Intracellular trafficking of the HCMV immunoevasin UL16 depends on elements present in both its cytoplasmic and transmembrane domains

Expression of the UL16 glycoprotein leads to down-regulation of NKG2D-ligands from the surface of the human cytomegalovirus (HCMV)-infected cell. The molecular elements responsible for UL16 trafficking and intracellular localization were investigated by preparing various chimeric proteins and mutants, using CD8 as a reporter molecule. A YQRL motif, present in UL16's cytoplasmic tail was functional for internalization, but the presence of the transmembrane domain modified the fate of the molecule after internalization. Various elements of the transmembrane domain that affected the trafficking of the protein were identified; however, their influence was modified in turn by the presence of the cytoplasmic tail of UL16. Strikingly, the extremely slow maturation rate of the native viral protein was only reproduced by the chimera that contained both transmembrane and cytoplasmic regions of UL16. These findings add data to a topic of increasing interest and importance: the role of the transmembrane domain of a protein in controlling its intracellular trafficking. In addition, they provide a new insight into the mechanism of action of the viral immunoevasin UL16.     

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.     

Discovery of facile amides-functionalized rhodanine-3-acetic acid derivatives as potential anticancer agents by disrupting microtubule dynamics

Microtubule dynamics are crucial for multiple cell functions, and cancer cells are particularly sensitive to microtubule-modulating agents. Here, we describe the design and synthesis of a series of (Z)-2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-N-phenylacetamide derivatives and evaluation of their microtubule-modulating and anticancer activities in vitro. Proliferation assays identified I₂₀ as the most potent of the antiproliferative compounds, with 50% inhibitory concentrations ranging from 7.0 to 20.3 µM with A549, PC-3, and HepG2 human cancer cell lines. Compound I₂₀ also disrupted cancer A549 cell migration in a concentration-dependent manner. Immunofluorescence microscopy, transmission electron microscopy, and tubulin polymerisation assays suggested that compound I₂₀ promoted protofilament assembly. In support of this possibility, computational docking studies revealed a strong interaction between compound I₂₀ and tubulin Arg β369, which is also the binding site for the anticancer drug Taxol. Our results suggest that (Z)-2-(5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-N-phenylacetamide derivatives could have utility for the development of microtubule-stabilising therapeutic agents.     

Genetic and Mapping Analysis of Arabidopsis thaliana Male Sterile Mutant filament no elongation

To identify new genes important for anther development, we screened for male sterile mutants among a population of Arabidopsis ecotype Columbia (Col) mutagenized by T DNA insertion (provided by ARBC). A male sterile mutant line with normal vegetative and flora development but no seed yield was isolated from Salk_118481 line. T DNA insertion site identification showed that there were no T DNA sequences in the genome of the mutants. Genetic analysis indicated that the mutant was controlled by a single recessive nuclear gene named filament no elongation because the filament of the mutant remains very short at the 13-14 stage of anther development. The fne gene was mapped to a region of 97kb between the molecular makers MBD2 and MMG4 on chromosome 5 using map based cloning technique. No genes involved filament elongation were reported in this region, so we believe that FNE gene could be a new gene controlling filament elongation in Arabidopsis.     

Do calmodulin binding IQ motifs have built‐in capping domains?

Most calmodulin (CaM) targets are α‐helices. It is not clear if CaM induces the adoption of an α‐helix configuration to its targets or if those targets are selected as they spontaneously adopt an α‐helical conformation. Other than an α‐helix propensity, there is a great variety of CaM targets with little more in common. One exception to this rule is the IQ site that can be recognized in a number of targets, such as those ion channels belonging to the KCNQ family. Although there is negligible sequence similarity between the IQ motif and the docking site on SK2 channels, both adopt a similar three‐dimensional disposition. The isolated SK2 target presents a pre‐folded core region that becomes fully α‐helical upon binding to CaM. The existence of this pre‐folded state suggests the occurrence of capping within CaM targets. In this review, we examine the capping properties within the residues flanking this core domain, and relate known IQ motifs and capping.     

Grass abundance maintains positive plant–arthropod diversity relationships in maize fields and margins in South Africa

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