Self-Assembly of Flagellin into Immunostimulatory Ring-like Nanostructures as an Antigen Delivery System.

Proteinaceous nanoparticles represent attractive antigen carriers for vaccination as their size and repetitive antigen displays that mimic most viral particles enable efficient immune processing. However, these nanocarriers are often unable to stimulate efficiently the innate immune system, requiring coadministration with adjuvants to promote long-lasting protective immunity. The protein flagellin, which constitutes the primary constituent of the bacterial flagellum, has been widely evaluated as an antigen carrier due to its intrinsic adjuvant properties involving activation of the innate immune receptor Toll-like receptor 5 (TLR5). Although flagellin is known for its ability to self-assemble into micron-scale length nanotubes, few studies have evaluated the potential usage of flagellin-based nanostructures as immunostimulatory antigen carriers. In this study, we reported for the first time a strategy to guide the self-assembly of a flagellin protein from Bacillus subtilis, Hag, into lower aspect ratio nanoparticles by hindering non-covalent interactions responsible for its elongation into nanotubes. We observed that addition of an antigenic sequence derived from the influenza A virus (3M2e) at the C-terminus of this flagellin, as opposed to positioning the epitope into mid-sequence, precluded filament elongation and resulted in low aspect ratio ring-like nanostructures upon salting-out-induced self-assembly. These nanostructures displayed the antigen at their surface and shared morphological and structural characteristics with flagellin nanotubes, with a diameter of approximately 12 nm, and an α-helix-rich secondary structure. Flagellin ring-like nanostructures were efficiently internalized by antigen-presenting cells, and avidly activated the TLR5 in vitro as well as the innate and adaptive immune responses. Intranasal immunization of mice with these nanostructures resulted in the potentiation of the antigen-specific antibody response and protection against a lethal infection with the influenza A virus, illustrating the potential of these intrinsically immunostimulatory nanostructures as antigen carriers.

Recombinant Bacillus subtilis flagellin Hag is a potent immunostimulant with reduced proinflammatory properties compared to Salmonella enterica serovar Typhimurium FljB.

Flagellin constitutes a potential adjuvant for vaccines owing to its robust immunostimulatory properties. However, clinical trials have revealed that flagellin derived from Salmonella enterica serovar Typhimurium induces high levels of proinflammatory markers and substantial adverse effects. The flagellin from Bacillus subtilis, Hag, shares high sequence homology with Salmonella FljB within the D0 and D1 domains responsible for TLR5 engagement, while the D2 and D3 domains associated with an off-target immune response are absent. Accordingly, we compared the immunostimulatory and proinflammatory properties of Hag with FljB by harnessing an epitope from the matrix 2 protein (M2e) of the influenza virus. Both flagellins engaged TLR5, with FljB showing a 2.5-fold higher potency than Hag. Mice inoculation showed a robust FljB- or Hag-induced M2e-specific antibody response, with Hag demonstrating a decreased secretion of proinflammatory markers and reduced weight loss. This study revealed that flagellin Hag is a potent immunoadjuvant with reduced proinflammatory properties.

Identification of transmissible proteotoxic oligomer-like fibrils that expand conformational diversity of amyloid assemblies.

Protein misfolding and amyloid deposition are associated with numerous diseases. The detailed characterization of the proteospecies mediating cell death remains elusive owing to the (supra)structural polymorphism and transient nature of the assemblies populating the amyloid pathway. Here we describe the identification of toxic amyloid fibrils with oligomer-like characteristics, which were assembled from an islet amyloid polypeptide (IAPP) derivative containing an Asn-to-Gln substitution (N21Q). While N21Q filaments share structural properties with cytocompatible fibrils, including the 4.7 Å inter-strand distance and ß-sheet-rich conformation, they concurrently display characteristics of oligomers, such as low thioflavin-T binding, high surface hydrophobicity and recognition by the A11 antibody, leading to high potency to disrupt membranes and cause cellular dysfunction. The toxic oligomer-like conformation of N21Q fibrils, which is preserved upon elongation, is transmissible to naïve IAPP. These stable fibrils expanding the conformational diversity of amyloid assemblies represent an opportunity to elucidate the structural basis of amyloid disorders.

Self-assembled peptide nanorod vaccine confers protection against influenza A virus.

