The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation.

The developmental signalling protein Notch can be proteolytically activated following ligand-interaction at the cell surface, or can be activated independently of its ligands, following Deltex (Dx)-induced Notch endocytosis and trafficking to the lysosomal membrane. The means by which different pools of Notch are directed towards these alternative outcomes remains poorly understood. We found that the Drosophila ZO-1 protein Polychaetoid (Pyd) suppresses specifically the Dx-induced form of Notch activation both in vivo and in cell culture assays. In vivo we confirmed the physiological relevance and direction of the Pyd/Dx interaction by showing that the expanded ovary stem cell niche phenotypes of pyd mutants require the presence of functional Dx and other components that are specific to the Dx-induced Notch activation mechanism. In S2 cells we found that Pyd can form a complex with Dx and Notch at the cell surface and reduce Dx-induced Notch endocytosis. Similar to other known activities of ZO-1 family proteins, the action of Pyd on Dx-induced endocytosis and signalling was found to be cell density dependent. Thus, together, our results suggest an alternative means by which external cues can tune Notch signalling through Pyd regulation of Dx-induced Notch trafficking.

Development of a screening platform for directed evolution using the reef coral fluorescent protein ZsGreen as a solubility reporter.

Soluble proteins, with high expression levels, are preferred candidates for structural and functional studies. In cases of low expression, aggregation or inclusion body formation, time-consuming searches for optimal expression or refolding conditions are required. We have developed a high-throughput solubility engineering and screening platform for proteins that are expressed in an insoluble form in Escherichia coli with the aim of obtaining a broad spectrum of best hits with increased solubility in difficult to express target proteins. This process has been developed using error-prone PCR to introduce random base changes in genes of interest. Expression of mutated proteins in fusion with the reef coral fluorescent protein ZsGreen as a solubility marker has enabled the selection of more soluble variants. We have used a colony picker to achieve high-throughput selection of E.coli expressing more soluble target protein-ZsGreen fusions, with increased fluorescence. The whole process enables us to complete one round of mutation, screening and analysis of 20,000 potential soluble clones within approximately 8 weeks. We describe the development of the methods using different model proteins and show one example, the kinase domain from the human EphB2 receptor, as a successful application of the whole platform.

Regulation of the nuclear localization of the human Nedd4-related WWP1 protein by Notch.

Nedd4 family ubiquitin ligases regulate trafficking and degradation of numerous target substrates in different cellular compartments, including at the plasma membrane, in endosomes, in the secretory pathway and in the nucleus. WWP1 is a Nedd4 family protein closely related to mouse Itch and Drosophila Su(dx), both of which have been shown to regulate the Notch receptor. To investigate the possibility that WWP1 is also associated with Notch signalling we coexpressed human Notch1 and WWP1 in mouse myoblast cells. We found that WWP1 could localize to both the nucleus and cytoplasm in a context dependent manner. Coexpression of human Notch1 (hN1) depleted WWP1 from the nucleus to colocalise with hN1 in early endosomes, dependent on the presence of the C-terminal HECT domain. Furthermore we found that full-length expressed WWP1 could interact in vitro with the cytoplasmic domain of human Notch1. The Notch receptor has multiple roles in development, mediating a short-range signal that controls cell fate and pattern formation. The canonical Notch signal involves proteolytic release of the soluble Notch intracellular domain and the activation by the latter of the transcription factor Suppressor of Hairless/CBF-1 in the nucleus. This pathway does not however account for all of the activity of Notch. The ability of Notch to regulate the nuclear localization of WWP1 suggests a possible alternative mechanism by which Notch may communicate a signal to the nucleus. Drosophila Notch similarly regulated the nuclear localization of the Drosophila Nedd4 family protein, Suppressor of deltex, implying conservation of this mechanism during evolution.

Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein.

Human immunodeficiency virus type 1 Nef protein is N-terminally myristoylated, a modification reported to be required for the association of Nef with cytoplasmic membranes. As myristate alone is not sufficient to anchor a protein stably into a membrane, it has been suggested that N-terminal basic residues contribute to Nef membrane association via electrostatic interactions with acidic phospholipids. Here, data are presented pertaining to the role of the myristate and basic residues in Nef membrane association, subcellular localization and function. Firstly, by using a biochemical assay for membrane association it was shown that, whereas myristoylation of Nef was not essential, mutation of a cluster of four arginines between residues 17 and 22 reduced membrane association dramatically. Mutation of two lysines at residues 4 and 7 had negligible effect alone, but when combined with the arginine substitutions, abrogated membrane association completely. By using indirect immunofluorescence, it was demonstrated that mutation of either of the two basic clusters altered the subcellular distribution of Nef dramatically. Thirdly, the requirement of the arginine and lysine clusters for Nef-mediated CD4 down modulation was shown to correlate precisely with membrane association. These data suggest that membrane localization and subcellular targeting of Nef are controlled by a complex interplay of signals at the N terminus of the protein.

