Abeta protofibrils possess a stable core structure resistant to hydrogen exchange

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease.     

Amyloid in neurodegenerative diseases: friend or foe?

Accumulation of amyloid-like aggregates is a hallmark of numerous neurodegenerative disorders such as Alzheimer's and polyglutamine disease. Yet, whether the amyloid inclusions found in these diseases are toxic or cytoprotective remains unclear. Various studies suggest that the toxic culprit in the amyloid folding pathway is actually a soluble oligomeric species which might interfere with normal cellular function by a multifactorial mechanism including aberrant protein-protein interactions. Molecular chaperones suppress toxicity of amyloidogenic proteins by inhibiting aggregation of non-native disease substrates and targeting them for refolding or degradation. Paradoxically, recent studies also suggest a protective action of chaperones in their promotion of the assembly of large, tightly packed, benign aggregates that sequester toxic protein species.     

Induction,inhibition, and incorporation: Different roles for anionic and zwitterionic lysolipids in the fibrillation of the functional amyloid FapC

Amyloid proteins are widespread in nature both as pathological species involved in several diseases and as functional entities that can provide protection and storage for the organism. Lipids have been found in amyloid deposits from various amyloid diseases and have been shown to strongly affect the formation and structure of both pathological and functional amyloid proteins. Here, we investigate how fibrillation of the functional amyloid FapC from Pseudomonas is affected by two lysolipids, the zwitterionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine and the anionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPG). Small-angle X-ray scattering, circular dichroism, dynamic light scattering, and thioflavin T fluorescence measurements were performed simultaneously on the same sample to ensure reproducibility and allow a multimethod integrated analysis. We found that LPG strongly induces fibrillation around its critical micelle concentration (cmc) by promoting formation of large structures, which mature via accumulation of intermediate fibril structures with a large cross section. At concentrations above its cmc, LPG strongly inhibits fibrillation by locking FapC in a core–shell complex. In contrast, lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine induces fibrillation at concentrations above its cmc, not via strong interactions with FapC but by being incorporated during fibrillation and likely stabilizing the fibrillation nucleus to reduce the lag phase. Finally, we show that LPG is not incorporated into the fibril during assembly but rather can coat the final fibril. We conclude that lipids affect both the mechanism and outcome of fibrillation of functional amyloid, highlighting a role for lipid concentration and composition in the onset and mechanism of fibrillation in vivo.     

Structure-activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross-β-sheet structure are the pathological hallmarks of several diseases including Alzheimer’s disease, but are also associated with functional states such as the fungal HET-s prion. This review aims to summarize the recent high-resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross-β-sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity.     

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly

The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of “functional amyloids” expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.     

Amyloidogenic sequences in native protein structures

Numerous short peptides have been shown to form β‐sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 proteins containing 45 sequences known to form amyloid, to see how the proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen‐bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form β structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a β structure. Secondly, amyloidogenic sequences that are found in β structure are usually buried within the protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to protein aggregation via assembly of the amyloidogenic regions. The use of α‐helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for β structure, is thus a widespread mechanism to avoid protein aggregation.     

Functional amyloid formation within mammalian tissue

Amyloid is a generally insoluble, fibrous cross-β sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin—a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.     

Study of Exosomes Shed New Light on Physiology of Amyloidogenesis

Accumulation of toxic amyloid oligomers, a key feature in the pathogenesis of amyloid-related diseases, results from an imbalance between generation and clearance of amyloidogenic proteins. Cell biology has brought to light the key roles of multivesicular endosomes (MVEs) and their intraluminal vesicles (ILVs), which can be secreted as exosomes, in amyloid generation and clearance. To better understand these roles, we have investigated a relevant physiological model of amyloid formation in pigment cells. These cells have tuned their endosomes to optimize the formation of functional amyloid fibrils from the premelanosome protein (PMEL) and to avoid potential accumulation of toxic species. The functional amyloids derived from PMEL reveal striking analogies with the generation of Aβ peptides. We have recently strengthened these analogies using extracellular vesicles as reporters of the endosomal processes that regulate PMEL melanogenesis. We have shown that in pigmented cells, apolipoprotein E (ApoE) is associated with ILVs and exosomes, and regulates the formation of PMEL amyloid fibrils in endosomes. This process secures the generation of amyloid fibrils by exploiting ILVs as amyloid-nucleating platforms. This physiological model of amyloidogenesis could shed new light on the roles of MVEs and exosomes in conditions with pathological amyloid metabolism, such as Alzheimer’s disease.     

Functional Amyloids: Where Supramolecular Amyloid Assembly Controls Biological Activity or Generates New Functionality

Functional amyloids are a rapidly expanding class of fibrillar protein structures, with a core cross-β scaffold, where novel and advantageous biological function is generated by the assembly of the amyloid. The growing number of amyloid structures determined at high resolution reveal how this supramolecular template both accommodates a wide variety of amino acid sequences and also imposes selectivity on the assembly process. The amyloid fibril can no longer be considered a generic aggregate, even when associated with disease and loss of function. In functional amyloids the polymeric β-sheet rich structure provides multiple different examples of unique control mechanisms and structures that are finely tuned to deliver assembly or disassembly in response to physiological or environmental cues. Here we review the range of mechanisms at play in natural, functional amyloids, where tight control of amyloidogenicity is achieved by environmental triggers of conformational change, proteolytic generation of amyloidogenic fragments, or heteromeric seeding and amyloid fibril stability. In the amyloid fibril form, activity can be regulated by pH, ligand binding and higher order protofilament or fibril architectures that impact the arrangement of associated domains and amyloid stability. The growing understanding of the molecular basis for the control of structure and functionality delivered by natural amyloids in nearly all life forms should inform the development of therapies for amyloid-associated diseases and guide the design of innovative biomaterials.     

Curli biogenesis: Order out of disorder

Many bacteria assemble extracellular amyloid fibers on their cell surface. Secretion of proteins across membranes and the assembly of complex macromolecular structures must be highly coordinated to avoid the accumulation of potentially toxic intracellular protein aggregates. Extracellular amyloid fiber assembly poses an even greater threat to cellular health due to the highly aggregative nature of amyloids and the inherent toxicity of amyloid assembly intermediates. Therefore, temporal and spatial control of amyloid protein secretion is paramount. The biogenesis and assembly of the extracellular bacterial amyloid curli is an ideal system for studying how bacteria cope with the many challenges of controlled and ordered amyloid assembly. Here, we review the recent progress in the curli field that has made curli biogenesis one of the best-understood functional amyloid assembly pathways. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.     

Nonpathological extracellular amyloid is present during normal epididymal sperm maturation

Amyloids are aggregated proteins characterized by a specific cross-β-sheet structure and are typically associated with neurodegenerative diseases including Alzheimer's disease. Recently, however, several nonpathological amyloids have been found in intracellular organelles of normal mammalian tissues suggesting that amyloid may also carry out biological functions. We previously have shown that the epididymal cystatin CRES (cystatin-related epididymal spermatogenic), cst8, a reproductive-specific member of the cystatin superfamily of cysteine protease inhibitors, forms amyloid in vitro suggesting that CRES amyloid may also form in vivo within the epididymal lumen. Here we show that amyloid structures containing CRES are a component of the normal mouse epididymal lumen without any apparent cytotoxic effects on spermatozoa and that these structures change along the length of the tubule. These studies suggest the presence of a functional amyloid structure that may carry out roles in sperm maturation or maintenance of the luminal milieu and which itself may undergo maturational changes along the epididymis. In contrast to previous examples of functional amyloid which were intracellular, our studies now show that nonpathological/functional amyloid can also be extracellular. The presence of an extracellular and nonpathological amyloid in the epididymis suggests that similar amyloid structures may be present in other organ systems and may carry out distinctive tissue-specific functions.     

Consensus features in amyloid fibrils: sheet-sheet recognition via a (polar or nonpolar) zipper structure

Amyloid fibrils characterized as highly intractable thread-like species are associated with many neurodegenerative diseases. Although neither the mechanism of amyloid formation nor the origin of amyloid toxicity is currently completely understood, the detailed three-dimensional atomic structures of the yeast protein Sup35 and Abeta amyloid protein determined by recent experiments provide the first and important step towards the comprehension of the pathogenesis and aggregation mechanisms of amyloid diseases. By analyzing these two amyloid peptides which have available crystal structures and other amyloid sequences with proposed structures using computational simulations, we delineate three common features in amyloid organizations and amyloid structures. These could contribute to an improved understanding of the molecular mechanism of amyloid formation, the nature of the aggregation driving forces that stabilize these structures and the development of potential therapeutic agents against amyloid diseases.     

Diversity, biogenesis and function of microbial amyloids

Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimer's, Parkinson's and Huntington's diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.     

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.     

Role of ganglioside metabolism in the pathogenesis of Alzheimer's disease--a review

Gangliosides are expressed in the outer leaflet of the plasma membrane of the cells of all vertebrates and are particularly abundant in the nervous system. Ganglioside metabolism is closely associated with the pathology of Alzheimer's disease (AD). AD, the most common form of dementia, is a progressive degenerative disease of the brain characterized clinically by progressive loss of memory and cognitive function and eventually death. Neuropathologically, AD is characterized by amyloid deposits or "senile plaques," which consist mainly of aggregated variants of amyloid beta-protein (Abeta). Abeta undergoes a conformational transition from random coil to ordered structure rich in beta-sheets, especially after addition of lipid vesicles containing GM1 ganglioside. In AD brain, a complex of GM1 and Abeta, termed "GAbeta," has been found to accumulate. In recent years, Abeta and GM1 have been identified in microdomains or lipid rafts. The functional roles of these microdomains in cellular processes are now beginning to unfold. Several articles also have documented the involvement of these microdomains in the pathogenesis of certain neurodegenerative diseases, such as AD. A pivotal neuroprotective role of gangliosides has been reported in in vivo and in vitro models of neuronal injury, Parkinsonism, and related diseases. Here we describe the possible involvement of gangliosides in the development of AD and the therapeutic potentials of gangliosides in this disorder.     

