与人类疾病相关的淀粉样短肽

有些天然蛋白质可通过错误折叠形成淀粉样纤维,并进一步沉积导致淀粉样病变,被认为是许多重大人类疾病的病理基础。因此,阐明天然蛋白质错误折叠、聚集形成淀粉样纤维的分子机制,是预防、诊断和治疗相关疾病的关键。研究者们从天然蛋白质中鉴定出许多能够形成淀粉样纤维的关键短肽片段,即淀粉样短肽,对它们形成淀粉样纤维的能力及其在完整蛋白质聚集过程中的决定性作用进行深入研究。本文对近年来人类疾病相关淀粉样短肽的研究展开综述。首先,介绍鉴定淀粉样短肽的标准及其相应的研究方法和技术手段;并回顾近年来与一些重大人类疾病相关的淀粉样短肽,尤其是与神经退行性疾病相关淀粉样短肽的进展情况,对淀粉样短肽中出现频率较高的氨基酸残基及其可能的自组装原理进行总结分析;最后,展望这些淀粉样短肽作为靶点在相关疾病诊断和治疗方面的意义,并初步探讨它们作为新型生物材料在生物医学工程领域的应用前景。本文一方面为阐明天然蛋白质形成淀粉样沉淀的分子机制提供参考,另一方面也为相关疾病的治疗提供思路,同时也为新型生物材料的开发提出潜在的可能性。     

Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self-assembled amyloid fibrils are also involved in physiological processes in several micro-organisms. Yet, the molecular basis for the recognition and self-assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self-assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid-associated disorders.     

Solid-state NMR as a probe of amyloid structure

Solid state nuclear magnetic resonance (NMR) has developed into one of the most informative and direct experimental approaches to the characterization of the molecular structures of amyloid fibrils, including those associated with Alzheimer's disease. In this article, essential aspects of solid state NMR methods are described briefly and results obtained to date regarding the supramolecular organization of amyloid fibrils and the conformations of peptides within amyloid fibrils are reviewed.     

Imaging Amyloid Fibrils within Cells Using a Se-Labelling Strategy

The process of aggregation leading to amyloid formation by peptides and proteins is associated with diseases ranging from systemic amyloidoses to neurodegenerative disorders such as Alzheimer's disease. A key question in understanding the link between amyloid formation and its pathological consequences is the ultrastructural localisation and morphological form of amyloid species within the cellular environment. The acquisition of such information has proven to be challenging, but we report here a novel approach that enables amyloid fibrils to be visualised directly within a cell. First, fibrils are assembled from selenium analogues of the sulfur-containing cysteine peptides, and then, atomic number contrast transmission electron microscopy is used to detect the selenium doped species selectively within the carbon-rich background of the cell. We demonstrate the power of this approach by imaging human monocyte-derived macrophage cells that have been exposed to fibrils from an amyloidogenic fragment of the disease-associated protein transthyretin. The ready incorporation of seleno-cysteine and methionine instead of their natural sulfur-containing analogues, a feature that is already commonly used in X-ray diffraction studies of proteins, suggests that this method can be used as a general strategy to image specific peptides and proteins within the cellular environment using electron microscopy.     

Molecular structure of amyloid fibrils: insights from solid-state NMR

Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the beta-amyloid peptide associated with Alzheimer's disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.     

Replica exchange molecular dynamics simulation of cross‐fibrillation of IAPP and PrP106‐126

Aggregation of proteins into amyloid is the central hallmark of a number of protein diseases. Most studies were carried out on the aggregation between proteins of similar species. However, it was observed that some patients with certain protein disease can easily acquire another unrelated protein disease. As such, it is also important to examine aggregation between proteins of different species. Usually aggregation between proteins of the same species can be attributed to the similarity between their respective amino acid sequences. In this article, we were motivated by an experimental study of aggregation between amylin (Islet Amyloid Polypeptide, IAPP) and prion_106‐126 (PrP106‐126) fragment (JACS, 2013, 135, 13582–9). It was found that the two non‐homologous peptides can aggregate quickly to form fibrils in the presence of negatively charged lipid bilayer. We attempted to elucidate the molecular mechanism of the early stage of dimerization of these two peptides through extensive replica exchange molecular dynamics simulations. Conformations consisting of various degrees of β‐sheets structures, both intra‐chain and inter‐chain, were found in the simulations. The conformations of the aggregated complex are very diverse, which suggests that the cross‐species fibrils formed between the two proteins are highly polymorphic. The driving forces are mainly hydrophobic interactions, including aromatic‐aliphatic interactions. The palindromic region of PrP106‐126 and SNNFGAIL region of IAPP were found to play important roles in the interaction. Our study sheds insight into the exciting research of protein cross‐fibrillation. Proteins 2016; 84:1134–1146. © 2016 Wiley Periodicals, Inc.     

