The Fusion Activity of HIV-1 gp41 Depends on Interhelical Interactions

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function

Group A Streptococcal S Protein Utilizes Red Blood Cells as Immune Camouflage and Is a Critical Determinant for Immune Evasion.

Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system

The ubiK protein is an accessory factor necessary for bacterial Ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.Agence Nationale de la Recherche ANR-15-CE11-0001-02Centre National de la Recherche Scientifique PICS07279French State Program "Investissements d'Avenir" ANR-11-LABX-001

In vitro toxicity of nanoceria: effect of coating and stability in biofluids

Due to the increasing use of nanometric cerium oxide in applications, concerns about the toxicity of these particles have been raised and have resulted in a large number of investigations. We report here on the interactions between 7 nm anionically charged cerium oxide particles and living mammalian cells. By a modification of the particle coating including low-molecular weight ligands and polymers, two generic behaviors are compared: particles coated with citrate ions that precipitate in biofluids and particles coated with poly(acrylic acid) that are stable and remain nanometric. We find that nanoceria covered with both coating agents are taken up by mouse fibroblasts and localized into membrane-bound compartments. However, flow cytometry and electron microscopy reveal that as a result of their precipitation, citrate-coated particles interact more strongly with cells. At cerium concentration above 1 mM, only citrate-coated nanoceria (and not particles coated with poly(acrylic acid)) display toxicity and moderate genotoxicity. The results demonstrate that the control of the surface chemistry of the particles and its ability to prevent aggregation can affect the toxicity of nanomaterials.Comment: 33 pages 10 figures, accepted at Nanotoxicolog

A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration

Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK) and Src, and that this signalling pathway lies downstream of fascin-microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and may have implications for the design of therapies to target fascin in metastatic disease

Suspension cell culture in microgravity and development of a space bioreactor

NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first space bioreactor has been designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small (500 ml) bioreactor is being constructed for flight experiments in the Shuttle middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption, and control of low shear stress on cells

HFE and transferrin directly compete for transferrin receptor in solution and at the cell surface

Transferrin receptor (TfR) is a dimeric cell surface protein that binds both the serum iron transport protein transferrin (Fe-Tf) and HFE, the protein mutated in patients with the iron overload disorder hereditary hemochromatosis. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This apparent affinity reduction could result from direct competition between HFE and Fe-Tf for their overlapping binding sites on each TfR polypeptide chain, from negative cooperativity, or from a combination of both. To explore the mechanism of the affinity reduction, we constructed a heterodimeric TfR that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Binding studies using a heterodimeric form of soluble TfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of the effects of HFE on iron homeostasis result from competition with Fe-Tf for TfR binding. Experiments using transfected cell lines demonstrate a physiological role for this competition in altering HFE trafficking patterns

Interactions between sub-10 nm iron and cerium oxide nanoparticles and 3T3 fibroblasts : the role of the coating and aggregation state

Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of the cerium and iron oxide sub-10 nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol-1). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease of the mitochondrial activity for cerium concentrations above 5 mM (equivalent to 0.8 g L-1). We also observe that the citrate-coated particles are internalized by the cells in large amounts, typically 250 pg per cell after a 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (< 30 pg per cell). The strong uptake shown by the citrate-coated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.Comment: 9 figures, 2 table