Structural Determinants of Skeletal Muscle Ryanodine Receptor Gating

Ryanodine receptor type 1 (RyR1) releases Ca²⁺ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca²⁺. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912–Glu-4948) and a linker helix (Val-4830–Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.     

Glycosphingolipids and mitochondria: role in apoptosis and disease

Glycosphingolipids (GSLs) comprise a class of lipids with important structural and signaling functions. Synthesized from ceramide in the Golgi, they are subsequently distributed to different compartments, most predominantly in the plasma membrane where they integrate signaling platforms. A recently characterized trafficking of ganglioside GD3 (GD3), a GSLs with two sialic-acid residues, to mitochondria has revealed a novel function of this lipid as a death effector. In addition to the interaction of GD3 with mitochondria recruiting these organelles to apoptotic pathways, GD3 disables survival paths dependent on NF-kappaB, thus favoring the balance towards cell death. The present review gathers the evidence documenting this emerging function of GSLs in cell death and their involvement in pathological states.     

Flexible oviposition behavior in the golden egg bug (Phyllomorpha laciniata) and its implications for offspring survival

Female golden egg bugs show unique flexibility in their oviposition behavior. Females can lay eggs on plants, where they are leftunattended, or on the back of conspecifics, where they remainuntil hatching. In this article we show that eggs have greatersurvival rates when carried by an adult than when laid on plants.The main causes of egg mortality are predators and a parasitoidwasp. Our results suggest that, although predation pressureis similar, fewer eggs are attacked by parasitoid wasps whencarried by an adult than when laid on plants. In addition,we show that, when given a choice, females prefer to lay eggson the backs of conspecifics than on plants. Thus, female ovipositionchoice is adaptive and minimizes individual offspring mortality.The factors that may maintain such behavioral variation in current populations are discussed.     

Protamine 3 shows evidence of weak, positive selection in mouse species (genus Mus)--but it is not a protamine

Protamines are short and highly basic sperm-specific nuclear proteins that replace somatic histones during spermiogenesis in a process that is crucial for sperm formation and function. Many mammals have two protamine genes (PRM1 and PRM2) located in a gene cluster, which appears to evolve fast. Another gene in this cluster (designated protamine 3 [PRM3]) encodes a protein that is conserved among mammals but that does not seem to be involved in chromatin condensation. We have compared protein sequences and amino acid compositions of protamines in this gene cluster, searched for evidence of positive selection of PRM3, and examined whether sexual selection (sperm competition) may drive the evolution of the PRM3 gene. Nucleotide and amino acid analyses of mouse sequences revealed that PRM3 was very different from PRM1 and from both the precursor and the mature sequences of PRM2. Among 10 mouse species, PRM3 showed weak evidence of positive selection in two species, but there was no clear association with levels of sperm competition. In analyses from among mammalian species, no evidence of positive selection was found in PRM3. We conclude that PRM3 exhibits several clear differences from other protamines and, furthermore, that it cannot be regarded as a true protamine.     

Molecular evolution of the ligands of the insulin-signaling pathway: dilp genes in the genus Drosophila

Drosophila melanogaster, unlike mammals, has seven insulin-like peptides (DILPS). In Drosophila, all seven genes (dilp1-7) are single copy in the 12 species studied, except for D. grimshawi with two tandem copies of dilp2. Our comparative analysis revealed that genes dilp1-dilp7 exhibit differential functional constraint, which is indicative of some functional divergence. Species of the subgenera Sophophora and Drosophila differ in some traits likely affected by the insulin-signaling pathway, such as adult body size. It is in the branch connecting the two subgenera that we found the footprint left by positive selection driving nonsynonymous changes at some dilp1 codons to fixation. Finally, the similar rate at which the two dilp2 copies of D. grimshawi have evolved since their duplication and the presence of a putative regulatory region highly conserved between the two paralogs would suggest that both copies were preserved either because of subfunctionalization or dose dependency rather than by the neofunctionalization of one of the two copies.     

