The eIF3 complex of Leishmania—subunit composition and mode of recruitment to different cap-binding complexes

Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit.     

Hierarchical tree snipping: clustering guided by prior knowledge

MOTIVATION: Hierarchical clustering is widely used to cluster genes into groups based on their expression similarity. This method first constructs a tree. Next this tree is partitioned into subtrees by cutting all edges at some level, thereby inducing a clustering. Unfortunately, the resulting clusters often do not exhibit significant functional coherence. RESULTS: To improve the biological significance of the clustering, we develop a new framework of partitioning by snipping--cutting selected edges at variable levels. The snipped edges are selected to induce clusters that are maximally consistent with partially available background knowledge such as functional classifications. Algorithms for two key applications are presented: functional prediction of genes, and discovery of functionally enriched clusters of co-expressed genes. Simulation results and cross-validation tests indicate that the algorithms perform well even when the actual number of clusters differs considerably from the requested number. Performance is improved compared with a previously proposed algorithm. AVAILABILITY: A java package is available at http://www.cs.bgu.ac.il/~dotna/ TreeSnipping     

Dysfunctional CD8 T Cells Form a Proliferative,Dynamically Regulated Compartment within Human Melanoma

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Is dinitrogen fixation significant in the Levantine Basin,East Mediterranean Sea?

We report N(2) fixation rates measured from two stations monitored monthly off the Mediterranean coast of Israel during 2006 and 2007, and along a transect from Israel to Crete in September 2008. Analyses of time-series data revealed expression of nifH genes from diazotrophs in nifH clusters I and II, including cyanobacterial bloom-formers Trichodesmium and diatom-Richelia intracellularis associations. However, nifH gene abundance and rates of N(2) fixation were very low in all size fractions measured (> 0.7 μm). Volumetric (15) N uptake ranged from below detection (～ 36% of > 300 samples) to a high of 0.3 nmol N l(-1) d(-1) and did not vary distinctly with depth or season. Areal N(2) fixation averaged ～ 1 to 4 μmol N m(-2) d(-1) and contributed only ～ 1% and 2% of new production and ～ 0.25% and 0.5% of primary production for the mixed (winter) and stratified (spring-fall) periods respectively. N(2) fixation rates along the 2008 east-west transect were also extremely low (0-0.04 nmol N l(-1) d(-1), integrated average 2.6 μmol N m(-2) d(-1) ) with 37% of samples below detection and no discernable difference between stations. We demonstrate that diazotrophy and N(2) fixation contribute only a minor amount of new N to the P impoverished eastern Mediterranean Sea.     

Inhibitory NK Receptor Recognition of HLA-G: Regulation by Contact Residues and by Cell Specific Expression at the Fetal-Maternal Interface

The non-classical HLA-G protein is distinguished from the classical MHC class I molecules by its expression pattern, low polymorphism and its ability to form complexes on the cell surface. The special role of HLA-G in the maternal-fetal interface has been attributed to its ability to interact with specific receptors found on maternal immune cells. However this interaction is restricted to a limited number of receptors. In this study we elucidate the reason for this phenomenon by comparing the specific contact residues responsible for MHC-KIR interactions. This alignment revealed a marked difference between the HLA-G molecule and other MHC class I molecules. By mutating these residues to the equivalent classical MHC residues, the HLA-G molecule regained an ability of interacting with KIR inhibitory receptors found on NK cells derived either from peripheral blood or from the decidua. Functional NK killing assays further substantiated the binding results. Furthermore, double immunofluorescent staining of placental sections revealed that while the conformed form of HLA-G was expressed in all extravillous trophoblasts, the free heavy chain form of HLA-G was expressed in more distal cells of the column, the invasion front. Overall we suggest that HLA-G protein evolved to interact with only some of the NK inhibitory receptors thus allowing a control of inhibition, while permitting appropriate NK cell cytokine and growth factor production necessary for a viable maternal fetal interface.     

Elastic network normal mode dynamics reveal the GPCR activation mechanism

G‐protein‐coupled receptors (GPCR) are a family of membrane‐embedded metabotropic receptors which translate extracellular ligand binding into an intracellular response. Here, we calculate the motion of several GPCR family members such as the M2 and M3 muscarinic acetylcholine receptors, the A_2A adenosine receptor, the β₂‐adrenergic receptor, and the CXCR4 chemokine receptor using elastic network normal modes. The normal modes reveal a dilation and a contraction of the GPCR vestibule associated with ligand passage, and activation, respectively. Contraction of the vestibule on the extracellular side is correlated with cavity formation of the G‐protein binding pocket on the intracellular side, which initiates intracellular signaling. Interestingly, the normal modes of rhodopsin do not correlate well with the motion of other GPCR family members. Electrostatic potential calculation of the GPCRs reveal a negatively charged field around the ligand binding site acting as a siphon to draw‐in positively charged ligands on the membrane surface. Altogether, these results expose the GPCR activation mechanism and show how conformational changes on the cell surface side of the receptor are allosterically translated into structural changes on the inside. Proteins 2014; 82:579–586. © 2013 Wiley Periodicals, Inc.