Proteinaceous nanostructures have emerged as a promising strategy to develop safe and efficient subunit vaccines. The ability of synthetic ß-sheet self-assembling peptides to stabilize antigenic determinants and to potentiate the epitope-specific immune responses have highlighted their potential as an immunostimulating platform for antigen delivery. Nonetheless, the intrinsic polymorphism of the resulting cross-ß fibrils, their length in the microscale and their close structural similarity with pathological amyloids could limit their usage in vaccinology. In this study, we harnessed electrostatic capping motifs to control the self-assembly of a chimeric peptide comprising a 10-mer ß-sheet sequence and a highly conserved epitope derived from the influenza A virus (M2e). Self-assembly led to the formation of 100-200 nm long uniform nanorods (NRs) displaying the M2e epitope on their surface. These cross-ß assemblies differed from prototypical amyloid fibrils owing to low polydispersity, short length, non-binding to thioflavin T and Congo Red dyes, and incapacity to seed homologous amyloid assembly. M2e-NRs were efficiently uptaken by antigen presenting cells and the cross-ß quaternary architecture activated the Toll-like receptor 2 and stimulated dendritic cells. Mice subcutaneous immunization revealed a robust M2e-specific IgG response, which was dependent on self-assembly into NRs. Upon intranasal immunization in combination with the polymeric adjuvant montanide gel, M2e-NRs conferred complete protection with absence of clinical signs against a lethal experimental infection with the H1N1 influenza A virus. These findings indicate that by acting as an immunostimulator and delivery system, synthetic peptide-based NRs constitute a versatile self-adjuvanted nanoplatform for the delivery of subunit vaccines.

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-ß Fibrils to Design Adjuvanted Nanovaccines.

Protein fibrils characterized with a cross-ß-sheet quaternary structure have gained interest as nanomaterials in biomedicine, including in the design of subunit vaccines. Recent studies have shown that by conjugating an antigenic determinant to a self-assembling ß-peptide, the resulting supramolecular assemblies act as an antigen delivery system that potentiates the epitope-specific immune response. In this study, we used a ten-mer self-assembling sequence (I10) derived from an amyloidogenic peptide to biophysically and immunologically characterize a nanofibril-based vaccine against the influenza virus. The highly conserved epitope from the ectodomain of the matrix protein 2 (M2e) was elongated at the N-terminus of I10 by solid phase peptide synthesis. The chimeric M2e-I10 peptide readily self-assembled into unbranched, long, and twisted fibrils with a diameter between five and eight nm. These cross-ß nanoassemblies were cytocompatible and activated the heterodimeric Toll-like receptor (TLR) 2/6. Upon mice subcutaneous immunization, M2e-fibrils triggered a robust anti-M2e specific immune response, which was dependent on self-assembly and did not require the use of an adjuvant. Overall, this study describes the efficacy of cross-ß fibrils to activate the TLR 2/6 and to stimulate the epitope-specific immune response, supporting usage of these proteinaceous assemblies as a self-adjuvanted delivery system for antigens.

Protein Supramolecular Structures: From Self-Assembly to Nanovaccine Design.

Life-inspired protein supramolecular assemblies have recently attracted considerable attention for the development of next-generation vaccines to fight against infectious diseases, as well as autoimmune diseases and cancer. Protein self-assembly enables atomic scale precision over the final architecture, with a remarkable diversity of structures and functionalities. Self-assembling protein nanovaccines are associated with numerous advantages, including biocompatibility, stability, molecular specificity and multivalency. Owing to their nanoscale size, proteinaceous nature, symmetrical organization and repetitive antigen display, protein assemblies closely mimic most invading pathogens, serving as danger signals for the immune system. Elucidating how the structural and physicochemical properties of the assemblies modulate the potency and the polarization of the immune responses is critical for bottom-up design of vaccines. In this context, this review briefly covers the fundamentals of supramolecular interactions involved in protein self-assembly and presents the strategies to design and functionalize these assemblies. Examples of advanced nanovaccines are presented, and properties of protein supramolecular structures enabling modulation of the immune responses are discussed. Combining the understanding of the self-assembly process at the molecular level with knowledge regarding the activation of the innate and adaptive immune responses will support the design of safe and effective nanovaccines.

Guiding the Morphology of Amyloid Assemblies by Electrostatic Capping: from Polymorphic Twisted Fibrils to Uniform Nanorods.

