Super-resolution imaging as a method to study GPCR dimers and higher-order oligomers

The study of G protein-coupled receptor (GPCR) dimers and higher-order oligomers has unveiled mechanisms for receptors to diversify signaling and potentially uncover novel therapeutic targets. The functional and clinical significance of these receptor–receptor associations has been facilitated by the development of techniques and protocols, enabling researchers to unpick their function from the molecular interfaces, to demonstrating functional significance in vivo, in both health and disease. Here we describe our methodology to study GPCR oligomerization at the single-molecule level via super-resolution imaging. Specifically, we have employed photoactivated localization microscopy, with photoactivatable dyes (PD-PALM) to visualize the spatial organization of these complexes to <10 nm resolution, and the quantitation of GPCR monomer, dimer, and oligomer in both homomeric and heteromeric forms. We provide guidelines on optimal sample preparation, imaging parameters, and necessary controls for resolving and quantifying single-molecule data. Finally, we discuss advantages and limitations of this imaging technique and its potential future applications to the study of GPCR function

Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots

G-protein-coupled receptors mediate the biological effects of many hormones and neurotransmitters and are important pharmacological targets. They transmit their signals to the cell interior by interacting with G proteins. However, it is unclear how receptors and G proteins meet, interact and couple. Here we analyse the concerted motion of G-protein-coupled receptors and G proteins on the plasma membrane and provide a quantitative model that reveals the key factors that underlie the high spatiotemporal complexity of their interactions. Using two-colour, single-molecule imaging we visualize interactions between individual receptors and G proteins at the surface of living cells. Under basal conditions, receptors and G proteins form activity-dependent complexes that last for around one second. Agonists specifically regulate the kinetics of receptor-G protein interactions, mainly by increasing their association rate. We find hot spots on the plasma membrane, at least partially defined by the cytoskeleton and clathrin-coated pits, in which receptors and G proteins are confined and preferentially couple. Imaging with the nanobody Nb37 suggests that signalling by G-protein-coupled receptors occurs preferentially at these hot spots. These findings shed new light on the dynamic interactions that control G-protein-coupled receptor signalling

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

Multi-epitope vaccine against cystic echinococcosis using immunodominant epitopes from EgA31 and EgG1Y162 antigens

Cystic echinococcosis (CE) is a near-cosmopolitan public health concern, especially in developing countries. Parasite ova shed via definitive host feces are responsible for livestock and occasionally human infection, leading to hydatid cysts. Immunization using proper antigens is a good immunoprophylactic strategy. This study was aimed to design a multi-epitope vaccine using EgA31 and EgG1Y162 antigens. Top high-ranked B-cell epitopes and major histocompatibility complex (MHC)-binding epitopes were predicted and selected to construct the vaccine model. Then, physico-chemical features, secondary and tertiary structures, refinement and validations were all evaluated using web servers for the multi-epitope vaccine construct. Finally, non-linear B-cell epitopes were determined for vaccine-antibody interactions. Moreover, the vaccine construct was subjected to disulfide engineering, molecular docking with human MHC-I and MHC-II molecules as well as codon adaptation and in silico cloning processes. In conclusion, this multimeric CE vaccine needs experimental and clinical confirmations to be considered as an actually immunogenic vaccine model. © 202

In silico design of a novel peptide-based vaccine against the ubiquitous apicomplexan Toxoplasma gondii using surface antigens

Human toxoplasmosis is a global public health concern and a commercial vaccine is still lacking. The present in silico study was done to design a novel vaccine candidate using tachyzoite-specific SAG1-realted sequence (SRS) proteins. Overlapping B-cell and strictly-chosen human MHC-I binding epitopes were predicted and connected together using appropriate spacers. Moreover, a TLR4 agonist, human high mobility group box protein 1 (HMGB1), and His-tag were added to the N- and C-terminus of the vaccine sequence. The final vaccine had 442 residues and a molecular weight of 47.71 kDa. Physico-chemical evaluation showed a soluble, highly antigenic and non-allergen protein, with coils and helices as secondary structures. The vaccine 3D model was predicted by ITASSER server, subsequently refined and was shown to possess significant interactions with human TLR4. As well, potent stimulation of cellular and humoral immunity was demonstrated upon chimeric vaccine injection. Finally, the outputs showed that this vaccine model possesses top antigenicity, which could provoke significant cell-mediated immune profile including IFN-gamma, and can be utilized towards prophylactic purposes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-023-00140-w

Application of polysaccharide-based biopolymers as supports in photocatalytic treatment of water and wastewater: a review

Rising health issues of Worldwide pollution by fossil fuel products are Fostering the development of safer materials such as biopolymers in many sectors such as food, pharmaceutical, medical and environmental industries. Indeed, biopolymers are often safe, biodegradable, cheap and easy to modify. Here we review photocatalysts based on polysaccharides for wastewater treatment. Polysaccharides include starch, cellulose, carrageenan, alginate, chitin, chitosan and gum. The main reasons for using biodegradable biopolymers are their ability to adsorb pollutants and to be modified with nanoparticles and semiconductors. © 2022, The Author(s), under exclusive licence to Springer Nature Switzerland AG

Development of a chimeric vaccine candidate based on Toxoplasma gondii major surface antigen 1 and apicoplast proteins using comprehensive immunoinformatics approaches

This study was aimed at designing and evaluation of a multimeric vaccine construct against Toxoplasma gondii via utilization of SAG1 along with apicoplast ribosomal proteins (S2, S5 and L11). Top-ranked MHC-I and MHC-II binding as well as shared, immunodominant linear B-cell epitopes were predicted and joined together via appropriate linkers. Also, TLR-4 agonist (RS-09 synthetic protein) and His-tag were added to the N- and C-terminal of the vaccine sequence. The finally-engineered chimeric vaccine had a length of 291 amino acids with a molecular weight of 31.46 kDa. Physico-chemical features showed a soluble, highly-antigenic and non-allergenic candidate. Secondary and tertiary structures were predicted, and subsequent analyses confirmed the construct stability that was capable to properly interact with human TLR-4. Immunoinformatics-based simulation displayed potent stimulation of T- and B-cell mediated immune responses upon vaccination with the proposed multi-epitope candidate. In conclusion, obtained information demonstrated a highly antigenic vaccine candidate, which could develop high levels of IFN-gamma and other components of cellular immune profile, and can be directed for toxoplasmosis prophylactic purposes