No abstract

The electrochemistry of elemental sulfur (S(8)) and the polysulfides Na(2)S(4) and Na(2)S(6) has been studied for the first time in nonchloroaluminate ionic liquids. The cyclic voltammetry of S(8) in the ionic liquids is different to the behavior reported in some organic solvents, with two reductions and one oxidation peak observed. Supported by in situ UV-vis spectro-electrochemical experiments, the main reduction products of S(8) in [C(4)mim][DCA] ([C(4)mim] = 1-butyl-3-methylimidazolium; DCA = dicyanamide) have been identified as S(6)(2-) and S(4)(2-), and plausible pathways for the formation of these species are proposed. Dissociation and/or disproportionation of the polyanions S(6)(2-) and S(4)(2-) appears to be slow in the ionic liquid, with only small amounts of the blue radical species S(3)(•-) formed in the solutions at r.t., in contrast with that observed in most molecular solvents.

The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.

E-cadherin-mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness is conferred by cadherin extracellular domains, and is regulated by an assembly of adaptors and enzymes associated with the cytoplasmic tail. Here, we employed proximity biotinylation and quantitative proteomics to isolate and identify 612 proteins in the vicinity of E-cadherin's cytoplasmic tail. We used a structureinformed database of protein-protein interactions to construct the most comprehensive E-cadherin interactome to date, containing 89 known E-cadhesome components and 346 novel proteins. Moreover, through cloning and expression of GFP-tagged fusion proteins we localized 26 of the novel proteins to adherens junctions. Finally, employing calcium depletion and myosin inhibition we show the E-cadherin interactome to be remarkably robust to perturbation and essentially independent of cell-cell junctions or actomyosin contractility.

No abstract

The MA domain of retroviral Gag proteins mediates association with the host cell membrane during assembly. The biochemical nature of this interaction is not well understood. We have used an in vitro flotation assay to directly measure Rous sarcoma virus (RSV) MA-membrane interaction in the absence of host cell factors. The association of purified MA and MA-containing proteins with liposomes of defined composition was electrostatic in nature and depended upon the presence of a biologically relevant concentration of negatively charged lipids. A mutant MA protein known to be unable to promote Gag membrane association and budding in vivo failed to bind to liposomes. These results were supported by computational modeling. The intrinsic affinity of RSV MA for negatively charged membranes appears insufficient to promote efficient plasma membrane binding during assembly. However, an artificially dimerized form of MA bound to liposomes by at least an order of magnitude more tightly than monomeric MA. This result suggests that the clustering of MA domains, via Gag-Gag interactions during virus assembly, drives membrane association in vivo.

The matrix domain (MA) of Gag polyproteins performs multiple functions throughout the retroviral life cycle. MA structures have an electropositive surface patch that is implicated in membrane association. Here, we use computational methods to demonstrate that electrostatic control of membrane binding is a central characteristic of all retroviruses. We are able to explain a wide range of experimental observations and provide a level of quantitative and molecular detail that has been inaccessible to experiment. We further predict that MA may exist in a variety of oligomerization states and propose mechanistic models for the effects of phosphoinositides and phosphorylation. The calculations provide a conceptual model for how non-myristoylated and myristoylated MAs behave similarly in assembly and disassembly. Hence, they provide a unified quantitative picture of the structural and energetic origins of the entire range of MA function and thus enhance, extend, and integrate previous observations on individual stages of the process.

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.