RNA tailing machinery drives amyloidogenic phase transition.

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.

Adsorption of Tannic Acid onto Gold Surfaces.

Thin film coatings are widely applicable in materials for consumer products, electronics, optical coatings, and even biomedical applications. Wet coating can be an effective method to obtain thin films of functional materials, and this technique has recently been studied in depth for the formation of bioinspired polyphenolic films. Naturally occurring polyphenols such as tannic acid (TA) have garnered interest due to their roles in biological processes and their applicability as antioxidants, antibacterial agents, and corrosion inhibitors. Understanding the adsorption of polyphenols to surfaces is a core aspect in the fabrication processes of thin films of these materials. In this work, the adsorption of TA to gold surfaces is measured using a quartz crystal microbalance with dissipation monitoring (QCMD) and surface plasmon resonance (SPR) for a wide range of TA concentrations. The adsorption kinetics, aggregation, and stability of TA solutions in physiological-like conditions are studied. Unexpectedly, it is found that the adsorption rates depend only weakly on concentration because of the presence of TA aggregates that do not adsorb. The mechanism of layer formation is also investigated, finding that TA monolayers readily adsorb onto gold with flat or edge-on molecular orientations dependent on the solution concentration. A mix of orientations in the intermediate case leads to slow multilayer adsorption.

Semiconductive and Biocompatible Nanofibrils from the Self-Assembly of Amyloid π-Conjugated Peptides.

Owing to their capacity to self-assemble into organized nanostructures, amyloid polypeptides can serve as scaffolds for the design of biocompatible semiconductive materials. Herein, symmetric and asymmetric amyloid π-conjugated peptides were prepared through condensation of perylene diimide (PDI) with a natural amyloidogenic sequence derived from the islet amyloid polypeptide. These PDI-bioconjugates assembled into long and linear nanofilaments in aqueous solution, which were characterized by a cross-ß-sheet quaternary organization. Current-voltage curves exhibited a clear signature of semiconductors, whereas the cellular assays revealed cytocompatibility and potential application in fluorescence microscopy. Although the incorporation of a single amyloid peptide appeared sufficient to drive the self-assembly into organized fibrils, the incorporation of two peptide sequences at the PDI's imide positions significantly enhanced the conductivity of nanofibril-based films. Overall, this study exposes a novel strategy based on amyloidogenic peptide to guide the self-assembly of π-conjugated systems into robust, biocompatible, and optoelectronic nanofilaments.

Bionanocomposites with Enhanced Physical Properties from Curli Amyloid Assemblies and Cellulose Nanofibrils.

Proteinaceous amyloid fibrils are one of the stiffest biopolymers due to their extensive cross-ß-sheet quaternary structure, whereas cellulose nanofibrils (CNFs) exhibit interesting properties associated with their nanoscale size, morphology, large surface area, and biodegradability. Herein, CNFs were supplemented with amyloid fibrils assembled from the Curli-specific gene A (CsgA) protein, the main component of bacterial biofilms. The resulting composites showed superior mechanical properties, up to a 7-fold increase compared to unmodified CNF films. Wettability and thermogravimetric analyses demonstrated high surface hydrophobicity and robust thermal tolerance. Bulk spectroscopic characterization of CNF-CsgA films revealed key insights into the molecular organization within the bionanocomposites. Atomic force microscopy and photoinduced force microscopy revealed the high-resolution location of curli assemblies into the CNF films. This novel sustainable and cost-effective CNF-based bionanocomposites supplemented with intertwined bacterial amyloid fibrils opens novel directions for environmentally friendly applications demanding high mechanical, water-repelling properties, and thermal resistance.

Compartmentalized processing of catechols during mussel byssus fabrication determines the destiny of DOPA.

