Mechanism of amyloid β-protein aggregation mediated by GM1 ganglioside clusters

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ～0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ～0.013 and ～0.044), the helical species and aggregated β-sheets (～15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ～0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.     

Nucleation of β-rich oligomers and β-barrels in the early aggregation of human islet amyloid polypeptide

The self-assembly of human islet amyloid polypeptide (hIAPP) into β-sheet rich amyloid aggregates is associated with pancreatic β-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into β-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than β-sheets, consistent with ensemble-based experimental measurements. However, in ~4–6% independent simulations, β-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These β-rich oligomers could adopt β-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these β-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. β-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.     

Molecular dynamics simulation of the nanofibrils formed by amyloid-based peptide amphiphiles

Abstract

Atomistic molecular dynamics simulations have been performed on the peptide amphiphiles (PAs) with four amyloid beta peptide fragments as head groups. The stable structures were monitored by the root mean square deviation with respect to the energy minimised initial structures. Random coil and β-sheet structures with hydrogen bonds along and perpendicular to the long axis of the nanofibre were obtained due to the different nature of the head groups. Influences of pH and capping ends on the nanofibre structures were investigated through variation of the protonation states of the ionic amino acids in the peptides. The peptides with opposite charges on both sides were found to have the fewest β-sheet structures, and the charges on the outer terminal tended to destruct the β-sheets while those at the inner side did not. The isolated charge in the centre of peptides was found to be able to promote the formation of regular β-sheets, while multiple charged residues could not support ordered β-sheet structures. When charge neutralisation occurred between adjacent residues, regular β-sheet laminates might also occur for systems with charges at the outer terminal. With the increase of β-sheet structures formed, the original twisted structures found for random coil structures of the PAs could be diminished by the hydrogen bonds.     

Structural polymorphism of human islet amyloid polypeptide (hIAPP) oligomers highlights the importance of interfacial residue interactions

A 37-residue of human islet amyloid polypeptide (hIAPP or amylin) is a main component of amyloid plaques found in the pancreas of ～90% of type II diabetes patients. It is reported that hIAPP oligomers, rather than mature fibrils, are major toxic species responsible for pancreatic islet β-cell dysfunction and even cell death, but molecular structures of these oligomers remain elusive. In this work, on the basis of recent solid-state NMR and mass-per-length (MPL) data, we model a series of hIAPP oligomers with different β-layers (one, two, and three layers), symmetries (symmetry and asymmetry), and associated interfaces using molecular dynamics simulations. Three distinct interfaces formed by C-terminal β-sheet and C-terminal β-sheet (CC), N-terminal β-sheet and N-terminal β-sheet (NN), and C-terminal β-sheet and N-terminal β-sheet (CN) are identified to drive multiple cross-β-layers laterally associated together to form different amyloid organizations via different intermolecular interactions, in which the CC interface is dominated by polar interactions, the NN interface is dominated by hydrophobic interactions, and the CN interface is dominated by mixed polar and hydrophobic interactions. Overall, the structural stability of the proposed hIAPP oligomers is a result of delicate balance between maximization of favorable peptide-peptide interactions at the interfaces and optimization of solvation energy with globular structure. Different hIAPP oligomeric models indicate a general and intrinsic nature of amyloid polymorphism, driven by different interfacial side-chain interactions. The proposed models are compatible with recent experimental data in overall size, cross-section area, and molecular weight. A general hIAPP aggregation mechanism is proposed on the basis of our simulated models and experimental data.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Aggregation of amphipathic peptides at an aqueous–organic interface using coarse-grained simulations

The effect of an aqueous/organic interface on the folding and aggregation of amphipathic peptides is examined by applying discontinuous molecular dynamics (DMD) simulations combined with an intermediate resolution protein model, PRIME20, to a peptide/interface system. The systems contain 48 (KLLK)₄ peptides in random coil or α-helical conformations interacting with both strong and weak interfaces. In the absence of an interface, most of the oligomers form helical bundles, a small fraction of which convert to β-sheets when the temperature is above the folding transition. Adding a weak interface decreases oligomer formation above the folding temperature and increases it below. Little monolayer formation is observed at the weak interface; instead reversible adsorption increases the local peptide concentration near the interface, promoting helical bundle formation in the aqueous phase below the folding temperature and β-sheet formation above the folding temperature. Introducing a strong interface leads to irreversible adsorption, promoting formation of helical monolayers below the folding temperature and mixed β-sheet/amorphous monolayers above the folding temperature. The (KLLK)₄ peptide is more likely to adsorb to the interface when it is in an α-helical conformation, as opposed to a random coil, because of its larger hydrophobic moment.     

Structural studies of spider silk proteins in the fiber

Although spider silk has been studied for a number of years the structures of the proteins involved have yet to be definitely determined. X-ray diffraction and solid-state nuclear magnetic resonance (NMR) were used to study major ampullate (dragline) silk from Nephila clavipes. The silk was studied in its natural state, in the supercontacted state and in the restretched state following supercontraction. The natural silk structure is dominated by β-sheets aligned parallel to the fiber axis. Supercontraction is characterized by randomizing of the orientation of the β-sheet. When the fiber is restretched alignment is regained. However, the same reorientation was observed for wetting of minor ampullate silk which does not supercontract. Thus, the reorientation of β-sheets alone cannot explain the supercontraction in dragline silk. Cocoon silk showed very little β-sheet orientation in the natural state and there were no changes upon wetting. NMR and X-ray diffraction data are consistent with the β-sheets arising from the poly-alanine sequences known to be present in the proteins of major ampullate silk as has been proposed previously. © 1997 John Wiley & Sons, Ltd.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Role of cholesterol in functional association between K(+)-Cl(-) cotransporter-3a and Na+,K(+)-ATPase

The conformation of amyloid-beta peptide (Aβ) determines if toxic aggregates are formed. The peptide structure by its turn depends on the environment and molecule-molecule interactions. We characterized the secondary structure of Aβ-(1-40) in surfactant solutions and interacting with monolayers. The peptide adopts β-sheet structure in solutions of ionic surfactants at sub-micelle concentrations and α-helix in the presence of ionic micelles. Uncharged micelles induce β-sheets. Aβ-(1-40) alters the critical micelle concentration value of the non-ionic surfactant, underlining hydrophobic interactions. At ionic monolayers the peptide forms β-sheets when its concentration at the surface is high enough. These results suggest that only electrostatic interactions of charged micelles that surround completely the peptide are able to induce non-aggregated α-helix structure.     

Solvent-induced tuning of internal structure in a protein amyloid protofibril

An important goal in studies of protein aggregation is to obtain an understanding of the structural diversity that is characteristic of amyloid fibril and protofibril structures at the molecular level. In this study, what to our knowledge are novel assays based on time-resolved fluorescence anisotropy decay and dynamic quenching measurements of a fluorophore placed at different specific locations in the primary structure of a small protein, barstar, have been used to determine the extent to which the protein sequence participates in the structural core of protofibrils. The fluorescence measurements reveal the structural basis of how modulating solvent polarity results in the tuning of the protofibril conformation from a pair of parallel β-sheets in heat-induced protofibrils to a single parallel β-sheet in trifluorethanol-induced protofibrils. In trifluorethanol-induced protofibrils, the single β-sheet is shown to be built up from in-register β-strands formed by nearly the entire protein sequence, while in heat-induced protofibrils, the pair of β-sheets motif is built up from β-strands formed by only the last two-third of the protein sequence.     

The Role of Prefibrillar Structures in the Assembly of a Peptide Amyloid

The self-assembly of proteins into stable, fibrillar aggregates is a general property of polypeptides most notably associated with degenerative diseases termed amyloidoses. These nano- to micrometer scale structures are formed predominantly of β-sheets that self-assemble by a nucleation-dependent mechanism. The rate-limiting step of assembly involves stabilization of high-energy intermediates in a kinetic step termed nucleation. Determination of the structural characteristics of these high-energy intermediates has been elusive, as its members are the least populated states on the assembly pathway. Using a peptide derived from diabetes-related amyloid, we use electron paramagnetic resonance (EPR) spectroscopy and disulfide crosslinking to show that fibers are composed of parallel, in-register β-sheets. Kinetic studies are then used to infer the structural elements of the pre-nucleation intermediates. Notably, stabilization of this ensemble is shown to depend on the number but not the position of amide side chains within the primary sequence. Additionally, fiber formation is accelerated by constructs that mimic the intra-sheet structure of the fiber. Our data suggest that pre-nucleation intermediates sample intra- β-sheet structure and place bounds on the possible nucleation mechanisms for fiber assembly. Understanding the nucleation of fibrillogenesis is critical so that this process can be prevented in disease and productively controlled by design.     

