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Current poultry vaccines against influenza A viruses target the globular head region of the hemagglutinin (HA1), providing limited protection against antigenically divergent strains. Experimental subunit vaccines based on the conserved ectodomain of the matrix protein 2 (M2e) induce cross-reactive antibody responses, but fail to fully prevent virus shedding after low pathogenic avian influenza (LPAI) virus challenge, and are ineffective against highly pathogenic avian influenza (HPAI) viruses. This study assessed the benefits of combining nanoparticles bearing three tandem M2e repeats (NR-3M2e nanorings or NF-3M2e nanofilaments) with an HA1 subunit vaccine in protecting chickens against a heterologous HPAI H5N1 virus challenge. Chickens vaccinated with the combined formulations developed M2e and HA1-specific antibodies, were fully protected from clinical disease and mortality, and showed no histopathological lesions or virus shedding, unlike those given only HA1, NR-3M2e, or NF-3M2e. Thus, the combined vaccine formulations provided complete cross-protection against HPAI H5N1 virus, and prevented environmental virus shedding, crucial for controlling avian influenza outbreaks. [ABSTRACT FROM AUTHOR]

The RNA tailing machinery adds nucleotides to the 3'-end of RNA molecules that are implicated in various biochemical functions, including protein synthesis and RNA stability. Here, we report a role for the RNA tailing machinery as enzymatic modifiers of intracellular amyloidogenesis. A targeted RNA interference screen identified Terminal Nucleotidyl-transferase 4b (TENT4b/Papd5) as an essential participant in the amyloidogenic phase transition of nucleoli into solid-like Amyloid bodies. Full-length-and-mRNA sequencing uncovered starRNA, a class of unusually long untemplated RNA molecules synthesized by TENT4b. StarRNA consists of short rRNA fragments linked to long, linear mixed tails that operate as polyanionic stimulators of amyloidogenesis in cells and in vitro. Ribosomal intergenic spacer noncoding RNA (rIGSRNA) recruit TENT4b in intranucleolar foci to coordinate starRNA synthesis driving their amyloidogenic phase transition. The exoribonuclease RNA Exosome degrades starRNA and functions as a general suppressor of cellular amyloidogenesis. We propose that amyloidogenic phase transition is under tight enzymatic control by the RNA tailing and exosome axis. [ABSTRACT FROM AUTHOR]

The deposition of insoluble proteinaceous aggregates in the form of amyloid fibrils within the extracellular space of tissues is associated with numerous diseases. The development of molecular approaches to arrest amyloid formation and prevent cellular degeneration remains very challenging due to the complexity of the process of protein aggregation, which encompasses an infinite array of conformations and quaternary structures. Polyanionic biopolymers, such as glycosaminoglycans and RNAs, have been shown to modulate the self‐assembly of amyloidogenic polypeptides and to reduce the toxicity induced by the formation of oligomeric and/or pre‐fibrillar proteospecies. This study evaluates the effects of double‐stranded DNA (dsDNA) nanostructures (1D, 2D, and 3D) on amyloid self‐assembly, fibril disaggregation, and the cytotoxicity associated with amyloidogenesis. Using the islet amyloid polypeptide (IAPP) whose pancreatic accumulation is the hallmark of type 2 diabetes, it was observed that dsDNA nanostructures inhibit amyloid formation by inducing the formation of spherical complexes in which the peptide adopts a random coil conformation. Interestingly, the DNA nanostructures showed a persistent ability to disassemble enzymatically and thermodynamically stable amyloid fibrils into nanoscale DNA/IAPP entities that are fully compatible with β‐pancreatic cells and are biodegradable by proteolysis. Notably, dsDNA nanostructures avidly trapped highly toxic soluble oligomeric species in complete cell culture media and converted them into non‐toxic binary complexes. Overall, these results expose the potent modulatory effects of dsDNA on amyloidogenic pathways, and these DNA nanoscaffolds could be used as a source of inspiration for the design of molecules to fight amyloid‐related disorders. [ABSTRACT FROM AUTHOR]

Amyloid fibrils are proteinaceous nanostructures known for their ability to activate the innate immune system, which has been recently exploited for their use as self-adjuvanted antigen delivery systems for vaccines. Among mechanisms of immunostimulation, the activation of the heterodimeric toll-like receptor 2/6 (TLR2/TLR6) by the cross-β-sheet quaternary conformation appears important. Nonetheless, the lack of control over the process of self-assembly and the polydispersity of the resulting supramolecular architectures make it challenging to elucidate the molecular basis of TLR2/TLR6 engagement by amyloid assemblies. In this context, we harnessed the effects of N- and C-terminal modifications of a short 10-mer β-peptide derived from the islet amyloid polypeptide (I 10 ) to investigate the relationships between the morphology and physicochemical properties of amyloid assemblies and their TLR2/TLR6 activity. Chemical substitutions at the N- and C-termini of the I 10 peptide, including addition of charged residues at the N-terminus and α-amidation of C-terminus, allowed the controlled formation of a diversity of architectures, including belt-like filaments, rigid nanorods as well as flat and twisted fibrils. These fully cytocompatible peptide nanostructures showed different potencies to activate TLR2/TLR6, which correlated with the charge exposed on the surface. These results further demonstrate the potent modulatory effect of N- and C-terminal electrostatic capping on the self-assembly of short synthetic β-peptides. This study also indicates that self-assembly into cross-β-sheet nanostructures is essential for the activation of the TLR2/TLR6 by amyloidogenic peptides, albeit the structural requirements of the engagement of this promiscuous immune receptor by the nanostructures remain challenging to precisely untangle.

The islet amyloid polypeptide (IAPP), also known as amylin, is a hormone playing key physiological roles. However, its aggregation and deposition in the pancreatic islets are associated with type 2 diabetes. While this peptide adopts mainly a random coil structure in solution, its secondary conformational conversion into α-helix represents a critical step for receptor activation and contributes to amyloid formation and associated cytotoxicity. Considering the large conformational landscape and high amyloidogenicity of the peptide, as well as the complexity of the self-assembly process, it is challenging to delineate the delicate interplay between helical folding, peptide aggregation, and receptor activation. In the present study, we probed the roles of helical folding on the function-toxicity duality of IAPP by restricting its conformational ensemble through side chain-to-side chain stapling via azide-alkyne cycloaddition. Intramolecular macrocyclization ( i ; i + 4) constrained IAPP into α-helix and inhibited its aggregation into amyloid fibrils. These helical derivatives slowed down the self-assembly of unmodified IAPP. Site-specific macrocyclization modulated the capacity of IAPP to perturb lipid bilayers and cell plasma membrane and reduced, or even fully inhibited, the cytotoxicity associated with aggregation. Furthermore, the α-helical IAPP analogs showed moderate to high potency toward cognate G protein-coupled receptors. Overall, these results indicate that macrocyclization represents a promising strategy to protect an amyloidogenic peptide hormone from aggregation and associated toxicity, while maintaining high receptor activity.

In this work, we synthesized an hexasaccharide derived from the capsular polysaccharide of group B Streptococcus type III capsular polysaccharide. Our convergent 3 + 3 strategy avoided the use of benzyl protecting groups allowing the installation of an azide anchoring group and providing a high yield for the final deprotection steps. Moreover, the minimal hexasaccharidic epitope was conjugated to CRM 197 and BSA via copper-catalyzed azide-alkyne cycloaddition for the preparation of a semisynthetic carbohydrate-based vaccine.