Inspired largely by the role of the posttranslationally modified amino acid dopa (DOPA) in mussel adhesion, catechol functional groups have become commonplace in medical adhesives, tissue scaffolds, and advanced smart polymers. Yet, the complex redox chemistry of catechol groups complicates cross-link regulation, hampering fabrication and the long-term stability/performance of mussel-inspired polymers. Here, we investigated the various fates of DOPA residues in proteins comprising mussel byssus fibers before, during, and after protein secretion. Utilizing a combination of histological staining and confocal Raman spectroscopy on native tissues, as well as peptide-based cross-linking studies, we have identified at least two distinct DOPA-based cross-linking pathways during byssus fabrication, achieved by oxidative covalent cross-linking or formation of metal coordination interactions under reducing conditions, respectively. We suggest that these end states are spatiotemporally regulated by the microenvironments in which the proteins are stored prior to secretion, which are retained after formation-in particular, due to the presence of reducing moieties. These findings provide physicochemical pathways toward greater control over properties of synthetic catechol-based polymers and adhesives.

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes.

Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ 31P and 2H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by 31P and 2H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.

Cell surface glycosaminoglycans exacerbate plasma membrane perturbation induced by the islet amyloid polypeptide.

Glycosaminoglycans (GAGs) are long and unbranched anionic heteropolysaccharides that have been associated with virtually all amyloid deposits. Soluble sulfated GAGs are known for their propensity to promote the self-assembly of numerous amyloidogenic proteins and to modulate their cytotoxicity. Nonetheless, although GAGs are prevalent on the outer leaflet of eukaryotic cell plasma membrane as part of proteoglycans, their contributions in the perturbation of lipid bilayer induced by amyloid polypeptides remain unknown. Herein, we investigate the roles of GAGs in the cytotoxicity and plasma membrane perturbation induced by the islet amyloid polypeptide (IAPP), whose deposition in the pancreatic islets is associated with type II diabetes. Cellular assays using GAG-deficient cells reveal that GAGs exacerbate IAPP-induced cytotoxicity and permeabilization of the plasma membrane. Confocal microscopy and flow cytometry analyses show that IAPP sequestration at the cell surface is dependent of GAGs and of the aggregation propensity of the peptide. Using giant plasma membrane vesicles (GPMVs) prepared from GAG-deficient cells, we investigate the direct contributions of membrane-embedded proteoglycans in IAPP-induced membrane disassembly. In sharp contrast to soluble sulfated GAGs, kinetics of amyloid self-assembly expose that the presence of GAGs on GPMVs does not significantly modulate in vitro amyloid formation. Overall, this study indicates that cell surface GAGs increase the local concentration of IAPP in the vicinity of the plasma membrane, promoting lipid bilayer perturbation and cell death.

Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity.

The overall impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on our society is unprecedented. The identification of small natural ligands that could prevent the entry and/or replication of the coronavirus remains a pertinent approach to fight the coronavirus disease (COVID-19) pandemic. Previously, we showed that the phenolic compounds corilagin and 1,3,6-tri-O-galloyl-ß-D-glucose (TGG) inhibit the interaction between the SARS-CoV-2 spike protein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 target receptor on the cell membrane of the host organism. Building on these promising results, we now assess the effects of these phenolic ligands on two other crucial targets involved in SARS-CoV-2 cell entry and replication, respectively: transmembrane protease serine 2 (TMPRSS2) and 3-chymotrypsin like protease (3CLpro) inhibitors. Since corilagin, TGG, and tannic acid (TA) share many physicochemical and structural properties, we investigate the binding of TA to these targets. In this work, a combination of experimental methods (biochemical inhibition assays, surface plasmon resonance, and quartz crystal microbalance with dissipation monitoring) confirms the potential role of TA in the prevention of SARS-CoV-2 infectivity through the inhibition of extracellular RBD/ACE2 interactions and TMPRSS2 and 3CLpro activity. Moreover, molecular docking prediction followed by dynamic simulation and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) free energy calculation also shows that TA binds to RBD, TMPRSS2, and 3CLpro with higher affinities than TGG and corilagin. Overall, these results suggest that naturally occurring TA is a promising candidate to prevent and inhibit the infectivity of SARS-CoV-2.

