Amyloid Conformation Research Articles

Familial Alzheimer’s Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloid-β Peptide

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.

NPT088 reduces both amyloid-β and tau pathologies in transgenic mice

IntroductionAlzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-β (Aβ) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein (“NPT088”) consisting of the active fragment of g3p and human-IgG1-Fc. MethodsAged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically. ResultsNPT088-lowered Aβ plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice. DiscussionThese observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aβ and tau, the hallmark pathologies of AD.

Key Points Concerning Amyloid Infectivity and Prion-Like Neuronal Invasion.

Amyloid aggregation has been related to an increasing number of human illnesses, from Alzheimer’s and Parkinson’s diseases (AD/PD) to Creutzfeldt-Jakob disease. Commonly, only prions have been considered as infectious agents with a high capacity of propagation. However, recent publications have shown that many amyloid proteins, including amyloid β-peptide, α-synuclein (α-syn) and tau protein, also propagate in a “prion-like” manner. Meanwhile, no link between propagation of pathological proteins and neurotoxicity has been demonstrated. The extremely low infectivity under natural conditions of most non-prion amyloids is far below the capacity to spread exhibited by prions. Nonetheless, it is important to elucidate the key factors that cause non-prion amyloids to become infectious agents. In recent years, important advances in our understanding of the amyloid processes of amyloid-like proteins and unrelated prions (i.e., yeast and fungal prions) have yielded essential information that can shed light on the prion phenomenon in mammals and humans. As shown in this review, recent evidence suggests that there are key factors that could dramatically modulate the prion capacity of proteins in the amyloid conformation. The concentration of nuclei, the presence of oligomers, and the toxicity, resistance and localization of these aggregates could all be key factors affecting their spread. In short, those factors that favor the high concentration of extracellular nuclei or oligomers, characterized by small size, with a low toxicity could dramatically increase prion propensity; whereas low concentrations of highly toxic intracellular amyloids, with a large size, would effectively prevent infectivity.

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS

The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils.

Ultra rapid in vivo screening for anti-Alzheimer anti-amyloid drugs.

More than 46 million people worldwide suffer from Alzheimer’s disease. A large number of potential treatments have been proposed; among these, the inhibition of the aggregation of amyloid β-peptide (Aβ), considered one of the main culprits in Alzheimer’s disease. Limitations in monitoring the aggregation of Aβ in cells and tissues restrict the screening of anti-amyloid drugs to in vitro studies in most cases. We have developed a simple but powerful method to track Aβ aggregation in vivo in real-time, using bacteria as in vivo amyloid reservoir. We use the specific amyloid dye Thioflavin-S (Th-S) to stain bacterial inclusion bodies (IBs), in this case mainly formed of Aβ in amyloid conformation. Th-S binding to amyloids leads to an increment of fluorescence that can be monitored. The quantification of the Th-S fluorescence along the time allows tracking Aβ aggregation and the effect of potential anti-aggregating agents.

Membrane-Induced Dichotomous Conformation of Amyloid β with the Disordered N-Terminal Segment Followed by the Stable C-Terminal β Structure.

Various neurodegenerative disorders are ascribed to pathogenic molecular processes involving conformational transitions of amyloidogenic proteins into toxic aggregates characterized by their β structures. Accumulating evidence indicates that neuronal cell membranes provide platforms for such conformational transitions of pathogenic proteins as best exemplified by amyloid β (Aβ). Therefore, membrane-bound Aβ species can be promising targets for the development of novel drugs for Alzheimer’s disease. In the present study, solid-state nuclear magnetic resonance spectroscopy has elucidated the membrane-induced conformation of Aβ, in which the disordered N-terminal segment is followed by the stable C-terminal β strand. The data provides an insight into the molecular processes of the conformational transition of Aβ coupled with its assembly into parallel β structures.

Prions are affected by evolution at two levels.

Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals.

