Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Lipid core peptide/poly(lactic-co-glycolic acid) as a highly potent intranasal vaccine delivery system against Group A streptococcus

Rheumatic heart disease represents a leading cause of mortality caused by Group A Streptococcus (GAS) infections transmitted through the respiratory route. Although GAS infections can be treated with antibiotics these are often inadequate. An efficacious GAS vaccine holds more promise, with intranasal vaccination especially attractive, as it mimics the natural route of infections and should be able to induce mucosal IgA and systemic IgG immunity. Nanoparticles were prepared by either encapsulating or coating lipopeptide-based vaccine candidate (LCP-1) on the surface of poly(lactic-co-glycolic acid) (PLGA). In vitro study showed that encapsulation of LCP-1 vaccine into nanoparticles improved uptake and maturations of antigen-presenting cells. The immunogenicity of lipopeptide incorporated PLGA-based nanoparticles was compared with peptides co-administered with mucosal adjuvant cholera toxin B in mice upon intranasal administration. Higher levels of J14-specific salivary mucosal IgA and systemic antibody IgG titres were observed for groups immunized with encapsulated LCP-1 compared to LCP-1 coated nanoparticles or free LCP-1. Systemic antibodies obtained from LCP-1 encapsulated PLGA NPs inhibited the growth of bacteria in six different GAS strains. Our results show that PLGA-based lipopeptide delivery is a promising approach for rational design of a simple, effective and patient friendly intranasal GAS vaccine resulting in mucosal IgA response

Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct

Background & objectives: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved C-region. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. Methods: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. Results: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. Interpretation & conclusion: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases

Donkey milk fermentation by lactococcus lactis subsp. Cremoris and lactobacillus rhamnosus affects the antiviral and antibacterial milk properties

Background: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. Methods: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. Results: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. Conclusions: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health

ASSEMBLY AND DISPLAY OF SURFACE PROTEINS IN ACTINOMYCES ORIS

Bacteria are an integral part of human health and disease. In the human host, dental plaques form as a result of up to 700 individual bacterial species colonizing oral surfaces and forming a multispecies biofilm. These biofilms are the cause of prevalent human diseases such as dental caries, gingivitis, and periodontitis. The microbes present in the oral biofilm are highly spatially and temporally structured and require a primary colonizing species to adhere to host tissue. As an important primary colonizer of the oral biofilm, the actinobacterium Actinomyces oris utilizes cell wall anchored proteins and glycoconjugates to initiate adherence to host surfaces, recruit additional bacterial species that could not bind otherwise, and maintain the structural integrity of the oral biofilm. In this thesis, I reveal mechanisms involved in the assembly and display of surface proteins that are central to these processes in A. oris. Cell wall-anchored proteins contain a signal peptide to direct their secretion to the exoplasmic side of the membrane, where they are liberated from the secretion machine by signal peptidases. Cell wall-anchored proteins also contain a cell wall sorting signal, which is required for their covalent attachment to peptidoglycan by transpeptidase enzymes called sortases. Furthermore, a subset of cell wall-anchored proteins are polymerized to form pili prior to being anchored. I found that pilin proteins require a distinct signal peptidase for their maturation and function and uncovered residues required for adherence in a minor pilin protein. In certain cases after translocation, proteins are modified by the addition of glycopolymers, and I characterized a phosphotransferase enzyme with a novel role in protein glycosylation. These studies contribute to the understanding of the role of A. oris as a primary colonizer in the oral biofilm. Additionally, using A. oris as a model for general processes has led to findings which are applicable to principles of biofilm formation, interspecies interactions, glycoconjugate formation, and bacterial pathogenesis

Raman Spectroscopy of Synthetic Antimicrobial Frog Peptides Magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus lueuis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 Å upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides

Transmembrane Complexes of DAP12 Crystallized in Lipid Membranes Provide Insights into Control of Oligomerization in Immunoreceptor Assembly

The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces