Amyloid State Research Articles

Amyloid fibers deriving from the aromatic core of C-terminal domain of nucleophosmin 1

Nucleophosmin (NPM1) is the most frequently mutated gene in Acute Myeloid Leukemia (AML) patients and mutations lead to its aberrant cytoplasmatic accumulation in leukemic cells. Its C-terminal domain (CTD) is endowed with a globular structure consisting of a three-helix bundle in the wild type form that is disrupted by AML mutations. Our recent results demonstrate, unexpectedly and unequivocally, that regions of the CTD of NPM1 are prone to aggregate to amyloid states. Here we present novel studies, at solution as well as fibrillar states of a nonapeptide covering the 264–272 region of NPM1: this small fragment is the most amyloidogenic stretch of the entire protein and its conformational and aggregation properties were investigated through Circular Dichroism, Fluorescence spectroscopies, amyloid seeding assay (ASA), isothermal titration calorimetry (ITC) and electrospray ionization (ESI) mass analyses. Structural features of fibrils were investigated by means of Scanning Electron Microscopy (SEM) and Wide-Angle X-ray Scattering (WAXS).This study deepens the amyloid fibrillization process of a short stretch of the CTD likely involved in the propagative mechanism for NPMc+ cytoplasmatic accumulation in leukemogenesis.

Formation of Heterotypic Amyloids: α-Synuclein in Co-Aggregation.

Protein misfolding resulting in the formation of ordered amyloid aggregates is associated with a number of devastating human diseases. Intrinsically disordered proteins (IDPs) do not autonomously fold up into a unique stable conformation and remain as an ensemble of rapidly fluctuating conformers. Many IDPs are prone to convert into the β-rich amyloid state. One such amyloidogenic IDP is α-synuclein that is involved in Parkinson's disease. Recent studies have indicated that other neuronal proteins, especially IDPs, can co-aggregate with α-synuclein in many pathological ailments. This article describes several such observations highlighting the role of heterotypic protein-protein interactions in the formation of hetero-amyloids. It is believed that the characterizations of molecular cross talks between amyloidogenic proteins as well as the mechanistic studies of heterotypic protein aggregation will allow us to decipher the role of the interacting proteins in amyloid proteomics.

Exposure of Aggregation-Prone Segments is the Requirement for Amyloid Fibril Formation.

Arranging into well-organized fibrillar aggregate, commonly known as amyloid fibril is an inherent property of any polypeptide chain. Amyloid fibrils are associated with a number of severe human pathologies like the Alzheimer's disease, Parkinson's disease, type2 diabetes and many more. Recent studies suggest that most of the fibrils are inert and extremely stable, thus could be used for the bionanotechnological applications. As the native state is protected by evolution from aggregation under physiological condition, understanding the structure of aggregation precursor state (APS) will be of extreme importance to decode mechanism of its formation and prevention. This review article includes the recent studies of identification and characterization of possible conformations of proteins which can act as APS. The literature regarding the research in this field revealed that any conformation ranging from native-like state to completely unfolded state could be an APS. The structural characteristics of the APS depend on the protein and on its surrounding environment. From this review of literatures, we conclude that exposure of aggregation-prone segments is the requirement for amyloid fibril formation and the amyloid state seems to be the most stable known physical state of the proteins. This means all conformations of proteins with exposed aggregation-prone segments can promote intermolecular interactions and channel to amyloid fibril pathway to acquire their minimum energy state.

Discovering Putative Prion-Like Proteins in Plasmodium falciparum: A Computational and Experimental Analysis.

Prions are a singular subset of proteins able to switch between a soluble conformation and a self-perpetuating amyloid state. Traditionally associated with neurodegenerative diseases, increasing evidence indicates that organisms exploit prion-like mechanisms for beneficial purposes. The ability to transit between conformations is encoded in the so-called prion domains, long disordered regions usually enriched in glutamine/asparagine residues. Interestingly, Plasmodium falciparum, the parasite that causes the most virulent form of malaria, is exceptionally rich in proteins bearing long Q/N-rich sequence stretches, accounting for roughly 30% of the proteome. This biased composition suggests that these protein regions might correspond to prion-like domains (PrLDs) and potentially form amyloid assemblies. To investigate this possibility, we performed a stringent computational survey for Q/N-rich PrLDs on P. falciparum. Our data indicate that ∼10% of P. falciparum protein sequences have prionic signatures, and that this subproteome is enriched in regulatory proteins, such as transcription factors and RNA-binding proteins. Furthermore, we experimentally demonstrate for several of the identified PrLDs that, despite their disordered nature, they contain inner short sequences able to spontaneously self-assemble into amyloid-like structures. Although the ability of these sequences to nucleate the conformational conversion of the respective full-length proteins should still be demonstrated, our analysis suggests that, as previously described for other organisms, prion-like proteins might also play a functional role in P. falciparum.

Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape

Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on- or off-pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon↛crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape.

Minimalist Prion-Inspired Polar Self-Assembling Peptides.

Nature provides copious examples of self-assembling supramolecular nanofibers. Among them, amyloid structures have found amazing applications as advanced materials in fields such as biomedicine and nanotechnology. Prions are a singular subset of proteins able to switch between a soluble conformation and an amyloid state. The ability to transit between these two conformations is encoded in the so-called prion domains (PrDs), which are long and disordered regions of low complexity, enriched in polar and uncharged amino acids such as Gln, Asn, Tyr, Ser, and Gly. The polar nature of PrDs results in slow amyloid formation, which allows kinetic control of fiber assembly. This approach has been exploited for fabrication of multifunctional materials because in contrast to most amyloids, PrDs lack hydrophobic stretches that can nucleate their aggregation, their assembly depends on the establishment of a large number of weak interactions along the complete domain. The length and low complexity of PrDs make their chemical synthesis for applied purposed hardly affordable. Here, we designed four minimalist polar binary patterned peptides inspired in PrDs, which include the [Q/N/G/S]-Y-[Q/N/G/S] motif frequently observed in these domains: NYNYNYN, QYQYQYQ, SYSYSYS, and GYGYGYG. Despite their small size, they all recapitulate the properties of full-length PrDs, self-assembling into nontoxic amyloids under physiological conditions. Thus, they constitute small building blocks for the construction of tailored prion-inspired nanostructures. We exploited Tyr residues in these peptides to generate highly stable dityrosine cross-linked assemblies for the immobilization of metal nanoparticles in the fibrils surface and to develop an electrocatalytic amyloid scaffold. Moreover, we show that the shorter and more polar NYNNYN, QYQQYQ, and SYSSYS hexapeptides also self-assemble into amyloid-like structures, consistent with the presence of these tandem motifs in human prion-like proteins.

Intranasally Administered S100A9 Amyloids Induced Cellular Stress, Amyloid Seeding, and Behavioral Impairment in Aged Mice.

Amyloid formation and neuroinflammation are major features of Alzheimer's disease pathology. Proinflammatory mediator S100A9 was shown to act as a link between the amyloid and neuroinflammatory cascades in Alzheimer's disease, leading together with Aβ to plaque formation, neuronal loss and memory impairment. In order to examine if S100A9 alone in its native and amyloid states can induce neuronal stress and memory impairment, we have administered S100A9 species intranasally to aged mice. Single and sequential immunohistochemistry and passive avoidance behavioral test were conducted to evaluate the consequences. Administered S100A9 species induced widespread cellular stress responses in cerebral structures, including frontal lobe, hippocampus and cerebellum. These were manifested by increased levels of S100A9, Bax, and to a lesser extent activated caspase-3 immunopositive cells. Upon administration of S100A9 fibrils, the amyloid oligomerization was observed in the brain tissues, which can further exacerbate cellular stress. The cellular stress responses correlated with significantly increased training and decreased retention latencies measured in the passive avoidance test for the S100A9 treated animal groups. Remarkably, the effect size in the behavioral tests was moderate already in the group treated with native S100A9, while the effect sizes were large in the groups administered S100A9 amyloid oligomers or fibrils. The findings demonstrate the brain susceptibility to neurotoxic damage of S100A9 species leading to behavioral and memory impairments. Intranasal administration of S100A9 species proved to be an effective method to study amyloid induced brain dysfunctions, and S100A9 itself may be postulated as a target to allay early stage neurodegenerative and neuroinflammatory processes.