Site-Specific Alkylation of the Islet Amyloid Polypeptide Accelerates Self-Assembly and Potentiates Perturbation of Lipid Membranes.

The accumulation of insoluble amyloids in the pancreatic islets is a pathological hallmark of type II diabetes and correlates closely with the loss of ß-cell mass. The predominant component of these amyloid deposits is the islet amyloid polypeptide (IAPP). The factors contributing to the conversion of IAPP from a monomeric bioactive peptide hormone into insoluble amyloid fibrils remain partially elusive. In this study, we investigated the effect of the oxidative non-enzymatic post-translational modification induced by the reactive metabolite 4-hydroxynonenal (HNE) on IAPP aggregation and cytotoxicity. Incubation of IAPP with exogenous HNE accelerated its self-assembly into ß-sheet fibrils and led to the formation of a Michael adduct on the His-18 side chain. To model this covalent modification, the imidazole N(π) position of histidine was alkylated using a close analogue of HNE, the octyl chain. IAPP lipidated at His-18 showed a hastened random coil-to-ß-sheet conformational conversion into fibrillar assemblies with a distinct morphology, a low level of binding to thioflavin T, and a high surface hydrophobicity. Introducing an octyl chain on His-18 enhanced the ability of the peptide to perturb synthetic lipid vesicles, to permeabilize the plasma membrane, and to induce the death of pancreatic ß-cells. Alkylated IAPP triggered the self-assembly of unmodified IAPP by prompting primary nucleation and increased its capacity to perturb the plasma membrane, indicating that only a small proportion of the modified peptide is necessary to shift the balance toward the formation of proteotoxic species. This study underlines the importance of studying IAPP post-translational modifications induced by oxidative metabolites in the context of pancreatic amyloids.

Corilagin and 1,3,6-Tri-O-galloy-ß-D-glucose: potential inhibitors of SARS-CoV-2 variants.

The COVID-19 disease caused by the virus SARS-CoV-2, first detected in December 2019, is still emerging through virus mutations. Although almost under control in some countries due to effective vaccines that are mitigating the worldwide pandemic, the urgency to develop additional vaccines and therapeutic treatments is imperative. In this work, the natural polyphenols corilagin and 1,3,6-tri-O-galloy-ß-d-glucose (TGG) are investigated to determine the structural basis of inhibitor interactions as potential candidates to inhibit SARS-CoV-2 viral entry into target cells. First, the therapeutic potential of the ligands are assessed on the ACE2/wild-type RBD. We first use molecular docking followed by molecular dynamics, to take into account the conformational flexibility that plays a significant role in ligand binding and that cannot be captured using only docking, and then analyze more precisely the affinity of these ligands using MMPBSA binding free energy. We show that both ligands bind to the ACE2/wild-type RBD interface with good affinities which might prevent the ACE2/RBD association. Second, we confirm the potency of these ligands to block the ACE2/RBD association using a combination of surface plasmon resonance and biochemical inhibition assays. These experiments confirm that TGG and, to a lesser extent, corilagin, inhibit the binding of RBD to ACE2. Both experiments and simulations show that the ligands interact preferentially with RBD, while weak binding is observed with ACE2, hence, avoiding potential physiological side-effects induced by the inhibition of ACE2. In addition to the wild-type RBD, we also study numerically three RBD mutations (E484K, N501Y and E484K/N501Y) found in the main SARS-CoV-2 variants of concerns. We find that corilagin could be as effective for RBD/E484K but less effective for the RBD/N501Y and RBD/E484K-N501Y mutants, while TGG strongly binds at relevant locations to all three mutants, demonstrating the significant interest of these molecules as potential inhibitors for variants of SARS-CoV-2.

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.

Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†.

Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62.

