Structural analysis of HopPmaL reveals the presence of a second adaptor domain common to the HopAB family of Pseudomonas syringae type III effectors

HopPmaL is a member of the HopAB family of type III effectors present in the phytopathogen Pseudomonas syringae. Using both X-ray crystallography and solution nuclear magnetic resonance, we demonstrate that HopPmaL contains two structurally homologous yet functionally distinct domains. The N-terminal domain corresponds to the previously described Pto-binding domain, while the previously uncharacterised C-terminal domain spans residues 308-385. While structurally similar, these domains do not share significant sequence similarity and most importantly demonstrate significant differences in key residues involved in host protein recognition, suggesting that each of them targets a different host protein.     

Structural Basis of E2–25K/UBB+1 Interaction Leading to Proteasome Inhibition and Neurotoxicity

E2–25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB⁺¹) and has been identified as a crucial factor regulating amyloid-β neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2–25K-UBB⁺¹ interaction, we determined the three-dimensional structures of UBB⁺¹, E2–25K and the E2–25K/ubiquitin, and E2–25K/UBB⁺¹ complex. The structures revealed that ubiquitin or UBB⁺¹ is bound to E2–25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2–25K mutants showed that disrupting UBB⁺¹ binding markedly diminishes synthesis of neurotoxic UBB⁺¹-anchored polyubiquitin. These results suggest that the interaction between E2–25K and UBB⁺¹ is critical for the synthesis and accumulation of UBB⁺¹-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.     

Competitive environments induce shifts in host fidelity

Recent models support the idea of sympatric speciation as a result of the joint effects of disruptive selection and assortative mating. We present experimental data, testing models of speciation through frequency‐dependent selection. We show that under high competition on a mixture of resources/hosts, strains of the Seed beetle, Callosobruchus maculatus, change their host fidelity and evolve a more generalistic behaviour in resource utilization among females. The change in host fidelity did not result in disruptive selection and was not followed by assortative mating. This means that only one of three fundamental prerequisites for sympatric speciation evolved as a result of the frequency‐dependent selection. We conclude that for this process to work, a shift to a novel food resource as a result of selection must also lead to a loss of preference for the original resource such that individuals are only able to use either one of the two.     

Local selection modifies phenotypic divergence among Rana temporaria populations in the presence of gene flow

In ectotherms, variation in life history traits among populations is common and suggests local adaptation. However, geographic variation itself is not a proof for local adaptation, as genetic drift and gene flow may also shape patterns of quantitative variation. We studied local and regional variation in means and phenotypic plasticity of larval life history traits in the common frog Rana temporaria using six populations from central Sweden, breeding in either open‐canopy or partially closed‐canopy ponds. To separate local adaptation from genetic drift, we compared differentiation in quantitative genetic traits (Q_ST) obtained from a common garden experiment with differentiation in presumably neutral microsatellite markers (F_ST). We found that R. temporaria populations differ in means and plasticities of life history traits in different temperatures at local, and in F_ST at regional scale. Comparisons of differentiation in quantitative traits and in molecular markers suggested that natural selection was responsible for the divergence in growth and development rates as well as in temperature‐induced plasticity, indicating local adaptation. However, at low temperature, the role of genetic drift could not be separated from selection. Phenotypes were correlated with forest canopy closure, but not with geographical or genetic distance. These results indicate that local adaptation can evolve in the presence of ongoing gene flow among the populations, and that natural selection is strong in this system.     

Molecular evidence of the survival of subterranean amphipods (Arthropoda) during Ice Age underneath glaciers in Iceland

Two endemic groundwater arthropod crustacean species, Crangonyx islandicus and Crymostygius thingvallensis, were recently discovered on the mid‐Atlantic volcanic island of Iceland. The extent of morphological differences from closest relatives, endemism, along with the geographic isolation of Iceland and its complete coverage by glaciers 21 000 years ago, suggests that these two species have survived glaciation periods in sub‐glacial refugia. Here we provide strong support for this hypothesis by an analysis of mitochondrial genetic variation within Crangonyx islandicus. Our results show that the species is divided into several distinct monophyletic groups that are found along the volcanic zone in Iceland, which have been separated by 0.5 to around 5 million years. The genetic divergence between groups reflects geographic distances between sampling sites, indicating that divergence occurred after the colonization of Iceland. The genetic patterns, as well as the dependency of genetic variation on distances from the tectonic plate boundary and altitude, points to recent expansion from several refugia within Iceland. This presents the first genetic evidence of multicellular organisms as complex as crustacean amphipods which have survived glaciations beneath an ice sheet. This survival may be explained by geothermal heat linked to volcanic activities, which may have maintained favourable habitats in fissures along the tectonic plate boundary in Iceland during glaciations.     