Peptides that self-assemble into cross-ß-sheet amyloid structures constitute promising building blocks to construct highly ordered proteinaceous materials and nanoparticles. Nevertheless, the intrinsic polymorphism of amyloids and the difficulty of controlling self-assembly currently limit their usage. In this study, the effect of electrostatic interactions on the supramolecular organization of peptide assemblies is investigated to gain insights into the structural basis of the morphological diversities of amyloids. Different charged capping units are introduced at the N-terminus of a potent ß-sheet-forming sequence derived from the 20-29 segment of islet amyloid polypeptide, known to self-assemble into polymorphic fibrils. By tuning the charge and the electrostatic strength, different mesoscopic morphologies are obtained, including nanorods, rope-like fibrils, and twisted ribbons. Particularly, the addition of positive capping units leads to the formation of uniform rod-like assemblies, with lengths that can be modulated by the charge number. It is proposed that electrostatic repulsions between N-terminal positive charges hinder ß-sheet tape twisting, leading to a unique control over the size of these cytocompatible nanorods by protofilament growth frustration. This study reveals the high susceptibility of amyloid formation to subtle chemical modifications and opens to promising strategies to control the final architecture of proteinaceous assemblies from the peptide sequence.

Identification of a hinge residue controlling islet amyloid polypeptide self-assembly and cytotoxicity.

The islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone whose deposition as amyloid fibrils in the pancreatic islets is associated with type 2 diabetes. Previous studies have suggested that residue Asn-21 plays a critical role in the in vitro self-assembly of IAPP. Herein, we studied structure-self-assembly relationships focusing on position 21 to gain detailed insights into the molecular mechanisms of IAPP self-assembly and to probe the conformational nature of the toxic assemblies associated with ß-cell death. Thioflavin T (ThT) fluorescence, CD spectroscopy, and transmission EM analysis revealed that the Asn-21 amide side chain is not required for IAPP nucleation and amyloid elongation, as N21A and N21G variants assembled into prototypical fibrils. In contrast, Asn-21 substitution with the conformationally constrained and turn-inducing residue Pro accelerated IAPP self-assembly. Successive substitutions with hydrophobic residues led to the formation of ThT-negative ß-sheet-rich aggregates having high surface hydrophobicity. Cell-based assays revealed no direct correlation between the in vitro amyloidogenicity of these variants and their toxicity. In contrast, leakage of anionic lipid vesicles disclosed that membrane disruption is closely associated with cytotoxicity. We observed that the N21F variant self-assembles into worm-like aggregates, causing loss of lipid membrane structural integrity and inducing ß-cell apoptosis. These results indicate that specific intra- and intermolecular interactions involving Asn-21 promote IAPP primary nucleation events by modulating the conformational conversion of the oligomeric intermediates into amyloid fibrils. Our study identifies position 21 as a hinge residue that modulates IAPP amyloidogenicity and cytotoxicity.

Engineering and evaluation of amyloid assemblies as a nanovaccine against the Chikungunya virus.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform. Chimeric peptides, comprising the peptide antigen attached to the amyloid core by a short flexible linker, were prepared by solid phase synthesis. As observed using atomic force microscopy, these polypeptides self-assembled into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. A quaternary conformation rich in cross-ß-sheets characterized these assemblies, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. ELISA assays and transmission electronic microscopy of immunogold labeled-fibrils revealed a high density of the Chikungunya virus E2 glycoprotein derived epitope exposed on the fibril surface. These amyloid fibrils were cytocompatible and were efficiently uptaken by macrophages. Mice immunization revealed a robust IgG response against the E2EP3 epitope, which was dependent on self-assembly and did not require co-injection of the Alhydrogel adjuvant. These results indicate that cross-ß-sheet amyloid assemblies constitute suitable synthetic self-adjuvanted assemblies to anchor antigenic determinants and to increase the immunogenicity of peptide epitopes.

Characterization of a Novel Alkalophilic Lipase From Aneurinibacillus thermoaerophilus: Lid Heterogeneity and Assignment to Family I.5.

Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase α/ß hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 °C under neutral pH, but its maximal activity was detected at pH 10 and 45 °C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 °C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure-function relationships in lipases.

