Seeded strain‐like transmission of β‐amyloid morphotypes in APP transgenic mice

The polymorphic β‐amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral β‐amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop β‐amyloid depositions that differ in morphology, binding of amyloid conformation‐sensitive dyes, and Aβ40/Aβ42 peptide ratio. To determine the nature of such β‐amyloid morphotypes, β‐amyloid‐containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced β‐amyloid deposition with the morphological, conformational, and Aβ40/Aβ42 ratio characteristics of β‐amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced β‐amyloid deposits with the characteristics of β‐amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced β‐amyloid deposits, although less prominent, and the induced deposits were similar to the β‐amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aβ in APP transgenic mice can be maintained by seeded conversion.     

Flipping the switch: How cysteine oxidation directs tau amyloid conformations

Tau can adopt distinct fibril conformations in different human neurodegenerative diseases, which may invoke distinct pathological mechanisms. In a recent issue, Weismiller et al. showed that intramolecular disulfide links between cys291 and cys322 for a specific tau isoform containing four microtubule-binding repeats direct the formation of a structurally distinct amyloid polymorph. These findings have implications in how oxidative stress can flip switches of tau polymorphism in these diseases.     

A molecular dynamics approach to the structural characterization of amyloid aggregation

A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.     

High pressure promotes circularly shaped insulin amyloid

Amyloids, initially associated with certain degenerative diseases, and recently with the prions and prion-based inheritance in yeasts, are linearly-ordered beta-sheet-rich protein aggregates, presently thought to represent a rather common generic trait of proteins as polymers. Regardless of genetic origins and properties of precursor protein molecules, amyloids share many physicochemical properties, including the linear fibrillar morphology. Here, we show that under high hydrostatic pressure insulin forms amyloids of a unique circular morphology. Despite a degree of size-distribution, the smallest forms of the approximate radius of 340-420 nm are most abundant among the ring-shaped structures. The circular amyloid is accompanied by bent 20-100 nm long fibrils. The pressure-enhancement of a ring-like supramolecular fold suggests an anisotropic distribution of void volumes in regular amyloid fibres. While the ability of high pressure to evoke such drastic perturbations on an amyloidogenic pathway may help tune conformation of amyloid templates (e.g. inducing the PrP(Sc)-type infectivity in amyloids grown in vitro from recombinant PrP), the very finding raises new questions concerning possible consequences for high-pressure food processing.     

Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein

Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity.     

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.     

Truncated forms of the prion protein PrP demonstrate the need for complexity in prion structure

Abstract

Self-propagation of aberrant protein folds is the defining characteristic of prions. Knowing the structural basis of self-propagation is essential to understanding prions and their related diseases. Prion rods are amyloid fibrils, but not all amyloids are prions. Prions have been remarkably intractable to structural studies, so many investigators have preferred to work with peptide fragments, particularly in the case of the mammalian prion protein PrP. We compared the structures of a number of fragments of PrP by X-ray fiber diffraction, and found that although all of the peptides adopted amyloid conformations, only the larger fragments adopted conformations that modeled the complexity of self-propagating prions, and even these fragments did not always adopt the PrP structure. It appears that the relatively complex structure of the prion form of PrP is not accessible to short model peptides, and that self-propagation may be tied to a level of structural complexity unobtainable in simple model systems. The larger fragments of PrP, however, are useful to illustrate the phenomenon of deformed templating (heterogeneous seeding), which has important biological consequences.     

Different amyloid aggregation of smooth muscles titin in vitro

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300–500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8–10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-β-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.     

Thermodynamic analysis of amyloid fibril structures reveals a common framework for stability in amyloid polymorphs

1. Download : Download high-res image (329KB)
2. Download : Download full-size image

     

Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy

Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.     

Interaction of membrane-bound islet amyloid polypeptide with soluble and crystalline insulin

Islet amyloid polypeptide (IAPP, also known as amylin) is the major protein component of pancreatic amyloid fibers in type II diabetes and is normally cosecreted with insulin from the beta-cells of the pancreas. IAPP forms amyloid fibrils rapidly at concentrations well below those found in vivo, yet progression of type II diabetes occurs over many years. Insulin, a known inhibitor of IAPP fibrillogenesis, exists as a dense crystalline or near-crystalline core in the secretory vesicle, while IAPP localizes to the region between the crystal and the secretory vesicle membrane. In vitro, IAPP fibrillogenesis is both accelerated by lipid membranes and inhibited by monomeric insulin. In this work, we investigate insulin-IAPP-lipid interactions in vitro under conditions chosen to approximate native secretory vesicle physiology and the amyloid disease state. The effect of insulin on IAPP fibrillogenesis is investigated using fluorescence spectrometry. Additionally, interactions of IAPP and lipids with crystalline insulin are studied using fluorescence microscopy. We find that, while soluble states of insulin and IAPP do not interact significantly, large assemblies of either insulin (crystals) or IAPP (fibers) can lead to stable IAPP-insulin interactions. The results raise the possibility of multiple physiological interactions between these two beta-cell hormones.     

Silent prions lying in wait: a two-hit model of prion/amyloid formation and infection

Diseases such as type 2 diabetes, Alzheimer's and Parkinson's are associated with the formation of amyloid. The transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease, are believed to result from infectious forms of amyloid proteins termed prions. The ability of amyloid to initiate spontaneously and in the case of prions, to transfer successfully from one host to another, has been hard to fully rationalize. In this paper we use a mathematical model to explore the idea that it might be a combination of the presence of the prion/amyloid form and a change in the state of the host that allows the amyloid/prion to successfully initiate and propagate itself. We raise the intriguing possibility that potentially infectious amyloid may lie dormant in an apparently healthy individual awaiting a change in the state of the host or transmittal to a new more susceptible host. On this basis we make an analogy between prion/amyloid disease development and the two-hit model of cancer progression. We additionally raise the possibility that infectious amyloid strains may be characterized by a size distribution of length or radius.     

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

β-amyloid (Aβ) is the main constituent of senile plaques seen in Alzheimer's disease. Aβ is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases β- and β-secretase. In this study, we examined content and localization of β-secretase-cleaved APP (β-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular β-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. β-sAPP was found to be localized in astrocytes and in axons. We found the β-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal β-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. β-sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered β-sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.     

Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide

The islet amyloid polypeptide (hIAPP) is a 37 amino acid residue polypeptide that was found to accumulate as amyloid fibrils in the pancreas of individuals with type II diabetes. Previous studies identified various fragments of hIAPP that can form amyloid fibrils in vitro (e.g. hIAPP(8-20), hIAPP(23-27), and hIAPP(30-37)). However, no comparative and systematic information was available on the role of these structural domains (or others) in the process of molecular recognition that mediates fibrillization, in the context of the full-length polypeptide. To systematically map and compare potential recognition domains, we studied the ability of hIAPP to interact with an array of 28 membrane-spotted overlapping peptides that span the entire sequence of hIAPP (i.e. hIAPP(1-10), hIAPP(2-11...), hIAPP(28-37)). Our study clearly identified a major domain of molecular recognition within hIAPP, as the polypeptide was found to bind with high affinity to a defined linear group of peptides ranging from hIAPP(7-16) to hIAPP(12-21). The maximal binding of the full-length polypeptide was to the hIAPP(11-20) peptide fragment (with the sequence RLANFLVHSS). In order to define the minimal fragment, within this apparent recognition motif, that is capable of self-association and thus may serve as the core molecular recognition motif, we examined the ability of truncated analogs of the recognition sequence to self-assemble into amyloid fibrils. The shortest active fragments capable of self-assembly were found to be the pentapeptides FLVHS and NFLVH. The apparent role of this motif in the process of hIAPP self-assembly is consistent with the profile of the hIAAP-binding distribution to the peptide array. The identification of such short recognition motifs is extremely useful in the attempts to develop means to block amyloid fibril formation by hIAPP. It is worth mentioning that this is only the second time in which peptides as short as a pentapeptide were shown to form amyloid fibrils (the other pentapeptide is FGAIL).     

Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: Implications for the study of amyloid formation

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin(20-29)) is commonly used as a model system to study this process. Studies of amylin(20-29) and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin(20-29), which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.     

Molecular dynamics simulations of early steps in RNA‐mediated conversion of prions

The rate‐limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis‐folded state in which the protein not only forms cell‐toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA‐mediated mis‐folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.     

Conformation‐specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen

We engineered and employed a chaperone‐like amyloid‐binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross‐reacted with amyloid beta‐peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1‐hIAPP complex cross‐react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation‐specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation‐sensitive and sequence‐independent and can target more than one type of protofibril species.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Misfolding of the amyloid beta-protein: a molecular dynamics study

Amyloid beta-proteins spontaneously aggregate and build plaques in the brains of Alzheimer's disease patients. The polypeptide has been the subject of extensive in vitro and computational research. Still, the pathway to aggregational forms and their exact conformations remain largely unclear. Here we present an extensive molecular dynamics approach simulating the protein in various temperatures, pH conditions, and with different charge states of the N- and C-termini, thus exploring the conformational space of the protein at large. Our results show that the protein is able to sample different conformations, many of which are rich in beta structure content, and all characterized by a rapid loss of helix 1 that converts into a pi-helix, while helix 2 samples random and beta-rich structures. Moreover, a hydrophobic cluster is observed involving Val18, Phe19, Ala21, and Gly25. The results are carefully compared with recent NMR and spectroscopic data, and are in global agreement with the experimental findings.