Investigating Effects and Determinants of PAP248–286‐Membrane Interactions: Membrane Leakage and Peptide/Lipid Co‐Aggregation

Many amyloid‐forming peptides appear to possess potentially beneficial antimicrobial activities. Furthermore, it has been suggested that this previously unrecognized activity may have a role in innate immunity. Antimicrobial peptides, which are important effectors in the innate immune response, can act either directly by inducing membrane leakage and cytolysis, or indirectly through bacterial adhesion inhibition or enhanced phagocytosis via bacterial agglutination. Amyloid‐forming peptides themselves display a variety of membrane‐disrupting and membrane‐remodeling activities, including membrane leakage and vesicle aggregation. In turn, some membranes are known to catalyze amyloid formation. Peptide‐membrane interactions may thus underlie both the pathological activity of amyloid‐forming peptides through their cytotoxicity due to pre‐fibrillar oligomers and their beneficial functions through their direct or indirect antimicrobial activities. Therefore, studying the factors driving the interactions between amyloid‐forming peptides and membranes may be valuable in understanding their roles in both healthy and diseased states.To understand the general principles that govern the interactions of amyloid‐forming peptides with membranes, we use liposomes as model membranes to gain precise control over their size and composition. We have also chosen to focus our studies on an amyloidogenic fragment of prostatic acid phosphatase (PAP248–286). In its amyloid state, this peptide is named “semen‐derived enhancer of viral infection” (SEVI), and greatly increases HIV infectivity. Using spectroscopic techniques, we have discovered that PAP248–286 causes liposome disruption in a charge and dose dependent manner. This dependence is expected given the cationic nature of PAP248–286, and is consistent with similar activities of other pre‐amyloid toxins and antimicrobial peptides. In addition, we have discovered that PAP248–286 forms large, fibrillar lipid‐peptide co‐aggregates. These co‐aggregates have only been observed at specific peptide to lipid ratios with completely anionic liposomes. While this species, which we have termed “messicles”, looks somewhat fibrillar and is Thioflavin T positive, the morphology and kinetics of formation are more reminiscent of an amorphous aggregate than bona fide amyloid. We propose that messicle formation occurs via a mechanism wherein addition of the right amount of negatively charged liposomes completely or partially neutralizes the positive charges on PAP248–286, allowing coated liposomes to self‐associate, and eventually leading to flocculate formation. Since this process would be controlled by the binding affinity of the peptides for the liposomes, messicles might be able to form by this mechanism with other membrane compositions. Considering messicles as another structure in the diverse repertoire of states accessible to PAP248–286 may be important to understanding its function. As amyloid formation and amorphous aggregation occur for other disease‐associated peptides as well, this work could also guide further investigation into their membrane activity and potentially beneficial functions.Support or Funding InformationNSF Graduate Research Fellowship to E.V.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2.

Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.

PIN+]ing down the mechanism of prion appearance.

Prions are conformationally flexible proteins capable of adopting a native state and a spectrum of alternative states associated with a change in the function of the protein. These alternative states are prone to assemble into amyloid aggregates, which provide a structure for self-replication and transmission of the underlying conformer and thereby the emergence of a new phenotype. Amyloid appearance is a rare event in vivo, regulated by both the aggregation propensity of prion proteins and their cellular environment. How these forces normally intersect to suppress amyloid appearance and the ways in which these restrictions can be bypassed to create protein-only phenotypes remain poorly understood. The most widely studied and perhaps most experimentally tractable system to explore the mechanisms regulating amyloid appearance is the [PIN+] prion of Saccharomyces cerevisiae. [PIN+] is required for the appearance of the amyloid state for both native yeast proteins and for human proteins expressed in yeast. These observations suggest that [PIN+] facilitates the bypass of amyloid regulatory mechanisms by other proteins in vivo. Several models of prion appearance are compatible with current observations, highlighting the complexity of the process and the questions that must be resolved to gain greater insight into the mechanisms regulating these events.

Lipopolysaccharide-binding protein (LBP) can reverse the amyloid state of fibrin seen or induced in Parkinson's disease.

The thrombin-induced polymerisation of fibrinogen to form fibrin is well established as a late stage of blood clotting. It is known that Parkinson’s Disease (PD) is accompanied by dysregulation in blood clotting, but it is less widely known as a coagulopathy. In recent work, we showed that the presence of tiny amounts of bacterial lipopolysaccharide (LPS) in healthy individuals could cause clots to adopt an amyloid form, and this could be observed via scanning electron microscopy (SEM) or via the fluorescence of thioflavin-T. This could be prevented by the prior addition of lipopolysaccharide-binding protein (LBP). We had also observed by SEM this unusual clotting in the blood of patients with Parkinson’s Disease. We hypothesised, and here show, that this too can be prevented by LBP in the context of PD. This adds further evidence implicating inflammatory microbial cell wall products as an accompaniment to the disease, and may be part of its aetiology. This may lead to novel treatment strategies in PD designed to target microbes and their products.