Detection of amyloid-β fibrils using the DNA-intercalating dye YOYO-1: Binding mode and fibril formation kinetics

Identification of the chemical and biological properties of amyloid fibrils is important for understanding their roles in human diseases and to clarify the mechanisms that govern their formation. In pursuit of these goals, small molecule fluorescent dyes have received increasing attention as probes of amyloid conformations. In this study, we report on the ability of YOYO-1, a homodimeric derivative of oxazole yellow, to detect fibrils formed by the Alzheimer's disease related Aβ(1–42) peptide. We find that YOYO-1 binds to Aβ(1–42) fibrils with the long axes of its oxazole yellow moieties parallel to the fibril axis, resulting in a 200x emission enhancement; a result that shows that YOYO-1 is a sensitive amyloid probe. Further, YOYO-1 exhibits characteristic absorption shifts upon binding to the Aβ(1–42) fibrils that we attribute to a self-stacking to non-stacking transition in its homodimer configuration; herein we show how this phenomenon can be exploited to estimate the degree of dye binding. Furthermore, we show that YOYO-1 can be used to monitor the kinetics of amyloid formation reactions. Taken together, our results show that YOYO-1 is a sensitive amyloid probe that can operate with both absorption and fluorescence read-outs, and this suggests that this commercially available dye could become a useful complement to thioflavin-T for in vitro amyloid-sensing applications.

Mammalian prion protein (PrP) forms conformationally different amyloid intracellular aggregates in bacteria.

BackgroundAn increasing number of proteins are being shown to assemble into amyloid structures that lead to pathological states. Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious. The structural conversion of the cellular prion protein (PrPC), into its misfolded pathogenic form (PrPSc) is the central event of prion-driven pathologies. The study of the structural properties of intracellular amyloid aggregates in general and of prion-like ones in particular is a challenging task. In this context, the evidence that the inclusion bodies formed by amyloid proteins in bacteria display amyloid-like structural and functional properties make them a privileged system to model intracellular amyloid aggregation.ResultsHere we provide the first demonstration that recombinant murine PrP and its C-terminal domain (90–231) attain amyloid conformations inside bacteria. Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity.ConclusionsOverall, our results suggest that modelling PrP amyloid formation in microbial cell factories might open an avenue for a better understanding of the structural features modulating the pathogenic impact of this intriguing protein.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0361-y) contains supplementary material, which is available to authorized users.

Nucleation: The Birth of a New Protein Phase

Nucleation is a process that initiates phase transitions. Since the classical work of Gibbs on nucleation thermodynamics at the end of the nineteenth century, research into nucleation processes has been spread into a huge variety of scientific fields. To date, nucleation has been studied in disciplines ranging from biophysics to cosmology in systems spanning atomic to planetary scales and beyond (1). Proteins in solution are also known historically to form a variety of states and structures through nucleation (2,3).

Qualitative changes in human γ-secretase underlie familial Alzheimer’s disease

Presenilin (PSEN) pathogenic mutations cause familial Alzheimer's disease (AD [FAD]) in an autosomal-dominant manner. The extent to which the healthy and diseased alleles influence each other to cause neurodegeneration remains unclear. In this study, we assessed γ-secretase activity in brain samples from 15 nondemented subjects, 22 FAD patients harboring nine different mutations in PSEN1, and 11 sporadic AD (SAD) patients. FAD and control brain samples had similar overall γ-secretase activity levels, and therefore, loss of overall (endopeptidase) γ-secretase function cannot be an essential part of the pathogenic mechanism. In contrast, impaired carboxypeptidase-like activity (γ-secretase dysfunction) is a constant feature in all FAD brains. Significantly, we demonstrate that pharmacological activation of the carboxypeptidase-like γ-secretase activity with γ-secretase modulators alleviates the mutant PSEN pathogenic effects. Most SAD cases display normal endo- and carboxypeptidase-like γ-secretase activities. However and interestingly, a few SAD patient samples display γ-secretase dysfunction, suggesting that γ-secretase may play a role in some SAD cases. In conclusion, our study highlights qualitative shifts in amyloid-β (Aβ) profiles as the common denominator in FAD and supports a model in which the healthy allele contributes with normal Aβ products and the diseased allele generates longer aggregation-prone peptides that act as seeds inducing toxic amyloid conformations.

Prion aggregate structure in yeast cells is determined by the Hsp104-Hsp110 disaggregase machinery.

Prions consist of misfolded proteins that have adopted an infectious amyloid conformation. In vivo, prion biogenesis is intimately associated with the protein quality control machinery. Using electron tomography, we probed the effects of the heat shock protein Hsp70 chaperone system on the structure of a model yeast [PSI+] prion in situ. Individual Hsp70 deletions shift the balance between fibril assembly and disassembly, resulting in a variable shell of nonfibrillar, but still immobile, aggregates at the surface of the [PSI+] prion deposits. Both Hsp104 (an Hsp100 disaggregase) and Sse1 (the major yeast form of Hsp110) were localized to this surface shell of [PSI+] deposits in the deletion mutants. Elevation of Hsp104 expression promoted the appearance of this novel, nonfibrillar form of the prion aggregate. Moreover, Sse1 was found to regulate prion fibril length. Our studies reveal a key role for Sse1 (Hsp110), in cooperation with Hsp104, in regulating the length and assembly state of [PSI+] prion fibrils in vivo.

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.

Origin of life. Primordial genetics: Information transfer in a pre-RNA world based on self-replicating beta-sheet amyloid conformers

The question of the origin of life on Earth can largely be reduced to the question of what was the first molecular replicator system that was able to replicate and evolve under the presumably very harsh conditions on the early Earth. It is unlikely that a functional RNA could have existed under such conditions and it is generally assumed that some other kind of information system preceded the RNA world. Here, I present an informational molecular system that is stable, self-replicative, environmentally responsive, and evolvable under conditions characterized by high temperatures, ultraviolet and cosmic radiation. This postulated pregenetic system is based on the amyloid fold, a functionally unique polypeptide fold characterized by a cross beta-sheet structure in which the beta strands are arranged perpendicular to the fiber axis. Beside an extraordinary structural robustness, the amyloid fold possesses a unique ability to transmit information by a three-dimensional templating mechanism. In amyloidogenesis short peptide monomers are added one by one to the growing end of the fiber. From the same monomeric subunits several structural variants of amyloid may be formed. Then, in a self-replicative mode, a specific amyloid conformer can act as a template and confer its spatially encoded information to daughter molecular entities in a repetitive way. In this process, the specific conformational information, the spatially changed organization, is transmitted; the coding element is the steric zipper structure, and recognition occurs by amino acid side chain complementarity. The amyloid information system fulfills several basic requirements of a primordial evolvable replicator system: (i) it is stable under the presumed primitive Earth conditions, (ii) the monomeric building blocks of the informational polymer can be formed from available prebiotic compounds, (iii) the system is self-assembling and self-replicative and (iv) it is adaptive to changes in the environment and evolvable.

Insights into Chronic Wasting Disease and Bovine Spongiform Encephalopathy Species Barriers by Use of Real-Time Conversion.

The propensity for transspecies prion transmission is related to the structural characteristics of the enciphering and new host PrP, although the exact mechanism remains incompletely understood. The effects of variability in prion protein on cross-species prion transmission have been studied with animal bioassays, but the influence of prion protein structure versus that of host cofactors (e.g., cellular constituents, trafficking, and innate immune interactions) remains difficult to dissect. To isolate the effects of protein-protein interactions on transspecies conversion, we used recombinant PrP(C) and real-time quaking-induced conversion (RT-QuIC) and compared chronic wasting disease (CWD) and classical bovine spongiform encephalopathy (cBSE) prions. To assess the impact of transmission to a new species, we studied feline CWD (fCWD) and feline BSE (i.e., feline spongiform encephalopathy [FSE]). We cross-seeded fCWD and FSE into each species' full-length, recombinant PrP(C) and measured the time required for conversion to the amyloid (PrP(Res)) form, which we describe here as the rate of amyloid conversion. These studies revealed the following: (i) CWD and BSE seeded their homologous species' PrP best; (ii) fCWD was a more efficient seed for feline rPrP than for white-tailed deer rPrP; (iii) conversely, FSE more efficiently converted bovine than feline rPrP; (iv) and CWD, fCWD, BSE, and FSE all converted human rPrP, although not as efficiently as homologous sCJD prions. These results suggest that (i) at the level of protein-protein interactions, CWD adapts to a new species more readily than does BSE and (ii) the barrier preventing transmission of CWD to humans may be less robust than estimated. We demonstrate that bovine spongiform encephalopathy prions maintain their transspecies conversion characteristics upon passage to cats but that chronic wasting disease prions adapt to the cat and are distinguishable from the original prion. Additionally, we showed that chronic wasting disease prions are effective at seeding the conversion of normal human prion protein to an amyloid conformation, perhaps the first step in crossing the species barrier.

Distinguishing Loss of Structure from Subunit Dissociation for Protein Complexes with Variable Temperature Ion Mobility Mass Spectrometry

The thermal stability and strength of interactions in proteins are commonly measured using isothermal calorimetry and differential scanning calorimetry providing a measurement that averages over structural transitions that occur as the proteins melt and dissociate. Here, we apply variable temperature ion mobility mass spectrometry (VT-IM-MS) to study the effect of temperature on the stability and structure of four multimeric protein complexes. VT-IM-MS is used here to investigate the change in the conformation of model proteins, namely, transthyretin (TTR), avidin, concanavalin A (conA), and human serum amyloid P component (SAP) at elevated temperatures prior, during, and after dissociation up to 550 K. As the temperature of the buffer gas is increased from 300 to 350 K, a small decrease in the collision cross sections ((DT)CCS(He)) of protein complexes from the values at room temperature is observed, and is associated with complex compaction occurring close to the reported solution T(m). At significantly higher temperatures, each protein complex undergoes an increase in (DT)CCS(He) and in the width of arrival time distributions (ATD), which is attributed to extensive protein unfolding, prior to ejection of a highly charged monomer species. This approach allows us to decouple the distinct gas phase melting temperature (T(m)) from the temperature at which we see subunit dissociation. The thermally induced dissociation (TID) mechanism is observed to initially proceed via the so-called "typical" (CID) dissociation route. Interestingly, data collected at higher analysis temperature suggests that the TID process might be adapting more "atypical" dissociation route.

Structural Studies of Truncated Forms of the Prion Protein PrP

Prions are proteins that adopt self-propagating aberrant folds. The self-propagating properties of prions are a direct consequence of their distinct structures, making the understanding of these structures and their biophysical interactions fundamental to understanding prions and their related diseases. The insolubility and inherent disorder of prions have made their structures difficult to study, particularly in the case of the infectious form of the mammalian prion protein PrP. Many investigators have therefore preferred to work with peptide fragments of PrP, suggesting that these peptides might serve as structural and functional models for biologically active prions. We have used x-ray fiber diffraction to compare a series of different-sized fragments of PrP, to determine the structural commonalities among the fragments and the biologically active, self-propagating prions. Although all of the peptides studied adopted amyloid conformations, only the larger fragments demonstrated a degree of structural complexity approaching that of PrP. Even these larger fragments did not adopt the prion structure itself with detailed fidelity, and in some cases their structures were radically different from that of pathogenic PrPSc.

Could α-synuclein amyloid-like aggregates trigger a prionic neuronal invasion?

Parkinson's disease (PD), a progressive neurodegenerative disease primarily affecting voluntary and controlled movement, is characterized by abnormal accumulations of α-synuclein (α-syn) in intraneuronal Lewy bodies. In the last years, the increased number of evidences from both the in vitro and in vivo studies has shown the ability of α-syn to misfold in amyloid conformations and to spread via neuron-to-neuron transmission, suggesting a prion-like behaviour. However, in contrast to prion protein (PrP), α-syn transmission is far from neuronal invasion. The high neuronal toxicity of both mature fibres and oligomeric species, as well as the intracellular localization of the protein and the difficulty to be secreted, could be key factors impeding the prion ability of α-syn aggregates.

The comparative analysis of various amyloid models

Considered natural and experimental amyloidosis models in the existing theories context and known amyloidogenesis mechanisms. Available clinical and experimental observations indicate that the opinion of a fatal incurable amyloidosis wrong. It is shown that there is a significant amount of experimental easily replicable amyloidosis models, which may be used for practicing the treatment methods of this pathology. We offer an amyloidosis models classification: natural (animal models with generic amyloidosis), cell clones, artificial (infectious, protein, etc.). Based on the analysis of amyloidosis existing models concluded--none of the accepted in the scientific the theories community for amyloid building does not combine or explains all known facts about the amyloidogenesis mechanisms. It is assumed that there is a proteins group, the beta-sheet structure, which are potentially capable of amyloid conformation building. It is assumed that beta-sheets of these proteins have similar amino acid composition. The condition for the amyloid building conformation is getting too much protein in sufficient quantities in an uncharacteristic place where the ionic strength of the tissue fluid is such that it promotes the amyloid building conformation. It is assumed that an unfortunate amount of ionic strength environment amyloid protein is provided by polysaccharides, tubulins proteins and ionized silicon.

Characterization of amyloid-like properties in bacterial intracellular aggregates.

Protein aggregation into amyloid conformations is associated with more than 50 different human disorders. Recent studies demonstrate that the expression in bacteria of amyloid proteins results in the formation of intracellular aggregates structurally related to those underlying human diseases. The ease with which prokaryotic organisms can be genetically and biochemically manipulated makes them useful systems for studying how and why protein aggregates inside the cell, providing a tractable environment to rationally model in vivo amyloid formation. In this chapter we present an overview of the methods used to characterize the kinetic, structural, and functional properties of amyloid-like bacterial intracellular aggregates and how they can be employed to screen for lead compounds that might modulate amyloid deposition.