Liquid-liquid phase separation of type II diabetes-associated IAPP initiates hydrogelation and aggregation.

Amyloidoses (misfolded polypeptide accumulation) are among the most debilitating diseases our aging societies face. Amyloidogenesis can be catalyzed by hydrophobic-hydrophilic interfaces (e.g., air-water interface in vitro [AWI]). We recently demonstrated hydrogelation of the amyloidogenic type II diabetes-associated islet amyloid polypeptide (IAPP), a hydrophobic-hydrophilic interface-dependent process with complex kinetics. We demonstrate that human IAPP undergoes AWI-catalyzed liquid-liquid phase separation (LLPS), which initiates hydrogelation and aggregation. Insulin modulates these processes but does not prevent them. Using nonamyloidogenic rat IAPP, we show that, whereas LLPS does not require the amyloidogenic sequence, hydrogelation and aggregation do. Interestingly, both insulin and rat sequence delayed IAPP LLPS, which may reflect physiology. By developing an experimental setup and analysis tools, we show that, within the whole system (beyond the droplet stage), macroscopic interconnected aggregate clusters form, grow, fuse, and evolve via internal rearrangement, leading to overall hydrogelation. As the AWI-adsorbed gelled layer matures, its microviscosity increases. LLPS-driven aggregation may be a common amyloid feature and integral to pathology.

The kinetics of islet amyloid polypeptide phase-separated system and hydrogel formation are critically influenced by macromolecular crowding.

Many protein misfolding diseases (e.g. type II diabetes and Alzheimer's disease) are characterised by amyloid deposition. Human islet amyloid polypeptide (hIAPP, involved in type II diabetes) spontaneously undergoes liquid-liquid phase separation (LLPS) and a kinetically complex hydrogelation, both catalysed by hydrophobic-hydrophilic interfaces (e.g. air-water interface and/or phospholipids-water interfaces). Gelation of hIAPP phase-separated liquid droplets initiates amyloid aggregation and the formation of clusters of interconnected aggregates, which grow and fuse to eventually percolate the whole system. Droplet maturation into irreversible hydrogels via amyloid aggregation is thought to be behind the pathology of several diseases. Biological fluids contain a high volume fraction of macromolecules, leading to macromolecular crowding. Despite crowding agent addition in in vitro studies playing a significant role in changing protein phase diagrams, the mechanism underlying enhanced LLPS, and the effect(s) on stages beyond LLPS remain poorly or not characterised.We investigated the effect of macromolecular crowding and increased viscosity on the kinetics of hIAPP hydrogelation using rheology and the evolution of the system beyond LLPS by microscopy. We demonstrate that increased viscosity exacerbated the kinetic variability of hydrogelation and of the phase separated-aggregated system, whereas macromolecular crowding abolished heterogeneity. Increased viscosity also strengthened the gel meshwork and accelerated aggregate cluster fusion. In contrast, crowding either delayed cluster fusion onset (dextran) or promoted it (Ficoll). Our study highlights that an in vivo crowded environment would critically influence amyloid stages beyond LLPS and pathogenesis.

In vivo localization of human acetylcholinesterase-derived species in a ß-sheet conformation at the core of senile plaques in Alzheimer's disease.

Many neurodegenerative diseases are characterized by amyloid deposition. In Alzheimer's disease (AD), ß-amyloid (Aß) peptides accumulate extracellularly in senile plaques. The AD amyloid cascade hypothesis proposes that Aß production or reduced clearance leads to toxicity. In contrast, the cholinergic hypothesis argues for a specific pathology of brain cholinergic pathways. However, neither hypothesis in isolation explains the pattern of AD pathogenesis. Evidence suggests that a connection exists between these two scenarios: the synaptic form of human acetylcholinesterase (hAChE-S) associates with plaques in AD brains; among hAChE variants, only hAChE-S enhances Aß fibrillization in vitro and Aß deposition and toxicity in vivo Only hAChE-S contains an amphiphilic C-terminal domain (T40, AChE575-614), with AChE586-599 homologous to Aß and forming amyloid fibrils, which implicates T40 in AD pathology. We previously showed that the amyloid scavenger, insulin-degrading enzyme (IDE), generates T40-derived amyloidogenic species that, as a peptide mixture, seed Aß fibrillization. Here, we characterized 11 peptides from a T40-IDE digest for ß-sheet conformation, surfactant activity, fibrillization, and seeding capability. We identified residues important for amyloidogenicity and raised polyclonal antibodies against the most amyloidogenic peptide. These new antisera, alongside other specific antibodies, labeled sections from control, hAChE-S, hAPPswe, and hAChE-S/hAPPswe transgenic mice. We observed that hAChE-S ß-sheet species co-localized with Aß in mature plaque cores, surrounded by hAChE-S α-helical species. This observation provides the first in vivo evidence of the conformation of hAChE-S species within plaques. Our results may explain the role of hAChE-S in Aß deposition and aggregation, as amyloidogenic hAChE-S ß-sheet species might seed Aß aggregation.

Multiphasic effect of vinyl pyrrolidone polymers on amyloidogenesis, from macromolecular crowding to inhibition.

Deposition of misfolded amyloid polypeptides, associated with cell death, is the hallmark of many degenerative diseases (e.g. type II diabetes mellitus and Alzheimer's disease). In vivo, cellular and extracellular spaces are occupied by a high volume fraction of macromolecules. The resulting macromolecular crowding energetically affects reactions. Amyloidogenesis can either be promoted by macromolecular crowding through the excluded volume effect or inhibited due to a viscosity increase reducing kinetics. Macromolecular crowding can be mimicked in vitro by the addition of non-specific polymers, e.g. Ficoll, dextran and polyvinyl pyrrolidone (PVP), the latter being rarely used to study amyloid systems. We investigated the effect of PVP on amyloidogenesis of full-length human islet amyloid polypeptide (involved in type II diabetes) using fibrillisation and surface activity assays, ELISA, immunoblot and microscale thermophoresis. We demonstrate that high molecular mass PVP360 promotes amyloidogenesis due to volume exclusion and increase in effective amyloidogenic monomer concentration, like other crowders, but without the confounding effects of viscosity and surface activity. Interestingly, we also show that low molecular mass PVP10 has unique inhibitory properties as inhibition of fibril elongation occurs mainly in the bulk solution and is due to PVP10 directly and strongly interacting with amyloid species rather than the increase in viscosity typically associated with macromolecular crowding. In vivo, amyloidogenesis might be affected by the properties and proximity of endogenous macromolecular crowders, which could contribute to changes in associated pathogenesis. More generally, the PVP10 molecular backbone could be used to design small compounds as potential inhibitors of toxic species formation.

The air-water interface determines the outcome of seeding during amyloidogenesis.

Amyloid formation is a hallmark of protein misfolding diseases (e.g. Type II diabetes mellitus). The energetically unfavourable nucleation step of amyloidogenesis can be accelerated by seeding, during which pre-formed aggregates act as templates for monomer recruitment. Hydrophobic-hydrophilic interfaces [e.g. AWI (air-water interface)] can also catalyse amyloidogenesis due to the surfactant properties of amyloidogenic polypeptides. Using thioflavin T fluorescence and electron microscopy, we demonstrate that the outcome of seeding on human islet amyloid polypeptide amyloidogenesis is dependent upon whether the AWI is present or absent and is dictated by seed type. Seeding significantly inhibits (with AWI) or promotes (without AWI) plateau height compared with seedless controls; with short fibrils being more efficient seeds than their longer counterparts. Moreover, promotion of nucleation by increasing monomer concentrations can only be observed in the absence of an AWI. Using biophysical modelling, we suggest that a possible explanation for our results may reside in lateral interactions between seeds and monomers determining the fibril mass formed in seeded reactions at steady-state. Our results suggest that in vivo hydrophobic-hydrophilic interfaces (e.g. the presence of membranes and their turnover rate) may dictate the outcome of seeding during amyloidogenesis and that factors affecting the size of the pre-aggregate may be important.

Combined effects of agitation, macromolecular crowding, and interfaces on amyloidogenesis.

Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer's disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-ß peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD.

Enrichment of amyloidogenesis at an air-water interface.

The aggregation of proteins or peptides into amyloid fibrils is a hallmark of protein misfolding diseases (e.g., Alzheimer's disease) and is under intense investigation. Many of the experiments performed are in vitro in nature and the samples under study are ordinarily exposed to diverse interfaces, e.g., the container wall and air. This naturally raises the question of how important interfacial effects are to amyloidogenesis. Indeed, it has already been recognized that many amyloid-forming peptides are surface-active. Moreover, it has recently been demonstrated that the presence of a hydrophobic interface can promote amyloid fibrillization, although the underlying mechanism is still unclear. Here, we combine theory, surface property measurements, and amyloid fibrillogenesis assays on islet amyloid polypeptide and amyloid-ß peptide to demonstrate why, at experimentally relevant concentrations, the surface activity of the amyloid-forming peptides leads to enriched fibrillization at an air-water interface. Our findings indicate that the key that links these two seemingly different phenomena is the surface-active nature of the amyloid-forming species, which renders the surface concentration much higher than the corresponding critical fibrillar concentration. This subsequently leads to a substantial increase in fibrillization.

The erythrocyte membrane properties of beta thalassaemia heterozygotes and their consequences for Plasmodium falciparum invasion.

Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

Competing discrete interfacial effects are critical for amyloidogenesis.

Amyloid accumulation is associated with pathological conditions, including type II diabetes and Alzheimer's disease. Lipids influence amyloidogenesis and are themselves targets for amyloid-mediated cell membrane disruption. Amyloid precursors are surface-active, accumulating at hydrophobic-hydrophilic interfaces (e.g., air-water), where their biophysical and kinetic behaviors differ from those in the bulk solution with significant and underappreciated consequences. Biophysical modeling predicted the probability and rate of beta-sheet amyloid dimer formation to be higher and faster at the air-water interface (AWI) than in the bulk (by 14 and approximately 1500 times, respectively). Time-course staining experiments with a typical amyloid dye verified our predictions by demonstrating that without AWI, islet amyloid polypeptide (IAPP) fibrilization was abolished or slowed, depending on the conditions. Our controls included undisturbed IAPP reactions, and we ascertained that the AWI removal process (technical or material) did not itself affect the reaction. Furthermore, we showed that the role of membranes in amyloidogenesis has been previously underestimated; in an in vivo-like situation (with no AWI), anionic liposomes (containing dioleoylphosphatidylglycerol) enhanced IAPP fibrilogenesis far more than described previously in conventional assay conditions (in the presence of an AWI). These findings have implications for the protein misfolding field and in assay design to target toxic protein aggregation.

Reverse immunodynamics: a new method for identifying targets of protective immunity.

Despite a dramatic increase in our ability to catalogue variation among pathogen genomes, we have made far fewer advances in using this information to identify targets of protective immunity. Epidemiological models predict that strong immune selection can cause antigenic variants to structure into genetically discordant sets of antigenic types (e.g. serotypes). A corollary of this theory is that targets of immunity may be identified by searching for non-overlapping associations of amino acids among co-circulating antigenic variants. We propose a novel population genetics methodology that combines such predictions with phylogenetic analyses to identify genetic loci (epitopes) under strong immune selection. We apply this concept to the AMA-1 protein of the malaria parasite Plasmodium falciparum and find evidence of epitopes among certain regions of low variability which could render them ideal vaccine candidates. The proposed method can be applied to a myriad of multi-strain pathogens for which vast amounts of genetic data has been collected in recent years.

The Physiological and Pathological Implications of the Formation of Hydrogels, with a Specific Focus on Amyloid Polypeptides.

Hydrogels are water-swollen and viscoelastic three-dimensional cross-linked polymeric network originating from monomer polymerisation. Hydrogel-forming polypeptides are widely found in nature and, at a cellular and organismal level, they provide a wide range of functions for the organism making them. Amyloid structures, arising from polypeptide aggregation, can be damaging or beneficial to different types of organisms. Although the best-known amyloids are those associated with human pathologies, this underlying structure is commonly used by higher eukaryotes to maintain normal cellular activities, and also by microbial communities to promote their survival and growth. Amyloidogenesis occurs by nucleation-dependent polymerisation, which includes several species (monomers, nuclei, oligomers, and fibrils). Oligomers of pathological amyloids are considered the toxic species through cellular membrane perturbation, with the fibrils thought to represent a protective sink for toxic species. However, both functional and disease-associated amyloids use fibril cross-linking to form hydrogels. The properties of amyloid hydrogels can be exploited by organisms to fulfil specific physiological functions. Non-physiological hydrogelation by pathological amyloids may provide additional toxic mechanism(s), outside of membrane toxicity by oligomers, such as physical changes to the intracellular and extracellular environments, with wide-spread consequences for many structural and dynamic processes, and overall effects on cell survival.

Dynamics of the formation of a hydrogel by a pathogenic amyloid peptide: islet amyloid polypeptide.

Many chronic degenerative diseases result from aggregation of misfolded polypeptides to form amyloids. Many amyloidogenic polypeptides are surfactants and their assembly can be catalysed by hydrophobic-hydrophilic interfaces (an air-water interface in-vitro or membranes in-vivo). We recently demonstrated the specificity of surface-induced amyloidogenesis but the mechanisms of amyloidogenesis and more specifically of adsorption at hydrophobic-hydrophilic interfaces remain poorly understood. Thus, it is critical to determine how amyloidogenic polypeptides behave at interfaces. Here we used surface tensiometry, rheology and electron microscopy to demonstrate the complex dynamics of gelation by full-length human islet amyloid polypeptide (involved in type II diabetes) both in the bulk solution and at hydrophobic-hydrophilic interfaces (air-water interface and phospholipids). We show that the hydrogel consists of a 3D supramolecular network of fibrils. We also assessed the role of solvation and dissected the evolution over time of the assembly processes. Amyloid gelation could have important pathological consequences for membrane integrity and cellular functions.

Unique insertions within Plasmodium falciparum subtilisin-like protease-1 are crucial for enzyme maturation and activity.

Parasite serine proteases play essential roles in the asexual erythrocytic life cycle of the malaria parasite. The timing and location of expression of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) are consistent with a role in erythrocyte invasion. Maturation of PfSUB-1 involves two autocatalytic processing events in which an 82 kDa precursor is converted to a 54 kDa form, followed by further cleavage to produce a 47 kDa form. Here we have compared PfSUB-1 with a number of Plasmodium orthologues and the most closely related bacterial subtilase sequences and find that, like many malarial proteins, PfSUB-1 possesses both low and high complexity insertions. The latter take the form of six surface-associated strands or loops which are conserved in all SUB-1 orthologues but not present in any other subtilase. Several mutants of PfSUB-1 with deletions of all, or part, of each of the six loop insertions were produced in an insect cell expression system. Aside from loop III, which was dispensable, individual deletion of the loop insertions revealed a role in protein maturation and/or stability. Specific substitutions within loop II inhibited maturation and enzyme activity. Mutations in loops V and VI specifically inhibited the second step of autocatalytic maturation providing evidence that the two processing steps have distinct structural requirements and that conversion to p47 is not a prerequisite for proteolytic activity in trans.

Elongation dynamics of amyloid fibrils: a rugged energy landscape picture.

Protein amyloid fibrils are a form of linear protein aggregates that are implicated in many neurodegenerative diseases. Here, we study the dynamics of amyloid fibril elongation by performing Langevin dynamic simulations on a coarse-grained model of peptides. Our simulation results suggest that the elongation process is dominated by a series of local minimum due to frustration in monomer-fibril interactions. This rugged energy landscape picture indicates that the amount of recycling of monomers at the fibrils' ends before being fibrilized is substantially reduced in comparison to the conventional two-step elongation model. This picture, along with other predictions discussed, can be tested with current experimental techniques.

Structural elements regulating amyloidogenesis: a cholinesterase model system.

Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE), AChE(586-599), through the effect of single point mutations on beta-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE(586-599) was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE(586-599) assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high beta-strand propensity, for the conformational transition to beta-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to beta-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE(586-599) surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE(586-599) assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-pi, SH-aromatic, metal chelation and polar-polar) would maintain the beta-sheets together. We also propose that the stacking between the strands in the beta-sheets along the fiber axis could be stabilized through pi-pi interactions and metal chelation. The dissection of the specific molecular recognition driving AChE(586-599) amyloid assembly has provided further knowledge on such poorly understood and complicated process, which could be applied to protein folding and the targeting of amyloid diseases.

Heterologous amyloid seeding: revisiting the role of acetylcholinesterase in Alzheimer's disease.

Neurodegenerative diseases associated with abnormal protein folding and ordered aggregation require an initial trigger which may be infectious, inherited, post-inflammatory or idiopathic. Proteolytic cleavage to generate vulnerable precursors, such as amyloid-beta peptide (Abeta) production via beta and gamma secretases in Alzheimer's Disease (AD), is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Abeta in the central nervous system are regulated by several catabolic proteases, including insulysin (IDE) and neprilysin (NEP). The known association of human acetylcholinesterase (hAChE) with pathological aggregates in AD together with its ability to increase Abeta fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE(575-614)) is an exposed amphiphilic alpha-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR) with high propensity for conversion to non-native (hidden) beta-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE(586-599)) encompassing the CSR region shares homology with Abeta and forms beta-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE beta-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE(586-599), or versus Abeta, differed. Moreover, IDE digestion of T40 triggered the conformational switch from alpha- to beta-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers). Crucially, these CSR species significantly increased Abeta fibril formation both by seeding the energetically unfavorable formation of amyloid nuclei and by enhancing the rate of amyloid elongation. Hence, these results may offer an explanation for observations that implicate hAChE in the extent of Abeta deposition in the brain. Furthermore, this process of heterologous amyloid seeding by a proteolytic fragment from another protein may represent a previously underestimated pathological trigger, implying that the abundance of the major amyloidogenic species (Abeta in AD, for example) may not be the only important factor in neurodegeneration.

Subtilisin-like proteases of the malaria parasite.

Proteases play critical roles in the life cycle of the malaria parasite, Plasmodium spp. Within the asexual erythrocytic cycle, responsible for the clinical manifestations of malaria, substantial interest has focused on the role of parasite serine proteases as a result of indications that they are involved in red blood cell invasion. Over the past 6 years, three Plasmodium genes encoding serine proteases of the subtilisin-like clan, or subtilases, have been identified. All are expressed in the asexual blood stages and, in at least two cases, the gene products localize to secretory organelles of the invasive merozoite. They may have potential as novel drug targets. Here, we review progress in our understanding of the maturation, specificity, structure and function of these Plasmodium subtilases.

Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1.

Erythrocyte invasion by the malaria merozoite is prevented by serine protease inhibitors. Various aspects of the biology of Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1), including the timing of its expression and its apical location in the merozoite, suggest that this enzyme is involved in invasion. Recombinant PfSUB-1 expressed in a baculovirus system is secreted in the p54 form, noncovalently bound to its cognate propeptide, p31. To understand the role of p31 in PfSUB-1 maturation, we examined interactions between p31 and both recombinant and native enzymes. CD analyses revealed that recombinant p31 (rp31) possesses significant secondary structure on its own, comparable with that of folded propeptides of some bacterial subtilisins. Kinetic studies demonstrated that rp31 is a fast binding, high affinity inhibitor of PfSUB-1. Inhibition of two bacterial subtilisins by rp31 was much less effective, with inhibition constants 49-60-fold higher than that for PfSUB-1. Single (at the P4 or P1 position) or double (at P4 and P1 positions) point mutations of residues within the C-terminal region of rp31 had little effect on its inhibitory activity, and truncation of 11 residues from the rp31 C terminus substantially reduced, but did not abolish, inhibition. None of these modifications prevented binding to the PfSUB-1 catalytic domain or rendered the propeptide susceptible to proteolytic digestion by PfSUB-1. These studies provide new insights into the function of the propeptide in PfSUB-1 activation and shed light on the structural requirements for interaction with the catalytic domain.