Role of Site-Specific Asparagine Deamidation in Islet Amyloid Polypeptide Amyloidogenesis: Key Contributions of Residues 14 and 21.

Deamidation of an asparagine residue is a spontaneous non-enzymatic post-translational modification that results in the conversion of asparagine into a mixture of aspartic acid and isoaspartic acid. This chemical conversion modulates protein conformation and physicochemical properties, which could lead to protein misfolding and aggregation. In this study, we investigated the effects of site-specific Asn deamidation on the amyloidogenicity of the aggregation-prone peptide islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptidic hormone whose deposition as insoluble amyloid fibrils is closely associated with type 2 diabetes. Asn residues were successively substituted with an Asp or isoAsp, and amyloid formation was evaluated by a thioflavin T fluorescence assay, circular dichroism spectroscopy, atomic force microscopy, and transmission electron microscopy. Whereas deamidation at position 21 inhibited IAPP conformational conversion and amyloid formation, the N14D mutation accelerated self-assembly and led to the formation of long and thick amyloid fibrils. In contrast, IAPP was somewhat tolerant to the successive deamidation of Asn residues 22, 31, and 35. Interestingly, a small molar ratio of IAPP deamidated at position 14 promoted the formation of nucleating species and the elongation from unmodified IAPP. Besides, using the rat pancreatic ß cell line INS-1E, we observed that site-specific deamidation did not significantly alter IAPP-induced toxicity. These data indicate that Asn deamidation can modulate IAPP amyloid formation and fibril morphology and that the site of modification plays a critical role. Above all, this study reinforces the notion that IAPP amyloidogenesis is governed by precise intermolecular interactions involving specific Asn side chains.

Effects of oxidative post-translational modifications on structural stability and self-assembly of λ6 immunoglobulin light chain.

Light chain amyloidosis (AL) originates from the deposition of immunoglobulin light chains (LCs) as amyloid fibrils in the extracellular space of vital organs. Although non-enzymatic post-translational modifications (PTMs) have been shown to contribute to protein misfolding diseases, little is known about their contributions to LC amyloidogenicity. In this study, we investigated the effects of three oxidative PTMs, carbonylation by hydroxynonenal (HNE), oxidation and nitration, on the structure, thermodynamic stability and self-assembly propensity of a LC variable domain from the λ6 germline, Wil. We initially identified the specific residues that are susceptible to oxidative chemical modifications. HNE-conjugation at specific His residues and nitration of Tyr side chains modulated the conformational conversion driving Wil self-assembly and fibrillar aggregates formation. This study reinforces the notion that not only the thermodynamic stability, but also the chemical and structural properties, should be considered when evaluating the amyloidogenic potential of a LC.

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility.

Resveratrol protects DAergic PC12 cells from high glucose-induced oxidative stress and apoptosis: effect on p53 and GRP75 localization.

Resveratrol (RESV), a polyphenolic natural compound, has long been acknowledged to have cardioprotective and antiinflammatory actions. Evidence suggests that RESV has antioxidant properties that reduce the formation of reactive oxygen species leading to oxidative stress and apoptotic death of dopaminergic (DAergic) neurons in Parkinson's disease (PD). Recent literature has recognized hyperglycemia as a cause of oxidative stress reported to be harmful for the nervous system. In this context, our study aimed (a) to evaluate the effect of RESV against high glucose (HG)-induced oxidative stress in DAergic neurons, (b) to study the antiapoptotic properties of RESV in HG condition, and c) to analyze RESV's ability to modulate p53 and GRP75, a p53 inactivator found to be under expressed in postmortem PD brains. Our results suggest that RESV protects DAergic neurons against HG-induced oxidative stress by diminishing cellular levels of superoxide anion. Moreover, RESV significantly reduces HG-induced apoptosis in DAergic cells by modulating DNA fragmentation and the expression of several genes implicated in the apoptotic cascade, such as Bax, Bcl-2, cleaved caspase-3, and cleaved PARP-1. RESV also prevents the pro-apoptotic increase of p53 in the nucleus induced by HG. Such data strengthens the correlation between hyperglycemia and neurodegeneration, while providing new insight on the high occurrence of PD in patients with diabetes. This study enlightens potent neuroprotective roles for RESV that should be considered as a nutritional recommendation for preventive and/or complementary therapies in controlling neurodegenerative complications in diabetes.