Co-translational myristoylation alters the quaternary structure of HIV-1 Nef in solution.

We have studied the solution properties of Nef, a 24-kDa cotranslationally myristoylated protein produced by HIV-1 and other primate lentiviruses. Nef is found in the cytosol and also in association with cytoplasmic membranes, the latter, mediated in part by the myristoyl group attached to the N-terminal glycine. Recombinant Nef was coexpressed in Escherichia coli in tandem with N-myristoyl-transferase and is fully myristoylated. Analysis by circular dichroism showed the myristoylated form to contain a greater alpha-helical content than the nonmyristoylated form. Analysis of modified and unmodified Nef in solution using small angle X-ray scattering, dynamic laser light scattering and analytical ultracentrifugation consistently showed differences in the oligomeric states of the two forms of Nef. Myristoylated Nef is predominantly monomeric and small oligomers which are also present, can be converted to the monomeric form under reducing conditions. By contrast, the nonmyristoylated form exists as a stable hexadecamer in solution which disassociates into tetramers upon addition of reducing agents. Shape reconstructions from small angle scattering curves of nonmyristoylated Nef are compatible with a large disc-like structure in the hexadecameric oligomer consisting of four Nef tetramers. From these findings, we hypothesize that Nef undergoes a substantial conformational change from an "open" into a "closed" form whereby the myristate group is sequestered in a hydrophobic pocket. The myristoylated protein can switch to the open conformation by association of the N-terminal region of molecule with membranes. These changes would allow Nef to carry out various functions depending on the conformational and oligomeric states.

Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling.

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.

Regulation of notch endosomal sorting and signaling by Drosophila Nedd4 family proteins.

The Notch receptor mediates a short-range signal that regulates many cell fate decisions. The misregulation of Notch has been linked to cancer and to developmental disorders. Upon binding to its ligands, Delta (Dl) or Serrate (Ser), the Notch ectodomain is shed by the action of an ADAM protease. The Notch intracellular domain is subsequently released proteolytically from the membrane by Presenilin and translocates to the nucleus to activate the transcription factor, Suppressor of Hairless. We show in Drosophila that Notch signaling is limited by the activity of two Nedd4 family HECT domain proteins, Suppressor of deltex [Su(dx)] and DNedd4. We rule out models by which Su(dx) downregulates Notch through modulating Deltex or by limiting the adherens junction accumulation of Notch. Instead, we show that Su(dx) regulates the postendocytic sorting of Notch within the early endosome to an Hrs- and ubiquitin-enriched subdomain en route to the late endosome. We propose a model in which endocytic sorting of Notch mediates a decision between its activation and downregulation. Such intersections between trafficking routes may provide key points at which other signals can modulate Notch activity in both normal development and in the pathological misactivation of Notch.

The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation.

The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.

Down-regulation of Notch target gene expression by Suppressor of deltex.

In Drosophila, Suppressor of deltex (Su(dx)) mutations display a wing vein gap phenotype resembling that of Notch gain of function alleles. The Su(dx) protein may therefore act as a negative regulator of Notch but its activity on actual Notch signalling levels has not been demonstrated. Here we show that Su(dx) does regulate the level of Notch signalling in vivo, upstream of Notch target genes and in different developmental contexts, including a previously unknown role in leg joint formation. Overexpression of Su(dx) was capable of blocking both the endogenous activity of Notch and the ectopic Notch signalling induced by the overexpression of Deltex, an intracellular Notch binding protein. In addition, using the conditional phenotype of the Su(dx)(sp) allele, we show that loss of Su(dx) activity is rapidly followed by an up-regulation of E(spl)mbeta expression, the immediate target of Notch signal activation during wing vein development. While Su(dx) adult wing vein phenotypes are quite mild, only affecting the distal tips of the veins, we show that the initial consequence of loss of Su(dx) activity is more severe than previously thought. Using a time-course experiment we show that the phenotype is buffered by feedback regulation illustrating how signalling networks can make development robust to perturbation.