A Genome-wide RNAi Screen Reveals a Trio-Regulated Rho GTPase Circuitry Transducing Mitogenic Signals Initiated by G Protein-Coupled Receptors

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Highlights? Human cancers harbor frequent mutations in Gq-linked GPCRs and Gα_q subunits ? A genome-wide RNAi screen revealed that Trio is essential for activating AP-1 via Gα_q ? A network of Trio-regulated Rho GTPases and MAPKs links Gq to the nucleus ? A hard-wired Gq-Trio signaling axis promotes the growth of many human malignancies     

Lenalidomide overcomes suppression of human natural killer cell anti-tumor functions by neuroblastoma microenvironment-associated IL-6 and TGFβ1

Background

Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression.

Methods

Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma.

Results

CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide.

Conclusion

Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1.     

Design,construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Designed ankyrin repeat proteins (DARPins) are well‐established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin‐target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase‐7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.     

Does a cdc2 Kinase-Like Recognition Motif on the Core Protein of Hepadnaviruses Regulate Assembly and Disintegration of Capsids?

Hepadnaviruses are enveloped viruses, each with a DNA genome packaged in an icosahedral nucleocapsid, which is the site of viral DNA synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the absence of these proteins, nucleocapsids deliver viral DNA into the cell nucleus. Presumably, this step is identical to the delivery of viral DNA during the initiation of an infection. Unfortunately, the mechanisms triggering the disintegration of subviral core particles and delivery of viral DNA into the nucleus are not yet understood. We now report the identification of a sequence motif resembling a serine- or threonine-proline kinase recognition site in the core protein at a location that is required for the assembly of core polypeptides into capsids. Using duck hepatitis B virus, we demonstrated that mutations at this sequence motif can have profound consequences for RNA packaging, DNA replication, and core protein stability. Furthermore, we found a mutant with a conditional phenotype that depended on the cell type used for virus replication. Our results support the hypothesis predicting that this motif plays a role in assembly and disassembly of viral capsids.     

Effect of alpha interferon on the hepatitis C virus replicon

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.     

The netrin receptor frazzled is required in the target for establishment of retinal projections in the Drosophila visual system

Retinal axons in Drosophila make precise topographic connections with their target cells in the optic lobe. Here we investigate the role of the Netrins and their receptor Frazzled in the establishment of retinal projections. We find that the Netrins, although expressed in the target, are not required for retinal projections. Surprisingly, Frazzled, found on both retinal fibers and target cells, is required in the target for attracting retinal fibers, while playing at best a redundant role in the retinal fibers themselves; this finding demonstrates that target attraction is necessary for topographic map formation. Finally, we show that Frazzled is not required for the differentiation of cells in the target. Our data suggest that Frazzled does not function as a Netrin receptor in attracting retinal fibers to the target; nor does it seem to act as a homotypic cell adhesion molecule. We favor the possibility that Frazzled in the target interacts with a component on the surface of retinal fibers, possibly another Netrin receptor.     

Generation of the amyloid-beta peptide N terminus in Saccharomyces cerevisiae expressing human Alzheimer's amyloid-beta precursor protein

The Alzheimer's amyloid-beta precursor protein (betaAPP) is a type 1 membrane-spanning protein from which the Alzheimer's disease amyloid-beta peptide (Abeta) is proteolytically derived. To date, attempts to identify the enzymes responsible for Abeta generation have failed. Here we report the accumulation of Abeta-immunoreactive peptides in yeast expressing human betaAPP. Characterization of these peptides by metabolic labeling, immunoprecipitation with Abeta-specific antibodies, and N-terminal radiosequencing indicates that these peptides include the Abeta peptide at their N termini. The Abeta-like peptides generated in yeast were recovered predominantly as 8- and 12-14-kDa species. A 4-kDa species was recovered either when a protease-deficient strain was used to prevent breakdown or when the 8- and 12-14-kDa species were treated with disaggregating agents. The likely existence in yeast of enzymes generating the Abeta N terminus indicates that the molecular identification of yeast beta-secretase-like enzymes may be accomplished using genetic screens or empirical approaches based upon the sequenced genome of Saccharomyces cerevisiae.     

Fibrinolysis-inhibitory capacity of clot-embedded surfactant is enhanced by SP-B and SP-C

Incorporation of pulmonary surfactant into fibrin inhibits its plasmic degradation. In the present study we investigated the influence of surfactant proteins (SP)-A, SP-B, and SP-C on the fibrinolysis-inhibitory capacity of surfactant phospholipids. Plasmin-induced fibrinolysis was quantified by means of a (125)I-fibrin plate assay, and surfactant incorporation into polymerizing fibrin was analyzed by measuring the incorporation of (3)H-labeled L-alpha-dipalmitoylphosphatidylcholine into the insoluble clot material. Incorporation of a calf lung surfactant extract (Alveofact) and an organic extract of natural rabbit large surfactant aggregates (LSA) into a fibrin clot revealed a stronger inhibitory effect on plasmic cleavage of this clot than a synthetic phospholipid mixture (PLX) and unprocessed LSA. Reconstitution of PLX with SP-B and SP-C increased, whereas reconstitution with SP-A decreased, the fibrinolysis-inhibitory capacity of the phospholipids. The SP-B effect was paralleled by an increased incorporation of phospholipids into fibrin. We conclude that the inhibitory effect of surfactant incorporation into polymerizing fibrin on its susceptibility to plasmic cleavage is enhanced by SP-B and SP-C but reduced by SP-A. In the case of SP-B, increased phospholipid incorporation may underlie this finding.     

Short-time infusion of fish oil-based lipid emulsions, approved for parenteral nutrition, reduces monocyte proinflammatory cytokine generation and adhesive interaction with endothelium in humans

Potential impact of omega-3 fatty acids, as contained in fish oil, on immunological function has been suggested because observations of reduced inflammatory diseases in Greenland Inuit were published. A fish oil-based lipid emulsion has recently been approved for parenteral nutrition in many countries. We investigated the influence of a short infusion course of fish oil-based (omega-3) vs conventional (omega-6) lipid emulsion on monocyte function. In a randomized design, twelve healthy volunteers received omega-3 or omega-6 lipid infusion for 48 h, with cross-over repetition of the infusion course after 3 mo. Fatty acid profiles, monocyte cytokine release and adhesive monocyte-endothelium interaction were investigated. Resultant omega-6 lipid emulsion increased plasma-free fatty acids including arachidonic acid, whereas the omega-3/omega-6 fatty acid ratio in monocyte membranes remained largely unchanged. It also caused a tendency toward enhanced monocyte proinflammatory cytokine release and adhesive monocyte-endothelium interaction. In contrast, omega-3 lipid emulsion significantly increased the omega-3/omega-6 fatty acid ratio in the plasma-free fatty acid fraction and in monocyte membrane lipid pool, markedly suppressing monocyte generation of TNF-alpha, IL-1, IL-6, and IL-8 in response to endotoxin. In addition, it also significantly inhibited both monocyte-endothelium adhesion and transendothelial monocyte migration, although monocyte surface expression of relevant adhesive molecules (CD11b, CD18, CD49 days, CCR2) was unchanged. Although isocaloric, omega-3 and omega-6 lipid emulsions exert differential impact on immunological processes in humans. In addition to its nutritional value, fish oil-based omega-3 lipid emulsion significantly suppresses monocyte proinflammatory cytokine generation and features of monocyte recruitment.