Fusion activity of HIV gp41 fusion domain is related to its secondary structure and depth of membrane insertion in a cholesterol-dependent fashion

The human immunodeficiency virus (HIV) gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and the activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. The fusion domain formed an α-helix in membranes containing less than 30?mol% cholesterol and formed β-sheet secondary structure in membranes containing ≥30?mol% cholesterol. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical form and its β-sheet form. High cholesterol, which induced β-sheets, promoted fusion; however, acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided that their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion.     

A mechanistic link between oxidative stress and membrane mediated amyloidogenesis revealed by infrared spectroscopy

The fully developed lesion of Alzheimer's disease is a dense plaque composed of fibrillar amyloid β-proteins (Aβ) with a characteristic and well-ordered β-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form β-sheet structure, it is important to understand factors that induced Aβ to adopt this conformation. In this review, we describe the application of polarized attenuated total internal reflection infrared FT-IR spectroscopy for characterizing the conformation, orientation, and rate of accumulation of Aβ on lipid membranes. We also describe the application and yield of linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. Results have shown that membranes promote β-sheet formation under a variety of circumstances that may be significant to the pathogenesis of Alzheimer's disease.     

Solid-state NMR as a method to reveal structure and membrane-interaction of amyloidogenic proteins and peptides

It is important to understand the Amyloid fibril formation in view of numerous medical and biochemical aspects. Structural determination of amyloid fibril has been extensively studied using electron microscopy. Subsequently, solid state NMR spectroscopy has been realized to be the most important means to determine not only microscopic molecular structure but also macroscopic molecular packing. Molecular structure of amyloid fibril was first predicted to be parallel β-sheet structure, and subsequently, was further refined for Aβ(1-40) to be cross β-sheet with double layered in register parallel β-sheet structure by using solid state NMR spectroscopy. On the other hand, anti-parallel β-sheet structure has been reported to short fragments of Aβ-amyloid and other amyloid forming peptides. Kinetic study of amyloid fibril formation has been studied using a variety of methods, and two-step autocatalytic reaction mechanism used to explain fibril formation. Recently, stable intermediates or proto-fibrils have been observed by electron microscope (EM) images. Some of the intermediates have the same microscopic structure as the matured fibril and subsequently change to matured fibrils. Another important study on amyloid fibril formation is determination of the interaction with lipid membranes, since amyloid peptide are cleaved from amyloid precursor proteins in the membrane interface, and it is reported that amyloid lipid interaction is related to the cytotoxicity. Finally it is discussed how amyloid fibril formation can be inhibited. Firstly, properly designed compounds are reported to have inhibition ability of amyloid fibril formation by interacting with amyloid peptide. Secondly, it is revealed that site directed mutation can inhibit amyloid fibril formation. These inhibitors were developed by knowing the fibril structure determined by solid state NMR.     

Conformational entropy limits the transition from nucleation to elongation in amyloid aggregation

The formation of β-sheet-rich amyloid fibrils in Alzheimer’s disease and other neurodegenerative disorders is limited by a slow nucleation event. To understand the initial formation of β-sheets from disordered peptides, we used all-atom simulations to parameterize a lattice model that treats each amino acid as a binary variable with β- and non-β-sheet states. We show that translational and conformational entropy give the nascent β-sheet an anisotropic surface tension that can be used to describe the nucleus with 2D classical nucleation theory. Since translational entropy depends on concentration, the aspect ratio of the critical β-sheet changes with protein concentration. Our model explains the transition from the nucleation phase to elongation as the point where the β-sheet core becomes large enough to overcome the conformational entropy cost to straighten the terminal molecule. At this point the β-strands in the nucleus spontaneously elongate, which results in a larger binding surface to capture new molecules. These results suggest that nucleation is relatively insensitive to sequence differences in coaggregation experiments because the nucleus only involves a small portion of the peptide.     