Identification of human IKK-2 inhibitors of natural origin (part I): modeling of the IKK-2 kinase domain, virtual screening and activity assays

Background

Their large scaffold diversity and properties, such as structural complexity and drug similarity, form the basis of claims that natural products are ideal starting points for drug design and development. Consequently, there has been great interest in determining whether such molecules show biological activity toward protein targets of pharmacological relevance. One target of particular interest is hIKK-2, a serine-threonine protein kinase belonging to the IKK complex that is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Indeed, this has led to the development of synthetic ATP-competitive inhibitors for hIKK-2. Therefore, the main goals of this study were (a) to use virtual screening to identify potential hIKK-2 inhibitors of natural origin that compete with ATP and (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits.

Methodology/Principal Findings

We thus predicted that 1,061 out of the 89,425 natural products present in the studied database would inhibit hIKK-2 with good ADMET properties. Notably, when these 1,061 molecules were merged with the 98 synthetic hIKK-2 inhibitors used in this study and the resulting set was classified into ten clusters according to chemical similarity, there were three clusters that contained only natural products. Five molecules from these three clusters (for which no anti-inflammatory activity has been previously described) were then selected for in vitro activity testing, in which three out of the five molecules were shown to inhibit hIKK-2.

Conclusions/Significance

We demonstrated that our virtual-screening protocol was successful in identifying lead compounds for developing new inhibitors for hIKK-2, a target of great interest in medicinal chemistry. Additionally, all the tools developed during the current study (i.e., the homology model for the hIKK-2 kinase domain and the pharmacophore) will be made available to interested readers upon request.     

Major imprint of surface plankton on deep ocean prokaryotic structure and activity

Deep ocean microbial communities rely on the organic carbon produced in the sunlit ocean, yet it remains unknown whether surface processes determine the assembly and function of bathypelagic prokaryotes to a larger extent than deep‐sea physicochemical conditions. Here, we explored whether variations in surface phytoplankton assemblages across Atlantic, Pacific and Indian ocean stations can explain structural changes in bathypelagic (ca. 4,000 m) free‐living and particle‐attached prokaryotic communities (characterized through 16S rRNA gene sequencing), as well as changes in prokaryotic activity and dissolved organic matter (DOM) quality. We show that the spatial structuring of prokaryotic communities in the bathypelagic strongly followed variations in the abundances of surface dinoflagellates and ciliates, as well as gradients in surface primary productivity, but were less influenced by bathypelagic physicochemical conditions. Amino acid‐like DOM components in the bathypelagic reflected variations of those components in surface waters, and seemed to control bathypelagic prokaryotic activity. The imprint of surface conditions was more evident in bathypelagic than in shallower mesopelagic (200–1,000 m) communities, suggesting a direct connectivity through fast‐sinking particles that escape mesopelagic transformations. Finally, we identified a pool of endemic deep‐sea prokaryotic taxa (including potentially chemoautotrophic groups) that appear less connected to surface processes than those bathypelagic taxa with a widespread vertical distribution. Our results suggest that surface planktonic communities shape the spatial structure of the bathypelagic microbiome to a larger extent than the local physicochemical environment, likely through determining the nature of the sinking particles and the associated prokaryotes reaching bathypelagic waters.     

Arabidopsis thaliana Atrab28: a nuclear targeted protein related to germination and toxic cation tolerance

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues.Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.     

Palynological study of Veronica Sect. Veronica and Sect. Veronicastrum (Scrophulariaceae) and its taxonomic significance

Pollen grains from 30 taxa, mainly from the Western Mediterranean area belonging to the perennial sections of the genus Veronica (Scrophulariaceae), were studied by LM and SEM. A close relationship was found between pollen size and ploidy level. Palynological characters cannot be regarded as essential taxonomic tools within the groups studied, although they have systematic value in some specific cases. The monophyly of Veronica subsect. Multiflorae is supported by a common and exclusive sculpture of exine. Delimitation of the genus is discussed and hypothetical evolutionary trends concerning pollen characters are addressed.