Amyloid formation: interface influence

The causes of pathological conditions such as Alzheimer’s and Parkinson’s diseases are becoming better understood. Proteins that misfold from their native structure to form aggregates of β-sheet fibrils — termed amyloid — are known1,2 to be implicated in these ‘amyloid diseases’. Understanding the early steps of fibril formation is critical, and the conditions, mechanism and kinetics of protein and peptide aggregation are being widely investigated through a variety of in vitro studies. Kinetic aspects of the dispersion of the protein or peptide in solution are thought to influence the fibrillization process by mass-transfer effects. In addition, mixing also leads to shear forces, which can influence fibril growth by perturbing the equilibrium between the isolated and aggregated proteins, causing existing fibrils to fragment and create new nuclei3. Writing in the Journal of the American Chemical Society, David Talaga and co-workers have now highlighted4 an additional factor that can influence the fibrillization of amyloid-forming proteins — the presence of hydrophobic interfaces

Monitoring Alzheimer Amyloid Peptide Aggregation by EPR

Plaques containing the aggregated β-Amyloid (Aβ) peptide in the brain are the main indicators of Alzheimer’s disease. Fibrils, the building blocks of plaques, can also be produced in vitro and consist of a regular arrangement of the peptide. The initial steps of fibril formation are not well understood and could involve smaller aggregates (oligomers) of Aβ. Such oligomers have even been implicated as the toxic agents. Here, a method to study oligomers on the time scale of aggregation is suggested. We have labeled the 40 residue Aβ peptide variant containing an N-terminal cysteine (cys-Aβ) with the MTSL [1-oxyl-2,2,5,5-tetramethyl-Δ-pyrroline-3-methyl] methanethiosulfonate spin label (SL-Aβ). Fibril formation in solutions of pure SL-Aβ and of SL-Aβ mixed with Aβ was shown by Congo-red binding and electron microscopy. Continuous-wave 9 GHz electron paramagnetic resonance reveals three fractions of different spin-label mobility: one attributed to monomeric Aβ, one to a multimer (8–15 monomers), and the last one to larger aggregates or fibrils. The approach, in principle, allows detection of oligomers on the time scale of aggregation

Molecular Structures of Quiescently Grown and Brain-Derived Polymorphic Fibrils of the Alzheimer Amyloid Aβ9-40 Peptide: A Comparison to Agitated Fibrils

The presence of amyloid deposits consisting primarily of Amyloid-β (Aβ) fibril in the brain is a hallmark of Alzheimer's disease (AD). The morphologies of these fibrils are exquisitely sensitive to environmental conditions. Using molecular dynamics simulations combined with data from previously published solid-state NMR experiments, we propose the first atomically detailed structures of two asymmetric polymorphs of the Aβ9-40 peptide fibril. The first corresponds to synthetic fibrils grown under quiescent conditions and the second to fibrils derived from AD patients' brain-extracts. Our core structure in both fibril structures consists of a layered structure in which three cross-β subunits are arranged in six tightly stacked β-sheet layers with an antiparallel hydrophobic-hydrophobic and an antiparallel polar-polar interface. The synthetic and brain-derived structures differ primarily in the side-chain orientation of one β-strand. The presence of a large and continually exposed hydrophobic surface (buried in the symmetric agitated Aβ fibrils) may account for the higher toxicity of the asymmetric fibrils. Our model explains the effects of external perturbations on the fibril lateral architecture as well as the fibrillogenesis inhibiting action of amphiphilic molecules

The dynamical evolution of molecular clouds near the Galactic Centre - II. Spatial structure and kinematics of simulated clouds

The evolution of molecular clouds in galactic centres is thought to differ from that in galactic discs due to a significant influence of the external gravitational potential. We present a set of numerical simulations of molecular clouds orbiting on the 100-pc stream of the Central Molecular Zone (the central $\sim500$ pc of the Galaxy) and characterise their morphological and kinematic evolution in response to the background potential and eccentric orbital motion. We find that the clouds are shaped by strong shear and torques, by tidal and geometric deformation, and by their passage through the orbital pericentre. Within our simulations, these mechanisms control cloud sizes, aspect ratios, position angles, filamentary structure, column densities, velocity dispersions, line-of-sight velocity gradients, spin angular momenta, and kinematic complexity. By comparing these predictions to observations of clouds on the Galactic Centre 'dust ridge', we find that our simulations naturally reproduce a broad range of key observed morphological and kinematic features, which can be explained in terms of well-understood physical mechanisms. We argue that the accretion of gas clouds onto the central regions of galaxies, where the rotation curve turns over and the tidal field is fully compressive, is accompanied by transformative dynamical changes to the clouds, leading to collapse and star formation. This can generate an evolutionary progression of cloud collapse with a common starting point, which either marks the time of accretion onto the tidally-compressive region or of the most recent pericentre passage. Together, these processes may naturally produce the synchronised starbursts observed in numerous (extra)galactic nuclei

Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes

Background: Protein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-b peptide (QDAb) and its advanced applications. Methodology/Principal Findings: The QDAb construct retained Ab oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAb coaggregation with intact Ab42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Ab antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAb more significantly than oligomeric QDAb, and the ingested QDAb was mainly accumulated in the lysosome. Conclusions/Significance: These data demonstrate that QDAb is a novel nanoprobe for studying Ab oligomerization an

Kinematics of Galactic Centre clouds shaped by shear-seeded solenoidal turbulence

The Central Molecular Zone (CMZ; the central ∼500 pc of the Galaxy) is a kinematically unusual environment relative to the Galactic disc, with high-velocity dispersions and a steep size–linewidth relation of the molecular clouds. In addition, the CMZ region has a significantly lower star formation rate (SFR) than expected by its large amount of dense gas. An important factor in explaining the low SFR is the turbulent state of the star-forming gas, which seems to be dominated by rotational modes. However, the turbulence driving mechanism remains unclear. In this work, we investigate how the Galactic gravitational potential affects the turbulence in CMZ clouds. We focus on the CMZ cloud G0.253+0.016 (‘the Brick’), which is very quiescent and unlikely to be kinematically dominated by stellar feedback. We demonstrate that several kinematic properties of the Brick arise naturally in a cloud-scale hydrodynamics simulation, that takes into account the Galactic gravitational potential. These properties include the line-of-sight velocity distribution, the steepened size–linewidth relation, and the predominantly solenoidal nature of the turbulence. Within the simulation, these properties result from the Galactic shear in combination with the cloud’s gravitational collapse. This is a strong indication that the Galactic gravitational potential plays a crucial role in shaping the CMZ gas kinematics, and is a major contributor to suppressing the SFR, by inducing predominantly solenoidal turbulent modes

Thermodynamic Selection of Steric Zipper Patterns in the Amyloid Cross-β Spine

At the core of amyloid fibrils is the cross-β spine, a long tape of β-sheets formed by the constituent proteins. Recent high-resolution x-ray studies show that the unit of this filamentous structure is a β-sheet bilayer with side chains within the bilayer forming a tightly interdigitating “steric zipper” interface. However, for a given peptide, different bilayer patterns are possible, and no quantitative explanation exists regarding which pattern is selected or under what condition there can be more than one pattern observed, exhibiting molecular polymorphism. We address the structural selection mechanism by performing molecular dynamics simulations to calculate the free energy of incorporating a peptide monomer into a β-sheet bilayer. We test filaments formed by several types of peptides including GNNQQNY, NNQQ, VEALYL, KLVFFAE and STVIIE, and find that the patterns with the lowest binding free energy correspond to available atomistic structures with high accuracy. Molecular polymorphism, as exhibited by NNQQ, is likely because there are more than one most stable structures whose binding free energies differ by less than the thermal energy. Detailed analysis of individual energy terms reveals that these short peptides are not strained nor do they lose much conformational entropy upon incorporating into a β-sheet bilayer. The selection of a bilayer pattern is determined mainly by the van der Waals and hydrophobic forces as a quantitative measure of shape complementarity among side chains between the β-sheets. The requirement for self-complementary steric zipper formation supports that amyloid fibrils form more easily among similar or same sequences, and it also makes parallel β-sheets generally preferred over anti-parallel ones. But the presence of charged side chains appears to kinetically drive anti-parallel β-sheets to form at early stages of assembly, after which the bilayer formation is likely driven by energetics

Polymorphic Structures of Alzheimer's β-Amyloid Globulomers

Misfolding and self-assembly of Amyloid-β (Aβ) peptides into amyloid fibrils is pathologically linked to the development of Alzheimer's disease. Polymorphic Aβ structures derived from monomers to intermediate oligomers, protofilaments, and mature fibrils have been often observed in solution. Some aggregates are on-pathway species to amyloid fibrils, while the others are off-pathway species that do not evolve into amyloid fibrils. Both on-pathway and off-pathway species could be biologically relevant species. But, the lack of atomic-level structural information for these Aβ species leads to the difficulty in the understanding of their biological roles in amyloid toxicity and amyloid formation.Here, we model a series of molecular structures of Aβ globulomers assembled by monomer and dimer building blocks using our peptide-packing program and explicit-solvent molecular dynamics (MD) simulations. Structural and energetic analysis shows that although Aβ globulomers could adopt different energetically favorable but structurally heterogeneous conformations in a rugged energy landscape, they are still preferentially organized by dynamic dimeric subunits with a hydrophobic core formed by the C-terminal residues independence of initial peptide packing and organization. Such structural organizations offer high structural stability by maximizing peptide-peptide association and optimizing peptide-water solvation. Moreover, curved surface, compact size, and less populated β-structure in Aβ globulomers make them difficult to convert into other high-order Aβ aggregates and fibrils with dominant β-structure, suggesting that they are likely to be off-pathway species to amyloid fibrils. These Aβ globulomers are compatible with experimental data in overall size, subunit organization, and molecular weight from AFM images and H/D amide exchange NMR.Our computationally modeled Aβ globulomers provide useful insights into structure, dynamics, and polymorphic nature of Aβ globulomers which are completely different from Aβ fibrils, suggesting that these globulomers are likely off-pathway species and explaining the independence of the aggregation kinetics between Aβ globulomers and fibrils

Intrinsic Determinants of Aβ12–24 pH-Dependent Self-Assembly Revealed by Combined Computational and Experimental Studies

The propensity of amyloid- (A) peptide to self-assemble into highly ordered amyloid structures lies at the core of their accumulation in the brain during Alzheimer's disease. By using all-atom explicit solvent replica exchange molecular dynamics simulations, we elucidated at the atomic level the intrinsic determinants of the pH-dependent dimerization of the central hydrophobic segment A and related these with the propensity to form amyloid fibrils measured by experimental tools such as atomic force microscopy and fluorescence. The process of A dimerization was evaluated in terms of free energy landscape, side-chain two-dimensional contact probability maps, -sheet registries, potential mean force as a function of inter-chain distances, secondary structure development and radial solvation distributions. We showed that dimerization is a key event in A amyloid formation; it is highly prompted in the order of pH 5.02.98.4 and determines further amyloid growth. The dimerization is governed by a dynamic interplay of hydrophobic, electrostatic and solvation interactions permitting some variability of -sheets at each pH. These results provide atomistic insight into the complex process of molecular recognition detrimental for amyloid growth and pave the way for better understanding of the molecular basis of amyloid diseases

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 Alzheimer's 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