Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs

Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs

Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells

CD11c-specific bio-nanocapsule enhances vaccine immunogenicity by targeting immune cells

Abstract Background Various nanocarriers have been used to deliver subunit vaccines specifically to dendritic cells (DCs) for the improvement of immunogenicity. However, due to their insufficient DC priming ability, these vaccines could not elicit effective innate immunity. We have recently developed a DC-targeting bio-nanocapsule (BNC) by displaying anti-CD11c IgGs via protein A-derived IgG Fc-binding Z domain on the hepatitis B virus envelope L protein particles (α-DC-ZZ-BNC). Results After the chemical modification with antigens (Ags), the α-DC-ZZ-BNC-Ag complex could deliver Ags to DCs efficiently, leading to effective DC maturation and efficient endosomal escape of Ags, followed by Ag-specific T cell responses and IgG productions. Moreover, the α-DC-ZZ-BNC modified with Japanese encephalitis virus (JEV) envelope-derived D3 Ags could confer protection against 50-fold lethal dose of JEV injection on mice. Conclusion The α-DC-ZZ-BNC-Ag platform was shown to induce humoral and cellular immunities effectively without any adjuvant

Influence of Nivolumab for Intercellular Adhesion Force between a T Cell and a Cancer Cell Evaluated by AFM Force Spectroscopy

The influence of nivolumab on intercellular adhesion forces between T cells and cancer cells was evaluated quantitatively using atomic force microscopy (AFM). Two model T cells, one expressing high levels of programmed cell death protein 1 (PD-1) (PD-1high Jurkat) and the other with low PD-1 expression levels (PD-1low Jurkat), were analyzed. In addition, two model cancer cells, one expressing programmed death-ligand 1 (PD-L1) on the cell surface (PC-9, PD-L1+) and the other without PD-L1 (MCF-7, PD-L1−), were also used. A T cell was attached to the apex of the AFM cantilever using a cup-attached AFM chip, and the intercellular adhesion forces were measured. Although PD-1high T cells adhered strongly to PD-L1+ cancer cells, the adhesion force was smaller than that with PD-L1− cancer cells. After the treatment of PD-1high T cells with nivolumab, the adhesion force with PD-L1+ cancer cells increased to a similar level as with PD-L1− cancer cells. These results can be explained by nivolumab influencing the upregulation of the adhesion ability of PD-1high T cells with PD-L1+ cancer cells. These results were obtained by measuring intercellular adhesion forces quantitatively, indicating the usefulness of single-cell AFM analysis

Release of siRNA from Liposomes Induced by Curcumin

Liposomes are a potential carrier of small interfering RNA (siRNA) for drug delivery systems (DDS). In this study, we searched for a molecule capable of controlling the release of siRNA from a certain type of liposomes and found that curcumin could induce the release of siRNA from the liposomes encapsulating siRNA within 30 min. However, the release of siRNA from the liposomes by curcumin showed a unique dose-response (i.e., bell-shaped curve) with a maximal induction at around 60 μg/ml of curcumin. Liposomal lipid compositions and temperatures influenced the efficiency in the release of siRNA induced by curcumin. About 10% of curcumin at a 60 μg/ml dose was incorporated into the liposomes within 30 min under our experimental conditions. Our results suggest a possibility that curcumin is useful in controlling the permeability of liposomes carrying large molecules like siRNA

Evaluation of the CWTrAP system using α-factor peptide for yeast endogenous Ste2 receptor.

<p>(A) Pheromone signaling assays of α-factor-displaying yeast strains. Error bars represent the standard deviation of three independent experiments. (B) Immunofluorescence staining of α-factor displaying yeast strains. Anti-Flag antibody and Alexa Fluor 546-conjugated secondary antibody were used for detection of secreted α-factor or α-factor-anchor fusion proteins. IMG-4 was used as the host strain. The transformants used in these experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037136#pone.0037136.s008" target="_blank">Table S3</a>. Sec: free, secreted form of α-factor. AG: C-terminal half of α-agglutinin anchor.</p

Confirmation of protein expression.

<p>Western blots of extracts from somatostatin displaying yeast strains. Lane 1: Mock/Mock, 2: SSTR5/Mock, 3: SSTR5/Flag–Flo42, 4: SSTR5/S-14–Flag–Flo42, 5: Mock/S-14–Flag–Flo42. Anti-β-actin antibody was used as loading control. Anti-HA antibody was used for detection of SSTR5 receptor. Anti-Flag antibody was used for detection of Flag–Flo42 anchor or S-14–Flag–Flo42 fusion proteins. IMFD-70 was used as the host strain. The transformants used in these experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037136#pone.0037136.s008" target="_blank">Table S3</a>.</p