Probing the pressure-temperature stability of amyloid fibrils provides new insights into their molecular properties

A number of medical disorders, including Alzheimer's disease and type II diabetes, is characterised by the deposition of amyloid fibrils in tissue. The insolubility and size of the fibrils has largely precluded the determination of their structures at high resolution. Studies probing the stability of amyloid fibrils can reveal which non-covalent interactions are important in the formation and maintenance of the fibril structure. In particular, we review here the use of high hydrostatic pressure and high temperature as perturbation techniques. In general, small aggregates formed early in the assembly process can be dissociated by high pressure, but mature amyloid fibrils are highly pressure stable. This finding suggests that a temporal transition occurs during which side chain packing and hydrogen bond formation are optimised, whereas the hydrophobic effect and electrostatic interactions play a dominant role in the early stages of the aggregation. High temperatures, however, can disrupt most aggregates. Though the observed stability of amyloid fibrils is not unique to these structures, the notion that amyloid fibrils can represent the global minimum in free energy is supported by this type of investigations. Some implications regarding the nature of toxic species, associated with at least many of the amyloid disorders, and recently proposed structural models are discussed.     

蛋白质错误折叠相关疾病的多肽药物研究进展

蛋白质和多肽发生错误折叠形成不可溶的淀粉样纤维的过程,与阿尔茨海默病、帕金森病等多种神经退行性疾病密切相关。这些疾病可导致认知能力下降以及运动缺陷等症状。虽然已有多种相关治疗方案处于临床试验中,但目前仍无明确有效的方法可治愈或长期减缓疾病的进展。探寻和研究抑制淀粉样聚集、识别并促进毒性聚集物清除的抑制剂分子是药物研发的重要策略之一。在不同类型的抑制剂中,多肽类抑制剂因具有高特异性、低毒性、多样性,以及修饰后的抗水解稳定性和血脑屏障通透性,有望成为候选药物分子。本文总结了针对阿尔茨海默病相关的Aβ和Tau蛋白以及帕金森病相关的α-synuclein蛋白淀粉样纤维化的多肽抑制剂研究进展。基于淀粉样纤维化核心序列及纤维核心结构进行合理设计,或通过随机筛选,均可获得多肽抑制剂。这些天然和非天然的多肽分子大多具有抑制淀粉样纤维化、解聚成熟纤维和降低细胞毒性的作用,其中一些多肽在退行性疾病动物模型实验中,显示出降低脑损伤和缓解认知及运动障碍的效果。这些研究揭示了多肽作为蛋白质错误折叠和聚集相关疾病药物的特点,为研发一类新的有效药物奠定了基础。     

Generic inhibition of amyloidogenic proteins by two naphthoquinone-tryptophan hybrid molecules

Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs.     

Structures and dynamics of β-barrel oligomer intermediates of amyloid-beta16-22 aggregation

Accumulating evidence suggests that soluble oligomers are more toxic than final fibrils of amyloid aggregations. Among the mixture of inter-converting intermediates with continuous distribution of sizes and secondary structures, oligomers in the β-barrel conformation – a common class of protein folds with a closed β-sheet – have been postulated as the toxic species with well-defined three-dimensional structures to perform pathological functions. A common mechanism for amyloid toxicity, therefore, implies that all amyloid peptides should be able to form β-barrel oligomers as the aggregation intermediates. Here, we applied all-atom discrete molecular dynamics (DMD) simulations to evaluate the formation of β-barrel oligomers and characterize their structures and dynamics in the aggregation of a seven-residue amyloid peptide, corresponding to the amyloid core of amyloid-β with a sequence of ¹⁶KLVFFAE²² (Aβ16-22). We carried out aggregation simulations with various numbers of peptides to study the size dependence of aggregation dynamics and assembly structures. Consistent with previous computational studies, we observed the formation of β-barrel oligomers in all-atom DMD simulations. Using a network-based approach to automatically identify β-barrel conformations, we systematically characterized β-barrels of various sizes. Our simulations revealed the conformational inter-conversion between β-barrels and double-layer β-sheets due to increased structural strains upon forming a closed β-barrel while maximizing backbone hydrogen bonds. The potential of mean force analysis further characterized the free energy barriers between these two states. The obtained structural and dynamic insights of β-barrel oligomers may help better understand the molecular mechanism of oligomer toxicities and design novel therapeutics targeting the toxic β-barrel oligomers. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy