Létitia Jean

Combined effects of agitation, macromolecular crowding and interfaces on amyloidogenesis

Background: Macromolecular crowding and hydrophobic-hydrophilic interfaces promote amyloidogenesis. Results: The outcome of macromolecular crowding on Aβ amyloidogenesis depends on the spatial heterogeneity of the system. Conclusion: Viscosity dominates over the excluded volume effect only when the system contains a hydrophobic-hydrophilic interface. Significance: Studying both interfacial and macromolecular crowding effects together is crucial to understand amyloid systems in a physiological context. Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimers 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-β 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.

Competing discrete interfacial effects are critical for amyloidogenesis

Amyloid accumulation is associated with pathological conditions, including type II diabetes and Alzheimers 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 β‐sheet amyloid dimer formation to be higher and faster at the air‐water interface (AWI) than in the bulk (by 14 and ~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 amy‐loidogenesis has been previously underestimated;in an in vivo‐lke 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.—Jean, L., Lee, C. F., Lee, C., Shaw, M., Vaux, D. J. Competing discrete interfacial effects are critical for amyloidogenesis. FASEB J. 24, 309–317 (2010). www.fasebj.org

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., Alzheimers 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-β 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.

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-β peptide (Aβ) production via β and γ secretases in Alzheimers Disease (AD), is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Aβ 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 Aβ fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE575-614) is an exposed amphiphilic α-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR) with high propensity for conversion to non-native (hidden) β-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE586-599) encompassing the CSR region shares homology with Aβ and forms β-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE β-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE586-599, or versus Aβ, differed. Moreover, IDE digestion of T40 triggered the conformational switch from α- to β-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers). Crucially, these CSR species significantly increased Aβ 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 Aβ 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 (Aβ in AD, for example) may not be the only important factor in neurodegeneration.

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), AChE586-599, through the effect of single point mutations on β-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE586-599 was chosen due to its fibrilization tractability and AChE involvement in Alzheimers disease. The results revealed how specific regions and residues can control AChE586-599 assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high β-strand propensity, for the conformational transition to β-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 β-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE586-599 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 AChE586-599 assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-π, SH-aromatic, metal chelation and polar-polar) would maintain the β-sheets together. We also propose that the stacking between the strands in the β-sheets along the fiber axis could be stabilized through π-π interactions and metal chelation. The dissection of the specific molecular recognition driving AChE586-599 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.

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.

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.

Reverse immunodynamics: a new method to 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. We propose a novel methodology that combines predictions from epidemiological models with phylogenetic and structural analyses to identify such targets. Epidemiological models predict that strong immune selection can cause antigenic variants to exist in non-overlapping combinations. A corollary of this theory is that targets of immunity may be identified by searching for non-overlapping associations among antigenic variants. We applied this concept to the AMA-1 protein of the malaria parasite Plasmodium falciparum and found strong signatures of immune selection among certain regions of low variability which could render them ideal vaccine candidates.