The Signal Sequence Influences Post-Translational ER Translocation at Distinct Stages

The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.     

Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

Global downregulation of microRNAs (miRNAs) is a common feature of human tumors and has been shown to enhance cancer progression. Several components of the miRNA biogenesis machinery (XPO5, DICER and TRBP) have been shown to act as haploinsufficient tumor suppressors. How the deregulation of miRNA biogenesis promotes tumor development is not clearly understood. Here we show that loss of miRNA biogenesis increased resistance to endoplasmic reticulum (ER) stress-induced cell death. We observed that HCT116 cells with a DICER hypomorphic mutation (Exn5/Exn5) or where DICER or DROSHA were knocked down were resistant to ER stress-induced cell death. Extensive analysis revealed little difference in the unfolded protein response (UPR) of WT compared to Exn5/Exn5 HCT116 cells upon ER stress treatment. However, analysis of the intrinsic apoptotic pathway showed that resistance occurred upstream of the mitochondria. In particular, BAX activation and dissipation of mitochondrial membrane potential was attenuated, and there was altered expression of BCL-2 family proteins. These observations demonstrate a key role for miRNAs as critical modulators of the ER stress response. In our model, downregulation of miRNA biogenesis delays ER stress-induced apoptosis. This suggests that disrupted miRNA biogenesis may contribute to cancer progression by inhibiting ER stress-induced cell death.     

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for ¹⁵N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.     

Genotype‐environment associations support a mosaic hybrid zone between two tidal marsh birds

Local environmental features can shape hybrid zone dynamics when hybrids are bounded by ecotones or when patchily distributed habitat types lead to a corresponding mosaic of genotypes. We investigated the role of marsh‐level characteristics in shaping a hybrid zone between two recently diverged avian taxa – Saltmarsh (Ammodramus caudacutus) and Nelson's (A. nelsoni) sparrows. These species occupy different niches where allopatric, with caudacutus restricted to coastal marshes and nelsoni found in a broader array of wetland and grassland habitats and co‐occur in tidal marshes in sympatry. We determined the influence of habitat types on the distribution of pure and hybrid sparrows and assessed the degree of overlap in the ecological niche of each taxon. To do this, we sampled and genotyped 305 sparrows from 34 marshes across the hybrid zone and from adjacent regions. We used linear regression to test for associations between marsh characteristics and the distribution of pure and admixed sparrows. We found a positive correlation between genotype and environmental variables with a patchy distribution of genotypes and habitats across the hybrid zone. Ecological niche models suggest that the hybrid niche was more similar to that of A. nelsoni and habitat suitability was influenced strongly by distance from coastline. Our results support a mosaic model of hybrid zone maintenance, suggesting a role for local environmental features in shaping the distribution and frequency of pure species and hybrids across space.     

An improved neutral landscape model for recreating real landscapes and generating landscape series for spatial ecological simulations

Many studies have assessed the effect of landscape patterns on spatial ecological processes by simulating these processes in computer‐generated landscapes with varying composition and configuration. To generate such landscapes, various neutral landscape models have been developed. However, the limited set of landscape‐level pattern variables included in these models is often inadequate to generate landscapes that reflect real landscapes. In order to achieve more flexibility and variability in the generated landscapes patterns, a more complete set of class‐ and patch‐level pattern variables should be implemented in these models. These enhancements have been implemented in Landscape Generator (LG), which is a software that uses optimization algorithms to generate landscapes that match user‐defined target values. Developed for participatory spatial planning at small scale, we enhanced the usability of LG and demonstrated how it can be used for larger scale ecological studies. First, we used LG to recreate landscape patterns from a real landscape (i.e., a mountainous region in Switzerland). Second, we generated landscape series with incrementally changing pattern variables, which could be used in ecological simulation studies. We found that LG was able to recreate landscape patterns that approximate those of real landscapes. Furthermore, we successfully generated landscape series that would not have been possible with traditional neutral landscape models. LG is a promising novel approach for generating neutral landscapes and enables testing of new hypotheses regarding the influence of landscape patterns on ecological processes. LG is freely available online.     

Two different specific JNK activators are required to trigger apoptosis or compensatory proliferation in response to Rbf1 in Drosophila

The Jun Kinase (JNK) signaling pathway responds to diverse stimuli by appropriate and specific cellular responses such as apoptosis, differentiation or proliferation. The mechanisms that mediate this specificity remain largely unknown. The core of this signaling pathway, composed of a JNK protein and a JNK kinase (JNKK), can be activated by various putative JNKK kinases (JNKKK) which are themselves downstream of different adaptor proteins. A proposed hypothesis is that the JNK pathway specific response lies in the combination of a JNKKK and an adaptor protein upstream of the JNKK. We previously showed that the Drosophila homolog of pRb (Rbf1) and a mutant form of Rbf1 (Rbf1^D253A) have JNK-dependent pro-apoptotic properties. Rbf1^D253A is also able to induce a JNK-dependent abnormal proliferation. Here, we show that Rbf1-induced apoptosis triggers proliferation which depends on the JNK pathway activation. Taking advantage of these phenotypes, we investigated the JNK signaling involved in either Rbf1-induced apoptosis or in proliferation in response to Rbf1-induced apoptosis. We demonstrated that 2 different JNK pathways involving different adaptor proteins and kinases are involved in Rbf1-apoptosis (i.e. Rac1-dTak1-dMekk1-JNK pathway) and in proliferation in response to Rbf1-induced apoptosis (i.e., dTRAF1-Slipper-JNK pathway). Using a transient induction of rbf1, we show that Rbf1-induced apoptosis activates a compensatory proliferation mechanism which also depends on Slipper and dTRAF1. Thus, these 2 proteins seem to be key players of compensatory proliferation in Drosophila.     

Visual-Olfactory Integration in the Human Disease Vector Mosquito Aedes aegypti

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Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.