Effect of introducing a short amyloidogenic sequence from the Aβ peptide at the N‐terminus of 18‐residue amphipathic helical peptides

Fibril formation is the hallmark of pathogenesis in Alzheimer's disease and other amyloid disorders caused by conformational alterations leading to the aggregation of soluble monomers. Aβ40 self‐associates to form amyloid fibrils. Its central seven‐residue segment KLVFFAE (Aβ16–22), which is thought to be crucial for fibril formation of the full‐length peptide, forms fibrils even in isolation. Context‐dependent induction of amyloid formation by such sequences in peptides, which otherwise do not have that propensity, is of considerable interest. We have examined the effect of introducing the Aβ16–22 sequence at the N‐terminus of two amphipathic helical 18‐residue peptides Ac‐WYSEMKRNVQRLERAIEE‐am and Ac‐KQLIRFLKRLDRNLWGLA‐am, which have high average hydrophobic moment <μH> values but have net charges of 0 and +4, respectively, at neutral pH. Upon incubation in aqueous buffer, fibril‐like aggregates were discernible by transmission electron microscopy for the peptide with only 0 net charge, which also displayed ThT binding and β‐structure. Although both the sequences have been derived from amphipathic helical segments in globular proteins and possess high average hydrophobic moments, the +4 charge peptide lacks the ability to form fibrils, while the peptide with 0 charge has the tendency to form fibrillar structures. Variation in the net charge and the presence of several glutamic acids in the sequence of the peptide with net charge 0 appear to favor the formation of fibrils when the Aβ16–22 sequence is attached at the N‐terminus. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of amyloid core structure using chemical shifts

Amyloid fibrils are the pathological hallmark of a large variety of neurodegenerative disorders. The structural characterization of amyloid fibrils, however, is challenging due to their non‐crystalline, heterogeneous, and often dynamic nature. Thus, the structure of amyloid fibrils of many proteins is still unknown. We here show that the structure calculation program CS‐Rosetta can be used to obtain insight into the core structure of amyloid fibrils. Driven by experimental solid‐state NMR chemical shifts and taking into account the polymeric nature of fibrils CS‐Rosetta allows modeling of the core of amyloid fibrils. Application to the Y145X stop mutant of the human prion protein reveals a left‐handed β‐helix     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

Progress towards a molecular-level structural understanding of amyloid fibrils

The problem of determining and understanding the molecular structures of amyloid fibrils has attracted considerable attention and effort over the past several years. Although complete, high-resolution structures have not yet been obtained, key features of protein and peptide conformations and supramolecular organization within amyloid fibrils have been elucidated using a variety of novel experimental methods, including magnetic resonance spectroscopies, electron microscopy, X-ray and neutron scattering, and biochemical techniques. The experimental data are beginning to shed light on issues such as the nature of the intermolecular interactions that stabilize amyloid structures, the molecular structural basis for polymorphism in amyloid fibrils, the universality of amyloid structures, and the balance between structural order and disorder within amyloid fibrils. Recent structural data will contribute to an improved understanding of the mechanisms of amyloid formation and to the development of therapeutic agents for amyloid diseases.     

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

A partially structured region of a largely unstructured protein, Plasmodium falciparum merozoite surface protein 2 (MSP2), forms amyloid-like fibrils.

Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. It has been implicated in the process of erythrocyte invasion and is a leading vaccine candidate. MSP2 is an intrinsically unstructured protein (IUP), and recombinant MSP2 forms amyloid-like fibrils upon storage. We have examined synthetic peptides corresponding to sequences in the conserved N-terminal region of MSP2 for the presence of local structure and the ability to form fibrils related to those formed by full-length MSP2. In a 25-residue peptide corresponding to the entire N-terminal region of mature MSP2, structures calculated from NMR data show the presence of nascent helical and turn-like structures. An 8-residue peptide from the central region of the N-terminal domain (residues 8-15) also formed a turn-like structure. Both peptides formed fibrils that were similar but not identical to the amyloid-like fibrils formed by full-length MSP2. Notably, the fibrils formed by the peptides bound both Congo Red and Thioflavin T, whereas the fibrils formed by full-length MSP2 bound only Congo Red. The propensity of peptides from the N-terminal conserved region of MSP2 to form amyloid-like fibrils makes it likely that this region contributes to fibril formation by the full-length protein. Thus, in contrast to the more common pathway of amyloid formation by structured proteins, which proceeds via partially unfolded intermediates that then undergo beta-aggregation, MSP2 is an example of a largely unstructured protein with at least one small structured region that has an important role in fibril formation.     

Dichotomous versus palm-type mechanisms of lateral assembly of amyloid fibrils

Despite possessing a common cross-beta core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90-230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.     

Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides

Amyloid fibrils in which specific proteins have polymerized into a cross-beta-sheet structure are found in about 20 diseases. In contrast to the close structural similarity of fibrils formed in different amyloid diseases, the structures of the corresponding native proteins differ widely. We show here that peptides as short as 4 residues with the sequences KFFE or KVVE can form amyloid fibrils that are practically identical to fibrils formed in association with disease, as judged by electron microscopy and Congo red staining. In contrast, KLLE or KAAE do not form fibrils. The fibril-forming KFFE and KVVE show partial beta-strand conformation in solution, whereas the non-fibril-forming KLLE and KAAE show random structure only, suggesting that inherent propensity for beta-strand conformation promotes fibril formation. The peptides KFFK or EFFE do not form fibrils on their own but do so in an equimolar mixture. Thus, intermolecular electrostatic interactions, either between charged dipolar peptides or between complementary charges of co-fibrillating peptides favor fibril formation.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Amyloid character of self-assembling proteins based on adenovirus fiber shaft sequences

Fibrous proteins found in natural materials such as silk fibroins, spider silks, and viral spikes increasingly serve as a source of inspiration for the design of novel, artificial fibrous materials. The fiber protein from the adenovirus has previously served as a model for the design of artificial, self-assembling fibers. The fibrous shaft of this protein consists of 15-amino-acid sequence repeats that fold into a triple β-spiral motif in their native context. Recombinant proteins based on multimers of simplified consensus shaft repeats were previously reported to form self-assembling fibrils from which filaments could be spun. Here, we describe the structural characterization of these fibrils; X-ray fiber diffraction, Raman spectroscopy, and Congo Red binding strongly suggest an amyloid-type structure for these fibrils, with β-strands arranged perpendicular to the fibril axis. This amyloid structure is distinct from the native β-spiral fold, and similar to amyloid structures formed by short, synthetic peptides corresponding to shaft sequences. We discuss implications for the rational design of novel fibrous materials, based on crystal structure information and knowledge of folding and assembly pathways of natural fibrous proteins.     

Interactions between amyloidophilic dyes and their relevance to studies of amyloid inhibitors

Amyloid fibrils are filamentous aggregates of peptides and proteins implicated in a range of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. It has been known almost since their discovery that these β-sheet-rich proteinacious assemblies bind a range of specific dyes that, combined with other biophysical techniques, are convenient probes of the process of amyloid fibril formation. Two prominent examples of such dyes are Congo red (CR) and Thioflavin T (ThT). It has been reported that in addition to having a diagnostic role, CR is an inhibitor of the formation of amyloid structures, and these two properties have both been explained in terms of the same specific noncovalent interactions between the fibrils and the dye molecules. In this article, we show by means of quartz-crystal microbalance measurements that the binding of both ThT and CR to amyloid fibrils formed by the peptide whose aggregation is associated with Alzheimer's disease, Aβ(1-42), can be directly observed, and that the presence of CR interferes with the binding of ThT. Light scattering and fluorescence measurements confirm that an interaction exists between these dyes that can interfere with their ability to reflect accurately the quantity of amyloid material present in a given sample. Furthermore, we show that CR does not inhibit the process of amyloid fibril elongation, and therefore demonstrate the ability of the quartz-crystal microbalance method not only to detect and study the binding of small molecules to amyloid fibrils, but also to elucidate the mode of action of potential inhibitors.     

Tracking of nanoscale structural variations on a single amyloid fibril with tip‐enhanced Raman scattering

Amyloid fibrils are known to be responsible for diseases such as Alzheimer's disease. A detailed insight into the structure of amyloid fibrils is fundamental since it is not yet understood what triggers the misfolding of proteins to the fiber like structures. The molecular structure of fibril surfaces on a single amino acid level has not been revealed so far but would present a valuable contribution to this question. Here we demonstrate the direct molecular distinction of selected amino acids on insulin fibril surfaces with a lateral resolution better than 2 nm by applying tip‐enhanced Raman spectroscopy (TERS). This approach provides simultaneously a way to directly reveal conformational changes in the secondary structure, namely α‐helix, β‐sheet, on the fibril surface with nanometer resolution. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Inhibition of α-synuclein fibril assembly by small molecules: Analysis using epitope-specific antibodies

The conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases. Existing treatments are at best symptomatic. Accordingly, small molecule inhibitors of amyloid fibril formation and their mechanisms are of great interest. Here we report that the conformational changes undergone by α -synuclein as it assembles into amyloid fibrils can be detected by epitope-specific antibodies. We show that the conformations of polyphenol-bound α-synuclein monomers and dimers differ from those of unbound monomers and resemble amyloid fibrils. This strongly suggests that small molecule inhibitors bind and stabilize intermediates of amyloid fibril formation, consistent with the view that inhibitor-bound molecular species are on-pathway intermediates.