On the Aggregation of Apolipoprotein A-I

In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several diseases—such as atherosclerosis and amyloidosis—where insoluble aggregates containing ApoA-I are deposited in various organs or arteries. The deposited ApoA-I has been found in the form of amyloid fibrils, suggesting that amyloid formation may be involved in the development of these diseases. In the present study we investigated ApoA-I aggregation into amyloid fibrils and other aggregate morphologies. We studied the aggregation of wildtype ApoA-I as well as a disease-associated mutant, ApoA-I K107Δ, under different solution conditions. The aggregation was followed using thioflavin T fluorescence intensity. For selected samples the aggregates formed were characterized in terms of size, secondary structure content, and morphology using circular dichroism spectroscopy, dynamic light scattering, atomic force microscopy and cryo transmission electron microscopy. We find that ApoA-I may form globular protein-only condensates, in which the α-helical conformation of the protein is retained. The protein in its unmodified form appears resistant to amyloid formation; however, the conversion into amyloid fibrils rich in β-sheet is facilitated by oxidation or mutation. In particular, the K107Δ mutant shows higher amyloid formation propensity, and the end state appears to be a co-existence of β-sheet rich amyloid fibrils and α-helix-rich condensates.     

Free Cholesterol Accelerates Aβ Self-Assembly on Membranes at Physiological Concentration

The effects of membranes on the early-stage aggregation of amyloid β (Aβ) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer’s disease. We have shown that direct Aβ-membrane interactions dramatically enhance the Aβ aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aβ monomers and significantly changes the conformational sampling of Aβ monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aβ-lipid interaction is an important factor in the disease prone amyloid assembly process.     

The slowly aggregating salmon Calcitonin: a useful tool for the study of the amyloid oligomers structure and activity

Amyloid proteins of different aminoacidic composition share the tendency to misfold and aggregate in a similar way, following common aggregation steps. The process includes the formation of dimers, trimers, and low molecular weight prefibrillar oligomers, characterized by the typical morphology of globules less than 10 nm diameter. The globules spontaneously form linear or annular structures and, eventually, mature fibers. The rate of this process depends on characteristics intrinsic to the different proteins and to environmental conditions (i.e., pH, ionic strength, solvent composition, temperature). In the case of neurodegenerative diseases, it is now generally agreed that the pathogenic aggregates are not the mature fibrils, but the intermediate, soluble oligomers. However, the molecular mechanism by which these oligomers trigger neuronal damage is still unclear. In particular, it is not clear if there is a peculiar structure at the basis of the neurotoxic effect and how this structure interacts with neurons. This review will focus on the results we obtained using salmon Calcitonin, an amyloid protein characterized by a very slow aggregation rate, which allowed us to closely monitor the aggregation process. We used it as a tool to investigate the characteristics of amyloid oligomers formation and their interactions with neuronal cells. Our results indicate that small globules of about 6 nm could be the responsible for the neurotoxic effects. Moreover, our data suggest that the rich content in lipid rafts of neuronal cell plasma membrane may render neurons particularly vulnerable to the amyloid protein toxic effect.     

Effect of 1-Ethyl-3-methylimidazolium Tetrafluoroborate and Acetate Ionic Liquids on Stability and Amyloid Aggregation of Lysozyme

Amyloid fibrils draw attention as potential novel biomaterials due to their high stability, strength, elasticity or resistance against degradation. Therefore, the controlled and fast fibrillization process is of great interest, which raises the demand for effective tools capable of regulating amyloid fibrillization. Ionic liquids (ILs) were identified as effective modulators of amyloid aggregation. The present work is focused on the study of the effect of 1-ethyl-3-methyl imidazolium-based ILs with kosmotropic anion acetate (EMIM-ac) and chaotropic cation tetrafluoroborate (EMIM-BF₄) on the kinetics of lysozyme amyloid aggregation and morphology of formed fibrils using fluorescence and CD spectroscopy, differential scanning calorimetry, AFM with statistical image analysis and docking calculations. We have found that both ILs decrease the thermal stability of lysozyme and significantly accelerate amyloid fibrillization in a dose-dependent manner at concentrations of 0.5%, 1% and 5% (v/v) in conditions and time-frames when no fibrils are formed in ILs-free solvent. The effect of EMIM-BF₄ is more prominent than EMIM-ac due to the different specific interactions of the anionic part with the protein surface. Although both ILs induced formation of amyloid fibrils with typical needle-like morphology, a higher variability of fibril morphology consisting of a different number of intertwining protofilaments was identified for EMIM-BF_4.     

Molecular Simulations of Amyloid Structures,Toxicity, and Inhibition

The misfolding and aggregation of proteins and peptides into amyloid fibrils are believed to be responsible for the dysfunction and death of neuron cells in many neurodegenerative diseases. Resolving the atomic structures of amyloid peptides at different aggregation stages by molecular simulations has opened new ways to probe the molecular mechanisms of amyloid aggregation, toxicity, and inhibition, as well as to validate computational data with available experimental ones. In this review article, we summarize some recent and important findings on: 1) a number of atomic structures of amyloid oligomers with typical β-sheet-rich conformations, related to amyloid aggregation; 2) different amyloid peptide-induced membrane-disruption mechanisms, related to amyloid toxicity; and 3) rational design of different amyloid inhibitors capable of preventing amyloid aggregation and toxicity, related to amyloid inhibition. All these findings will provide some mechanistic implications for molecular mechanisms of amyloid aggregation, toxicity, and inhibition, which are fundamentally and practically important for the treatment of amyloid diseases.     

Retro‐Enantio N‐Methylated Peptides as β‐Amyloid Aggregation Inhibitors

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of β‐amyloid protein (Aβ). We applied the retro‐enantio concept to design an N‐methylated peptidic inhibitor of the Aβ42 aggregation process. This inhibitor, inrD, as well as the corresponding all‐L (inL) and all‐D (inD) analogues were assayed for inhibition of Aβ42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Aβ42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Aβ42 aggregation more effectively than inL, that inrD decreases Aβ42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro‐enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.     

Membrane disruption and early events in the aggregation of the diabetes related peptide IAPP from a molecular perspective

The aggregation of proteins is tightly controlled in living systems, and misfolded proteins are normally removed before aggregation of the misfolded protein can occur. But for reasons not clearly understood, in some individuals this degradation process breaks down, and misfolded proteins accumulate in insoluble protein aggregates (amyloid deposits) over time. Of the many proteins expressed in humans, a small but growing number have been found to form the long, highly ordered β-sheet protein fibers that comprise amyloid deposits. Despite a lack of obvious sequence similarity, the amyloid forms of diverse proteins are strikingly similar, consisting of long, highly ordered insoluble fibers with a characteristic crossed β-sheet pattern. Amyloidogenesis has been the focus of intense basic and clinical research, because a high proportion of amyloidogenic proteins have been linked to common degenerative diseases, including Alzheimer's disease, type II diabetes, and Parkinson's disease. The apparent link between amyloidogenic proteins and disease was initially attributed to the amyloid form of the protein; however, increasing evidence suggests that the toxicity is due to intermediates generated during the assembly of amyloid fibers. These intermediates have been proposed to attack cells in a variety of ways, such as by generating inflammation, creating reactive oxygen species, and overloading the misfolded protein response pathway. One common, well-studied mechanism is the disruption of the plasma and organelle membranes. In this Account, we examine the early molecular-level events in the aggregation of the islet amyloid polypeptide (IAPP, also called amylin) and its ensuing disruption of membranes. IAPP is a 37-residue peptide secreted in conjunction with insulin; it is highly amyloidogenic and often found in amyloid deposits in type II diabetics. IAPP aggregates are highly toxic to the β-cells that produce insulin, and thus IAPP is believed to be one of the factors involved in the transition from early to later stages of type II diabetes. Using variants of IAPP that are combinations of toxic or non-toxic and amyloidogenic or nonamyloidogenic forms, we have shown that formation of amyloid fibers is a sufficient but not necessary condition for the disruption of β-cells. Instead, the ability to induce membrane disruption in model membranes appears to be related to the peptide's ability to stabilize curvature in the membrane, which in turn is related to the depth of penetration in the membrane. Although many similarities exist between IAPP and other amyloidogenic proteins, one important difference appears to be the role of small oligomers in the assembly process of amyloid fibers. In many amyloidogenic proteins, small oligomers form a distinct metastable intermediate that is frequently the most toxic species; however, in IAPP, small oligomers appear to be transient and are rapidly converted to amyloid fibers. Moreover, the aggregation and toxicity of IAPP is controlled by other cofactors present in the secretory granule from which it is released, such as zinc and insulin, in a control mechanism that is somehow unbalanced in type II diabetics. Investigations into this process are likely to give clues to the mysterious origins of type II diabetes at the molecular level.     

Protein Folding and Aggregation into Amyloid: The Interference by Natural Phenolic Compounds

Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils) and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i) to stabilize toxic amyloid precursors; (ii) to prevent the growth of toxic oligomers or speed that of fibrils; (iii) to inhibit fibril growth and deposition; (iv) to disassemble preformed fibrils; and (v) to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.     

Polymorphism of Alpha-Synuclein Amyloid Fibrils Depends on Ionic Strength and Protein Concentration

Protein aggregate formation is linked with multiple amyloidoses, including Alzheimer‘s and Parkinson‘s diseases. Currently, the understanding of such fibrillar structure formation and propagation is still not sufficient, the outcome of which is a lack of potent, anti-amyloid drugs. The environmental conditions used during in vitro protein aggregation assays play an important role in determining both the aggregation kinetic parameters, as well as resulting fibril structure. In the case of alpha-synuclein, ionic strength has been shown as a crucial factor in its amyloid aggregation. In this work, we examine a large sample size of alpha-synuclein aggregation reactions under thirty different ionic strength and protein concentration combinations and determine the resulting fibril structural variations using their dye-binding properties, secondary structure and morphology. We show that both ionic strength and protein concentration determine the structural variability of alpha-synuclein amyloid fibrils and that sometimes even identical conditions can result in up to four distinct types of aggregates.     

Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer's disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting.     

Comparative Study of the Solid-Liquid Interfacial Adsorption of Proteins in Their Native and Amyloid Forms

The adhesive properties of amyloid fibers are thought to play a crucial role in various negative and positive aggregation processes, the study of which might help in their understanding and control. Amyloids have been prepared from two proteins, lysozyme and β-lactoglobulin, as well as an Exendin-4 derivative miniprotein (E5). Thermal treatment was applied to form amyloids and their structure was verified by thioflavin T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS) dye tests and electronic circular dichroism spectroscopy (ECD). Adsorption properties of the native and amyloid forms of the three proteins were investigated and compared using the mass-sensitive quartz crystal microbalance (QCM) technique. Due to the possible electrostatic and hydrophobic interactions, similar adsorbed amounts were found for the native or amyloid forms, while the structures of the adsorbed layers differed significantly. Native proteins formed smooth and dense adsorption layers. On the contrary, a viscoelastic, highly loose layer was formed in the presence of the amyloid forms, shown by increased motional resistance values determined by the QCM technique and also indicated by atomic force microscopy (AFM) and wettability measurements. The elongated structure and increased hydrophobicity of amyloids might contribute to this kind of aggregation.     

Super‐resolution Imaging of Amyloid Structures over Extended Times by Using Transient Binding of Single Thioflavin T Molecules

Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer's and other amyloid diseases. However, these oligomers are too small to be resolved by standard light microscopy. We have developed a simple and versatile tool to image amyloid structures by using thioflavin T without the need for covalent labeling or immunostaining. The dynamic binding of single dye molecules generates photon bursts that are used for fluorophore localization on a nanometer scale. Thus, photobleaching cannot degrade image quality, allowing for extended observation times. Super‐resolution transient amyloid binding microscopy promises to directly image native amyloid by using standard probes and record amyloid dynamics over minutes to days. We imaged amyloid fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed during different stages of amyloid‐β aggregation, as well as the structural remodeling of amyloid‐β fibrils by the compound epi‐gallocatechin gallate.     

Rapid Conversion of Amyloid-Beta 1-40 Oligomers to Mature Fibrils through a Self-Catalytic Bimolecular Process

The formation of fibrillar aggregates of the amyloid beta peptide (Aβ) in the brain is one of the hallmarks of Alzheimer’s disease (AD). A clear understanding of the different aggregation steps leading to fibrils formation is a keystone in therapeutics discovery. In a recent study, we showed that Aβ40 and Aβ42 form dynamic micellar aggregates above certain critical concentrations, which mediate a fast formation of more stable oligomers, which in the case of Aβ40 are able to evolve towards amyloid fibrils. Here, using different biophysical techniques we investigated the role of different fractions of the Aβ aggregation mixture in the nucleation and fibrillation steps. We show that both processes occur through bimolecular interplay between low molecular weight species (monomer and/or dimer) and larger oligomers. Moreover, we report here a novel self-catalytic mechanism of fibrillation of Aβ40, in which early oligomers generate and deliver low molecular weight amyloid nuclei, which then catalyze the rapid conversion of the oligomers to mature amyloid fibrils. This fibrillation catalytic activity is not present in freshly disaggregated low-molecular weight Aβ40 and is, therefore, a property acquired during the aggregation process. In contrast to Aβ40, we did not observe the same self-catalytic fibrillation in Aβ42 spheroidal oligomers, which could neither be induced to fibrillate by the Aβ40 nuclei. Our results reveal clearly that amyloid fibrillation is a multi-component process, in which dynamic collisions between different interacting species favor the kinetics of amyloid nucleation and growth.     

Light scattering study on the dynamic behaviour of cellulose inclusion complex in LiOH/urea aqueous solution

In our previous study, the rapid dissolution of cellulose in alkali/urea aqueous solution at low temperature induced by a dynamic self-assembly process has been demonstrated [1]. The cellulose solution was meta-stable, and its stability could be influenced by system fluctuations (temperature, concentration or time). In the present work, cellulose dissolved in 4.6wt% LiOH/15.0wt% urea aqueous solution pre-cooled to −12 °C was studied by means of dynamic light scattering (DLS). The results revealed that cellulose existed as single inclusion complexes (ICs) associated with LiOH and urea hydrate which could surround the ICs at their surface. And the ICs were stiff, as revealed by results from transmission electron microscopy (TEM) and light scattering (LS). When there was a system fluctuation, the self-association of cellulose with each other took place, resulted from the destruction of the urea shell, and leading to the aggregation of the ICs. For that reason, the ICs stability could be evaluated by the aggregation behaviour. In our findings, the hydrodynamic radius (R_h,app) for the cellulose dispersion in dilute solution shifted to higher values with an increase of the temperature, the concentration or the storage time, indicating an IC aggregation phenomenon.     

Exploring the early steps of amyloid peptide aggregation by computers

The assembly of normally soluble proteins into amyloid fibrils is a hallmark of neurodegenerative diseases. Because protein aggregation is very complex, involving a variety of oligomeric metastable intermediates, the detailed aggregation paths and structural characterization of the intermediates remain to be determined. Yet, there is strong evidence that these oligomers, which form early in the process of fibrillogenesis, are cytotoxic. In this paper, we review our current understanding of the underlying factors that promote the aggregation of peptides into amyloid fibrils. We focus here on the structural and dynamic aspects of the aggregation as observed in state-of-the-art computer simulations of amyloid-forming peptides with an emphasis on the activation-relaxation technique.     

Synthesis of New Tyrosol-Based Phosphodiester Derivatives: Effect on Amyloid β Aggregation and Metal Chelation Ability

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid β (Aβ) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aβ protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aβ₄₀ and a strong ability to chelate biometals such as Cu^II and Zn^II.     

Effects of Intramolecular Distance between Amyloidogenic Domains on Amyloid Aggregation

Peptide/protein aggregation is implicated in many amyloid diseases. Some amyloidogenic peptides/proteins, such as those implicated in Alzheimer’s and Parkinson’s diseases, contain multiple amyloidogenic domains connected by “linker” sequences displaying high propensities to form turn structures. Recent studies have demonstrated the importance of physicochemical properties of each amino acid contained in the polypeptide sequences in amyloid aggregation. However, effects on aggregation related to the intramolecular distance between amyloidogenic domains, which may be determined by a linker length, have yet to be examined. In the study presented here, we created peptides containing two copies of KFFE, a simple four-residue amyloidogenic domain, connected by GS-rich linker sequences with different lengths yet similar physicochemical properties. Our experimental results indicate that aggregation occurred most rapidly when KFFE domains were connected by a linker of an intermediate length. Our experimental findings were consistent with estimated entropic contribution of a linker length toward formation of (partially) structured intermediates on the aggregation pathway. Moreover, inclusion of a relatively short linker was found to inhibit formation of aggregates with mature fibril morphology. When the results are assimilated, our study demonstrates that intramolecular distance between amyloidogenic domains is an important yet overlooked factor affecting amyloid aggregation.     

Quantifying Prefibrillar Amyloids in vitro by Using a “Thioflavin‐Like” Spectroscopic Method

In Alzheimer's disease (AD) and other neurodegenerative disorders, proteins accumulate into ordered aggregates, called amyloids. Recent evidence suggests that these structures include both large, insoluble fibrils and smaller, prefibrillar structures, such as dimers, oligomers, and protofibrils. Recently, focus has shifted to the prefibrillar aggregates because they are highly neurotoxic and their levels appear to correlate with cognitive impairment. Thus, there is interest in finding methods for specifically quantifying these structures. One of the classic ways of detecting amyloid formation is through the fluorescence of the benzothiazole dye, thioflavin T (ThT). This reagent has been a “workhorse” of the amyloid field because it is robust and inexpensive. However, one of its limitations is that it does not distinguish between prefibrillar and fibrillar aggregates. We screened a library of 37 indoles for those that selectively change fluorescence in the presence of prefibrillar amyloid‐β (Aβ). From this process, we selected the most promising example, tryptophanol (TROL), to use in a quantitative “thioflavin‐like” assay. Using this probe in combination with electron microscopy, we found that prefibrils are largely depleted during Aβ aggregation in vitro but that they remain present after the apparent saturation of the ThT signal. These results suggest that a combination of TROL and ThT provides greater insight into the process of amyloid formation by Aβ. In addition, we found that TROL also recognizes other amyloid‐prone proteins, including ataxin‐3, amylin, and CsgA. Thus, this assay might be an inexpensive spectroscopic method for quantifying amyloid prefibrils in vitro.     

Structural Biology of Calcitonin: From Aqueous Therapeutic Properties to Amyloid Aggregation

Under appropriate conditions, peptides and proteins can assemble from their native state into prefibrillar oligomers and then mature into fibrillar aggregates. This transition forms the molecular basis of several pathologies, often related to the deposition of these amyloid fibrils. Several hormone peptides involved in fundamental biological processes have the tendency to self-assemble into amyloid fibrils, resulting in a loss of their native functions, and more importantly, entailing devastating consequences, such as the formation of amyloid depositions. Calcitonin is a 32 amino-acid hormone peptide that can be considered a molecular paradigm for the central events associated with hormone misfolding. Calcitonin in its native form is involved in various physiological functions, including mediating calcium homeostasis and maintaining bone structure. It is the latter function that has motivated the use of calcitonin as an aqueous therapeutic agent for the treatment of bone-related pathologies such as osteoporosis and Paget's disease. Despite some success as a therapeutic, calcitonin's ability to control these diseases is limited by its aggregation along the canonical amyloid aggregation pathway, compromising its long-term stability as a therapeutic agent. A better understanding of the misfolding process would not only provide the structural basis to improve calcitonin's long-term stability and activity as a therapeutic, but also provide valuable insights into pathological aggregation of other amyloids. In this work, we review the physiological roles of calcitonin, its structure, and aggregation process, and consider the effects of calcitonin's structure on its role as a therapeutic.     

Thiophene-Based Dual Modulators of Aβ and Tau Aggregation

Alzheimer's disease is characterized by the accumulation of amyloid beta (Aβ) and Tau aggregates in the brain, which induces various pathological events resulting in neurodegeneration. There have been continuous efforts to develop modulators of the Aβ and Tau aggregation process to halt or modify disease progression. A few small-molecule-based inhibitors that target both Aβ and Tau pathology have been reported. Here, we report the screening of a targeted library of small molecules to modulate Aβ and Tau aggregation together with their in vitro, in silico and cellular studies. In vitro ThT fluorescence assay, dot blot assay, gel electrophoresis and transmission electron microscopy (TEM) results have shown that thiophene-based lead molecules effectively modulate Aβ aggregation and inhibit Tau aggregation. In silico studies performed by employing molecular docking, molecular dynamics and binding-free energy calculations have helped in understanding the mechanism of interaction of the lead thiophene compounds with Aβ and Tau fibril targets. In cellulo studies revealed that the lead candidate is biocompatible and effectively ameliorates neuronal cells from Aβ and Tau-mediated amyloid toxicity.