Structural and functional comparison of the RING domains of two p53 E3 ligases, Mdm2 and Pirh2

The tumor suppressor p53 maintains genome stability and prevents malignant transformation by promoting cell cycle arrest and apoptosis. Both Mdm2 and Pirh2 have been shown to ubiquitylate p53 through their RING domains, thereby targeting p53 for proteasomal degradation. Using structural and functional analyses, here we show that the Pirh2 RING domain differs from the Mdm2 RING domain in its oligomeric state, surface charge distribution, and zinc coordination scheme. Pirh2 also possesses weaker E3 ligase activity toward p53 and directs ubiquitin to different residues on p53. NMR and mutagenesis studies suggest that whereas Pirh2 and Mdm2 share a conserved E2 binding site, the seven C-terminal residues of the Mdm2 RING directly contribute to Mdm2 E3 ligase activity, a feature unique to Mdm2 and absent in the Pirh2 RING domain. This comprehensive analysis of the Pirh2 and Mdm2 RING domains provides structural and mechanistic insight into p53 regulation by its E3 ligases.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Hierarchical regulation of pelagic invertebrates in lakes of the northern Great Plains: a novel model for interdecadal effects of future climate change on lakes

Endorheic lakes of the northern Great Plains encompass a wide range of environmental parameters (e.g., salinity, pH, DOC, Ca, nutrients, depth) that vary 1000‐fold among sites and through the past 2000 years due to variation in basin hydrology and evaporative forcing. However, while many environmental parameters are known to individually influence zooplankton diversity and taxonomic composition, relatively little is known of the hierarchical relationships among potential controls or of how regulatory mechanisms may change in response to climate variation on diverse scales. To address these issues, we surveyed 70 lakes within a 100 000 km² prairie region to simulate the magnitude of environmental change expected to occur over 100–1000 years and to quantify the unique and interactive effects of diverse environmental parameters in regulating pelagic invertebrate community structure at that scale. Multivariate analyses showed that salinity was the principal correlate of changes in invertebrate composition among lakes, with a sequential loss of taxa between salinities of 4 and 50 g total dissolved solids L^?1 until one to two species predominated in highly saline systems. In contrast, changes in the concentrations of Ca²⁺ and other mineral nutrients exerted secondary controls of invertebrate assemblages independent of salinity, whereas lake depth provided a tertiary regulatory mechanism structuring species composition. In contrast to these large‐scale hierarchical patterns, seasonal surveys (May, July, September) of a subset of 21 lakes in each of 2003–2005 revealed that annual meteorological variation had no measurable effect on pelagic invertebrates, despite large differences in temperature, precipitation, and evaporation arising from regional droughts. Together these findings show that pelagic invertebrate communities in saline lakes are resilient to interannual variability in climate, but suggest that lakes of the northern Great Plains may provide a sensitive model to forecast centennial effects of future climate change.     

Role of Pirh2 in mediating the regulation of p53 and c-Myc

Ubiquitylation is fundamental for the regulation of the stability and function of p53 and c-Myc. The E3 ligase Pirh2 has been reported to polyubiquitylate p53 and to mediate its proteasomal degradation. Here, using Pirh2 deficient mice, we report that Pirh2 is important for the in vivo regulation of p53 stability in response to DNA damage. We also demonstrate that c-Myc is a novel interacting protein for Pirh2 and that Pirh2 mediates its polyubiquitylation and proteolysis. Pirh2 mutant mice display elevated levels of c-Myc and are predisposed for plasma cell hyperplasia and tumorigenesis. Consistent with the role p53 plays in suppressing c-Myc-induced oncogenesis, its deficiency exacerbates tumorigenesis of Pirh2(-/-) mice. We also report that low expression of human PIRH2 in lung, ovarian, and breast cancers correlates with decreased patients' survival. Collectively, our data reveal the in vivo roles of Pirh2 in the regulation of p53 and c-Myc stability and support its role as a tumor suppressor.