The existence of the antisense transcript-encoded HIV-1 antisense protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blotting, flow cytometry, and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Here, our goals were to assess autophagy induction by ASP from different clades and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino-terminal region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASPs from different clades (A, B, C, D, and G) were tested next and were detected in monomeric and multimeric forms at various levels, and all induced autophagy (clade A ASP was less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knockout of ATG5, ATG7, and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP colocalized with p62 and LC3-II in autophagosome-like structures. Coimmunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported the idea that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function.IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramifications in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated neurological disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies hence demonstrate the importance in conducting further studies on this protein as it could represent a new interesting target for antiretroviral therapies and vaccine design.

Glycosaminoglycans Induce Amyloid Self-Assembly of a Peptide Hormone by Concerted Secondary and Quaternary Conformational Transitions.

Amyloids are polypeptide supramolecular assemblies that have been historically associated with numerous pathologies. Nonetheless, recent studies have identified many amyloid structures that accomplish vital physiological functions. Interestingly, amyloid fibrils, either pathological or functional, have been reported to be consistently associated with other biomolecules such as RNA and glycosaminoglycans (GAGs). These linear polyanions, RNA and GAGs, have also demonstrated an inherent ability to accelerate and/or promote amyloid formation. GAGs, including heparan sulfate, are highly charged polysaccharides that may have essential roles in the storage of peptide hormones in the form of amyloids. In this study, we evaluated the ability of sulfated GAGs to promote the self-assembly of the peptide (neuro)hormone PACAP27 and investigated the secondary and quaternary conformational transitions associated with the amyloidogenic process. PACAP27 readily self-assembled into insoluble, α-helix-rich globular particulates in the presence of sulfated GAGs, which gradually condensed and disappeared as nontoxic ß-sheet-rich amyloid fibrils were formed. By designing a PACAP27 derivative for which helical folding was hindered, we observed that the α-helix-to-ß-sheet conformational transition within the amorphous particulates constitutes the rate-limiting step of primary nucleation events. The proposed mechanism of GAG-induced self-assembly within insoluble particulates appears to be fundamentally different from usual amyloidogenic systems, which commonly implicates the formation of soluble prefibrillar proteospecies. Overall, this study provides new insights into the mechanistic details involved in the formation of functional amyloids catalyzed by polyanions, such as the assembly of nuclear amyloid bodies and the storage of peptide hormones.

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.

Kinetic and Conformational Insights into Islet Amyloid Polypeptide Self-Assembly Using a Biarsenical Fluorogenic Probe.

Amyloid fibril formation and tissue deposition are associated with many diseases. Studies have shown that prefibrillar intermediates, such as oligomers, are the most toxic proteospecies of the amyloidogenic cascade. Thus, understanding the mechanisms of formation and the conformational ensemble of prefibrillar species is critical. Due to their transient and heterogeneous nature, detection and characterization of prefibrillar species remain challenging. The fluorogenic probe fluorescein arsenical hairpin (FlAsH), which recognizes a tetracysteine motif, has been recently used to detect the oligomerization of amyloidogenic peptides encompassing a Cys-Cys tag. In this study, we extended the FlAsH detection method to gain novel kinetic and conformational insights into the self-assembly of islet amyloid polypeptide (IAPP), a 37-residue peptide hormone whose deposition is associated with type II diabetes. By positional scanning of the Cys-Cys motif, the stability of the noncontiguous tetracysteine FlAsH-binding sites formed during self-assembly was evaluated and revealed rapid monomer self-recognition through the convergence of C-terminal domains. On the other hand, the N-terminal domains come close to each other only upon the formation of the cross-ß-sheet amyloid structure. We demonstrated that this method is well-suited to detect thioflavin T-negative fibrils and to screen inhibitors of amyloid formation. This study highlights that with positional scanning of the split-tetracysteine motif (Cys-Cys), the FlAsH detection method offers unique time-dependent conformational insights on the proteospecies assembled throughout the amyloidogenic pathway.

Identification of a conformational heparin-recognition motif on the peptide hormone secretin: key role for cell surface binding.

Secretin is a peptide hormone that exerts pleiotropic physiological functions by specifically binding to its cognate membrane-bound receptor. The membrane catalysis model of peptide-receptor interactions states that soluble peptidic ligands initially interact with the plasma membrane. This interaction increases the local concentration and structures the peptide, enhancing the rate of receptor binding. However, this model does not consider the dense network of glycosaminoglycans (GAGs) at the surface of eukaryotic cells. These sulfated polysaccharide chains are known to sequester numerous proteic signaling molecules. In the present study, we evaluated the interaction between the peptide hormone secretin and sulfated GAGs and its contribution to cell surface binding. Using GAG-deficient cells and competition experiments with soluble GAGs, we observed by confocal microscopy and flow cytometry that GAGs mediate the sequestration of secretin at the cell surface. Isothermal titration calorimetry and surface plasmon resonance revealed that secretin binds to heparin with dissociation constants ranging between 0.9 and 4 µM. By designing secretin derivatives with a restricted conformational ensemble, we observed that this interaction is mediated by the presence of a specific conformational GAG-recognition motif that decorates the surface of the peptide upon helical folding. The present study identifies secretin as a novel GAG-binding polypeptide and opens new research direction on the functional role of GAGs in the biology of secretin.

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.

Probing the role of λ6 immunoglobulin light chain dimerization in amyloid formation.

Light chain amyloidosis (AL) is a lethal disease associated with the deposition of misfolded immunoglobulin light chains (LC) as amyloid fibrils in the extracellular space of vital organs. The exact mechanisms of LC self-assembly and the molecular basis leading to cellular and organ failure still remain poorly understood. In this study, we investigated the relationship between the quaternary structure, the stability and the amyloidogenecity of LC variable domain (VL) from the λ6 germline. We observed that the amyloidogenic λ6 Wil and its non-amyloidogenic counterpart Jto dimerize in a concentration-dependent manner and that the dimer affinity is considerably decreased in the presence of a high ionic strength. Our results showed that the dimeric state delays the structural conversion associated with amyloid formation and that the monomer is critical to initiate amyloidogenesis. Thermal and chemical unfolding studies revealed that the dimeric state of VL λ6 has an equivalent stability to the monomer. This indicates that the protective effect of dimerization is not related to thermodynamic stability but, most likely, resides in specific structural features. The toxicity of monomeric Jto and Wil as well as fibrillar aggregates was evaluated on cardiomyoblasts and ThT-negative proteospecies reduced cellular viability when employed at high concentration. This study provides novel insights into the complex process of LC amyloidogenesis and suggests that dimer stabilization constitutes a promising strategy to prevent self-assembly and amyloid deposition.

Modulation of amyloid assembly by glycosaminoglycans: from mechanism to biological significance.

Glycosaminoglycans (GAGs) are long and unbranched polysaccharides that are abundant in the extracellular matrix and basement membrane of multicellular organisms. These linear polyanionic macromolecules are involved in many physiological functions from cell adhesion to cellular signaling. Interestingly, amyloid fibrils extracted from patients afflicted with protein misfolding diseases are virtually always associated with GAGs. Amyloid fibrils are highly organized nanostructures that have been historically associated with pathological states, such as Alzheimer's disease and systemic amyloidoses. However, recent studies have identified functional amyloids that accomplish crucial physiological roles in almost all living organisms, from bacteria to insects and mammals. Over the last 2 decades, numerous reports have revealed that sulfated GAGs accelerate and (or) promote the self-assembly of a large diversity of proteins, both inherently amyloidogenic and non-aggregation prone. Despite the fact that many studies have investigated the molecular mechanism(s) by which GAGs induce amyloid assembly, the mechanistic elucidation of GAG-mediated amyloidogenesis still remains the subject of active research. In this review, we expose the contribution of GAGs in amyloid assembly, and we discuss the pathophysiological and functional significance of GAG-mediated fibrillization. Finally, we propose mechanistic models of the unique and potent ability of sulfated GAGs to hasten amyloid fibril formation.