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.

Toward a Soluble Model System for the Amyloid State.

The formation and deposition of amyloids is associated with many diseases. β-Sheet secondary structure is a common feature of amyloids, but the packing of sheets against one another is distinctive relative to soluble proteins. Standard methods that rely on perturbing a polypeptide's sequence and evaluating impact on folding can be problematic for amyloid aggregates because a single sequence can adopt multiple conformations and diverse packing arrangements. We describe initial steps toward a minimum-sized, soluble model system for the amyloid state that supports comparisons among sequence variants. Critical to this goal is development of a new linking strategy to enable intersheet association mediated by side chain interactions, which is characteristic of the amyloid state. The linker design we identified should ultimately support exploration of relationships between sequence and amyloid state stability for specific strand-association modes.

AmyPro: a database of proteins with validated amyloidogenic regions.

Soluble functional proteins may transform into insoluble amyloid fibrils that deposit in a variety of tissues. Amyloid formation is a hallmark of age-related degenerative disorders. Perhaps surprisingly, amyloid fibrils can also be beneficial and are frequently exploited for diverse functional roles in organisms. Here we introduce AmyPro, an open-access database providing a comprehensive, carefully curated collection of validated amyloid fibril-forming proteins from all kingdoms of life classified into broad functional categories (http://amypro.net). In particular, AmyPro provides the boundaries of experimentally validated amyloidogenic sequence regions, short descriptions of the functional relevance of the proteins and their amyloid state, a list of the experimental techniques applied to study the amyloid state, important structural/functional/variation/mutation data transferred from UniProt, a list of relevant PDB structures categorized according to protein states, database cross-references and literature references. AmyPro greatly improves on similar currently available resources by incorporating both prions and functional amyloids in addition to pathogenic amyloids, and allows users to screen their sequences against the entire collection of validated amyloidogenic sequence fragments. By enabling further elucidation of the sequential determinants of amyloid fibril formation, we hope AmyPro will enhance the development of new methods for the precise prediction of amyloidogenic regions within proteins.

Site-Specific Fluorescence Depolarization Kinetics Distinguishes the Amyloid Folds Responsible for Distinct Yeast Prion Strains.

The prion determinant of a yeast prion protein, Sup35NM, assembles into β-rich amyloid fibrils that switch the nonprion [psi-] state to the prion [PSI+] state of yeast. Previous studies showed that two distinct forms of amyloids (Sc4 and Sc37), generated in vitro at two different temperatures (4 and 37 °C), recapitulate the strain phenomenon in Saccharomyces cerevisiae. Sc4 demonstrates a strong [PSI+] phenotype, whereas Sc37 shows a weak phenotype. To discern the residue-specific structural and dynamical attributes associated with the amyloids that display strain diversity, we took advantage of the nonoccurrence of tryptophan (Trp) in the NM-domain and created 18 single-Trp variants spanning the entire polypeptide length. The fluorescence readouts from these locations reported the site-specific structural details in Sc4 and Sc37 fibrils. Highly sensitive picosecond fluorescence depolarization measurements at these positions allowed a conformational mobility map to be constructed. Nearly all of the residue positions demonstrated higher local flexibility in Sc4 amyloid, which exhibits a strong phenotype. The differences in the amplitude of local mobility were more pronounced at the two end segments of the N-domain than in the central region. The M-domain is partially exposed and exhibits a higher amplitude of local mobility, indicating a lower degree of chain packing in the amyloid state, as well as a higher mobility in the Sc4 state compared to the Sc37 state. The altered local conformational dynamics in these two distinct amyloid states provide molecular insights into the varied fragility and severing efficiency that govern the inheritance patterns of strong and weak prion strains.

Pathogenic Mutations Induce Partial Structural Changes in the Native β-Sheet Structure of Transthyretin and Accelerate Aggregation.

Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state nuclear magnetic resonance (NMR) structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective 13C labeling schemes to investigate structural features of β-structured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the 13C-13C correlation solid-state NMR spectra revealed that WT TTR aggregates contain an amyloid core consisting of nativelike CBEF and DAGH β-sheet structures, and the mutant TTR amyloids adopt a similar amyloid core structure with nativelike CBEF and AGH β-structures. However, the V30M mutant amyloid was shown to have a different DA β-structure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA β-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of the methionine side chain also revealed that the side chain of residue Met-30 pointing between strands D and A is not protected from oxidation in the V30M mutant, while protected in the native state, supporting the possibility that the DA β-structure might be disrupted in the V30M mutant amyloid.

Lipopolysaccharide-binding protein (LBP) reverses the amyloid state of fibrin seen in plasma of type 2 diabetics with cardiovascular co-morbidities

Type 2 diabetes (T2D) has many cardiovascular complications, including a thrombotic propensity. Many such chronic, inflammatory diseases are accompanied (and may be exacerbated, and possibly even largely caused) by amyloid fibril formation. Recognising that there are few strong genetic associations underpinning T2D, but that amyloidogenesis of amylin is closely involved, we have been seeking to understand what might trigger the disease. Serum levels of bacterial lipopolysaccharide are raised in T2D, and we recently showed that fibrin(ogen) polymerisation during blood clotting can be affected strongly by LPS. The selectivity was indicated by the regularisation of clotting by lipopolysaccharide-binding protein (LBP). Since coagulopathies are a hallmark of T2D, we wondered whether they might too be caused by LPS (and reversed by LBP). We show here, using SEM and confocal microscopy, that platelet-poor-plasma from subjects with T2D had a much greater propensity for hypercoagulability and for amyloidogenesis, and that these could both be reversed by LBP. These data imply that coagulopathies are an important feature of T2D, and may be driven by ‘hidden’ LPS. Given the prevalence of amyloid formation in the sequelae of diabetes, this opens up novel strategies for both the prevention and treatment of T2D.

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

ABSTRACTPrion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN+], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.

Overcoming electrostatic repulsions during amyloid assembly: Effect of pH and interaction with divalent metals using model peptides

Amyloids are polypeptide aggregates involved in many pathologies including Alzheimer's disease. Amyloid assembly is a complex process affected by different interactions including hydrogen bonding, van der Waals forces and electrostatic interactions. The highly regular amyloid structure allows for an arrangement of residues that forces side chains to be closely positioned, giving rise to potentially unfavorable interactions such as electrostatic repulsions. In these cases, amyloid assembly will depend on a balance between stabilizing versus unfavorable interactions. In this study, we rationally designed several amyloid-prone model peptides that had two acidic groups and tested their assembly into amyloids under different conditions. We found that at low pH (pH 4.0), most peptides spontaneously formed amyloids whereas no or little aggregation was observed at higher pHs (pH 8.0). When divalent metals with affinity for carboxylate groups were added at millimolar concentrations, most peptides exhibited a metal-dependent switch to the amyloid state at pH 8.0. Our results show that electrostatic repulsion between amyloid-prone sequences can be overcome in conditions that affect protonation of residue side chains. Moreover, the presence of divalent metals can contribute to electrostatic shielding through specific coordination with acidic groups and thus promote amyloid assembly.

Mapping the Broad Structural and Mechanical Properties of Amyloid Fibrils

Amyloids are fibrillar nanostructures of proteins that are assembled in several physiological processes in human cells (e.g., hormone storage) but also during the course of infectious (prion) and noninfectious (nonprion) diseases such as Creutzfeldt-Jakob and Alzheimer’s diseases, respectively. How the amyloid state, a state accessible to all proteins and peptides, can be exploited for functional purposes but also have detrimental effects remains to be determined. Here, we measure the nanomechanical properties of different amyloids and link them to features found in their structure models. Specifically, we use shape fluctuation analysis and sonication-induced scission in combination with full-atom molecular dynamics simulations to reveal that the amyloid fibrils of the mammalian prion protein PrP are mechanically unstable, most likely due to a very low hydrogen bond density in the fibril structure. Interestingly, amyloid fibrils formed by HET-s, a fungal protein that can confer functional prion behavior, have a much higher Young’s modulus and tensile strength than those of PrP, i.e., they are much stiffer and stronger due to a tighter packing in the fibril structure. By contrast, amyloids of the proteins RIP1/RIP3 that have been shown to be of functional use in human cells are significantly stiffer than PrP fibrils but have comparable tensile strength. Our study demonstrates that amyloids are biomaterials with a broad range of nanomechanical properties, and we provide further support for the strong link between nanomechanics and β-sheet characteristics in the amyloid core.