegans Strains by Whole-Genome Sequencing

Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.INDUCING molecular lesions in a genome is an effective approach to interrogate the genome for its functional elements. Molecular lesions can be induced using a variety of methods. Because of their efficiency and their ability to generate alleles with various different alterations in gene activity (e.g., amorphic, antimorphic, hypomorphic, and hypermorphic), chemical mutagens, such as ethyl methanesulfonate (EMS), are frequently used in genetic mutant screens (Anderson 1995). However, due to mutagen efficiency, a mutant animal selected for a single-locus phenotype invariably contains EMS-induced “background mutations” in its genome. Experimenters try to minimize the potential impact of background mutations through outcrossing to animals with a wild-type genome. Yet no full snapshots of genome sequences right after EMS mutagenesis and after outcrossing have so far been provided to illustrate the extent of background mutations and the extent to which they can indeed be eliminated.Another caveat of using base-changing chemical mutagens is the relative difficulty associated with identifying the phenotype-causing molecular lesion. In multicellular genetic model organisms, mutant identification involves time-consuming positional cloning approaches, usually involving breeding with genetically marked strains that allow pinpointing of the location of a molecular lesion. Even with rapid, SNP-based mapping approaches in animals with short generation times, such as Caenorhabditis elegans, substantial time hurdles, particularly in the final, fine-mapping stages, still exist. Conceptually similar problems in defining the location of a molecular lesion are encountered by human geneticists who attempt to identify disease-causing genetic lesions.Whole-genome sequencing (WGS) is beginning to emerge as an efficient and cost-effective tool to shortcut time-consuming mapping and positional cloning efforts (Hobert 2010). The sequencing of an entire genome and its ensuing comparison to a wild-type reference genome can potentially directly pinpoint the molecular lesion that results in the mutant phenotype the animal has been selected for. Proof-of-concept studies in bacteria, yeast, plants, worms, and flies have validated the applicability of this approach (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009; Flowers et al. 2010).Present-day deep sequencing platforms used for WGS generate relatively short sequence reads, thereby posing the bioinformatic challenge to align those reads to a reference genome. We previously described a software pipeline, MAQGene, which is based on the standard alignment program MAQ (Li et al. 2008) and facilitates this bioinformatic step by providing the end user with an extensively curated list of sequence variants from a WGS run of a mutated genome compared to a reference genome (Bigelow et al. 2009). This pipeline can be used for well-annotated, assembled genomes, such as C. elegans or Drosophila. In this article, we describe that this pipeline can identify not only point mutations but also deletions. We then use this pipeline to analyze a total of 17 EMS-mutagenized genomes. We find that EMS-mutagenized genomes carry a significant mutational load including presumptive loss-of-function alleles in several protein-coding genes that can lead to synthetic genetic interactions, one of which we describe here in more detail. We show that outcrossing to wild-type animals can lighten the mutational load; however, a substantial number of sequence variants are also introduced during outcrossing. Even though background mutations uncovered by WGS may complicate the interpretation of mutant phenotypes, they do provide a potentially useful source for functional studies of the affected genes.     

Gene expression in cortex and hippocampus during acute pneumococcal meningitis

Background  

Pneumococcal meningitis is associated with high mortality (~30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.     

Uncovering novel cell cycle players through the inactivation of securin in budding yeast

Budding yeast securin/Pds1p, an inhibitor of the anaphase activator separase/Esp1p, is involved in several checkpoint pathways and in promoting Esp1p's nuclear localization. Using a modified synthetic genetic array (SGA) screen for genes that become essential in the absence of Pds1p, we uncovered roles for uncharacterized genes in cell cycle processes, including Esp1p activation.     

VDAC is a conserved element of death pathways in plant and animal systems

Programmed cell death (PCD) is very much a part of plant life, although the underlying mechanisms are not so well understood as in animals. In animal cells, the voltage-dependent anion channel (VDAC), a major mitochondrial outer membrane transporter, plays an important role in apoptosis by participating in the release of intermembrane space proteins. To characterize plant PCD pathways by investigating the function of putative components in a mammalian apoptotic context, we have overexpressed a rice VDAC (osVDAC4) in the Jurkat T-cell line. Overexpression of osVDAC4 induces apoptosis, which can be blocked by Bcl-2 and the VDAC inhibitor DIDS. Modifying endogenous VDAC function by DIDS and hexokinase II (HxKII) in Jurkat cells inhibits mitochondria-mediated apoptotic pathways. Finally, we show that DIDS also abrogates heat-induced PCD in cucumber cotyledons. Our data suggest that VDAC is a conserved mitochondrial element of the death machinery in both plant and animal cells.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.