β-Barrel topology of Alzheimer's β-amyloid ion channels

Emerging evidence supports the ion channel mechanism for Alzheimer's disease pathophysiology wherein small β-amyloid (Aβ) oligomers insert into the cell membrane, forming toxic ion channels and destabilizing the cellular ionic homeostasis. Solid-state NMR-based data of amyloid oligomers in solution indicate that they consist of a double-layered β-sheets where each monomer folds into β-strand-turn-β-strand and the monomers are stacked atop each other. In the membrane, Aβ peptides are proposed to be β-type structures. Experimental structural data available from atomic force microscopy (AFM) imaging of Aβ oligomers in membranes reveal heterogeneous channel morphologies. Previously, we modeled the channels in a non-tilted organization, parallel with the cross-membrane normal. Here, we modeled a β-barrel-like organization. β-Barrels are common in transmembrane toxin pores, typically consisting of a monomeric chain forming a pore, organized in a single-layered β-sheet with antiparallel β-strands and a right-handed twist. Our explicit solvent molecular dynamics simulations of a range of channel sizes and polymorphic turns and comparisons of these with AFM image dimensions support a β-barrel channel organization. Different from the transmembrane β-barrels where the monomers are folded into a circular β-sheet with antiparallel β-strands stabilized by the connecting loops, these Aβ barrels consist of multimeric chains forming double β-sheets with parallel β-strands, where the strands of each monomer are connected by a turn. Although the Aβ barrels adopt the right-handed β-sheet twist, the barrels still break into heterogeneous, loosely attached subunits, in good agreement with AFM images and previous modeling. The subunits appear mobile, allowing unregulated, hence toxic, ion flux.     

Design, synthesis, and biophysical properties of a helical Abeta1-42 analog: Inhibition of fibrillogenesis and cytotoxicity

The aggregation of amyloid β-peptide (Aβ) into β-sheet-rich aggregates is a crucial step in the etiology of Alzheimer’s disease. Helical forms of Aβ have been suggested to be intermediates in the aggregation process of the peptide in aqueous phase, micelles and membranes. A stable helical Aβ analog would be useful to investigate the role of helical intermediates in fibrillization by Aβ. Here we designed a helical analog by simply cross-linking the Cys residues of A30C, G37C-Aβ1-42 with 1,6-bismaleimidohexane. The analog assumed a weak α-helical conformation in model membranes mimicking lipid raft microdomains of neuronal membranes under conditions in which the wild-type Aβ1-42 formed a β-sheet, indicating the cross-linking locally induced a helical conformation. Furthermore, addition of equimolar helical Aβ analog significantly reduced the amyloid formation and cytotoxicity by Aβ1-42. Thus, our helical Aβ1-42 is not only a model peptide to investigate the role of helical intermediates in fibrillization by Aβ, but also an inhibitor of Aβ-induced cytotoxicity.     

Relationship between digestibility and secondary structure of raw and thermally treated legume proteins: a Fourier transform infrared (FT-IR) spectroscopic study

The secondary structure of proteins in legumes, cereals, milk products and chicken meat was studied by diffuse reflectance infrared spectroscopy in the region of the amide I band. Major secondary structure components ( β-sheets, random coil, α-helix, turns), together with the low- and high-frequency side contributions, were resolved and related to the in vitro digestibility behaviour of the different foods. A strong inverse correlation between the relative spectral weights of the β-sheet structures and in vitro protein digestibility values was measured. Structural modifications in legume proteins induced by autoclaving were monitored by the changes in the amide I spectra. The results indicate that the β-sheet structures of raw legume proteins and the intermolecular β-sheet aggregates, arising upon heating, are primary factors in adversely affecting the digestibility.     

Concentration-dependent transitions govern the subcellular localization of islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a peptide hormone cosecreted with insulin by pancreatic β-cells. In type II diabetes, IAPP aggregates in a process that is associated with β-cell dysfunction and loss of β-cell mass. The relationship between IAPP's conformational landscape and its capacity to mediate cell death remains poorly understood. We have addressed these unknowns by comparing the cytotoxic effects of sequence variants with differing α-helical and amyloid propensities. IAPP was previously shown to oligomerize cooperatively on binding to lipid bilayers. Here, comparable transitions are evident in cell culture and are associated with a change in subcellular localization to the mitochondria under toxic conditions. Notably, we find that this toxic gain of function maps to IAPP's capacity to adopt aggregated membrane-bound α-helical, and not β-sheet, states. Our findings suggest that upon α-helical mediated oligomerization, IAPP acquires cell-penetrating peptide (CPP) properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.