Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion

We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 degrees C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently; however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth.     

Template-directed self-assembly and growth of insulin amyloid fibrils

The formation of amyloid aggregates in tissue is a pathological feature of many neurodegenerative diseases and type II diabetes. Amyloid deposition, the process of amyloid growth by the association of individual soluble amyloid molecules with a pre-existing amyloid template (i.e., plaque), is known to be critical for amyloid formation in vivo. The requirement for a natural amyloid template, however, has made amyloid deposition study difficult and cumbersome. In the present work, we developed a novel, synthetic amyloid template by attaching amyloid seeds covalently onto an N-hydroxysuccinimide-activated surface, where insulin was chosen as a model amyloidogenic protein. According to ex situ atomic force microscopy observations, insulin monomers in solution were deposited onto the synthetic amyloid template to form fibrils, like hair growth. The fibril formation on the template occurred without lag time, and its rate was highly accelerated than in the solution. The fibrils were long, over 2 mum, and much thinner than those in the solution, which was caused by limited nucleation sites on the template surface and lack of lateral twisting between fibrils. According to our investigations using thioflavin T-induced fluorescence, birefringent Congo red binding, and circular dichroism, fibrils grown on the template were identified to be amyloids that formed through a conformational rearrangement of insulin monomers upon interaction with the template. The amyloid deposition rate followed saturation kinetics with respect to insulin concentration in the solution. The characteristics of amyloid deposition on the synthetic template were in agreement with previous studies performed with human amyloid plaques. It is demonstrated that the synthetic amyloid template can be used for the screening of inhibitors on amyloid deposition in vitro.     

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.     

Detection and structural characterization of insulin prefibrilar oligomers using surface enhanced Raman spectroscopy

In vitro fibril formation typically exhibits a lag phase followed by a rapid elongation phase. Soluble prefibrilar oligomers form as multiple assembly states occur during the lag phase and, after forming a nucleus, rapidly propagate into amyloid aggregates and fibrils. The structure and morphology of amyloid fibrils have been extensively characterized over the last decades, while little is known about the structural organization of the prefibrilar oligomers or their multiple assembly states. The main difficulty in structural characterization of prefibrilar aggregates is their low concentration (pmolar) and their continual reactive conversion. Herein we overcome these difficulties by utilizing Surface‐Enhanced Raman Spectroscopy (SERS) with a model amyloid peptide, insulin. SERS is a powerful analytic tool that is able to provide detection of small molecules down to a single‐molecule level. Using SERS we found that during the 3 lag phase before the onset of insulin fibril formation, the amount of insulin oligomers increased more than twice after the first hour of incubation under fibrillation conditions (pH 1.6, 65°C) and then slowly decreased with time. The latter finding is kinetically linked to the conversion of the prefibrilar oligomers into fibril species. This study provides valuable new information about the time‐dependent structural organization of insulin oligomers and demonstrates the power and potential of SERS for detection and structural characterization of biological specimens present at low concentrations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:488–495, 2014     

Optical microscopy of growing insulin amyloid spherulites on surfaces in vitro

Amyloid fibrils are often found arranged into large ordered spheroid structures, known as spherulites, occurring in vivo and in vitro. The spherulites are predominantly composed of radially ordered amyloid fibrils, which self-assemble from protein in solution. We have observed and measured amyloid spherulites forming from heat-treated solutions of bovine insulin at low pH. The spherulites form in large numbers as semispherical dome-shaped objects on the cell surfaces, showing that surface defects or impurities, or the substrates themselves, can provide good nucleation sites for their formation. Using optical microscopy, we have measured the growth of individual spherulites as a function of time and in various conditions. There is a lag time before nucleation of the spherulites. Once they have nucleated, they grow, each with a radius increasing linearly, or faster than linearly, with time. Remarkably, this growth period has a sudden end, at which all spherulites in the system suddenly stop growing. A model of spherulite formation based on the polymerization of oriented fibrils around a nucleus, from a precursor in solution, quantitatively accounts for the observed growth kinetics. Seeding of native insulin solutions with preformed spherulites led to the preformed spherulites growing without a lag time. This seeding behavior is evidence that the fibrils in the spherulites assemble from small protein species rather than fibrils. The density of the spherulites was also measured and found to be constant with respect to radius, indicating that the space fills as the spherulite grows.     

Determination of size of folding nuclei of fibrils formed from recombinant Aβ(1-40) peptide

We have developed a highly efficient method for purification of the recombinant product Aβ(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aβ(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one “monomer” and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures.     

Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I.

To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds. Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.     

Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism

In the search for the molecular mechanism of insulin fibrillation, the kinetics of insulin fibril formation were studied under different conditions using the fluorescent dye thioflavin T (ThT). The effect of insulin concentration, agitation, pH, ionic strength, anions, seeding, and addition of 1-anilinonaphthalene-8-sulfonic acid (ANS), urea, TMAO, sucrose, and ThT on the kinetics of fibrillation was investigated. The kinetics of the fibrillation process could be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). The addition of seeds eliminated the lag phase. An increase in insulin concentration resulted in shorter lag times and faster growth of fibrils. Shorter lag times and faster growth of fibrils were seen at acidic pH versus neutral pH, whereas an increase in ionic strength resulted in shorter lag times and slower growth of fibrils. There was no clear correlation between the rate of fibril elongation and ionic strength. Agitation during fibril formation attenuated the effects of insulin concentration and ionic strength on both lag times and fibril growth. The addition of ANS increased the lag time and decreased the apparent growth rate for insulin fibril formation. The ANS-induced inhibition appears to reflect the formation of amorphous aggregates. The denaturant, urea, decreased the lag time, whereas the stabilizers, trimethylamine N-oxide dihydrate (TMAO) and sucrose, increased the lag times. The results indicated that both nucleation and fibril growth were controlled by hydrophobic and electrostatic interactions. A kinetic model, involving the association of monomeric partially folded intermediates, whose concentration is stimulated by the air-water interface, leading to formation of the critical nucleus and thence fibrils, is proposed.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.     

Structural polymorphism and possible pathways of amyloid fibril formation on the example of insulin protein

In this review we analyze the main works on amyloid formation of insulin. There are many environmental factors affecting the formation of insulin amyloid fibrils (and other amyloidogenic proteins) such as: protein concentration, pH, ionic strength of solution, medium composition (anions, cations), presence of denaturants (urea, guanidine chloride) or stabilizers (saccharose), temperature regime, agitation. Since polymorphism is potentially crucial for human diseases and may underlie the natural variability of some amyloid diseases, in this review we focus attention on polymorphism that is an important biophysical difference between native protein folding suggesting correspondence between the amino acid sequence and unique folding state, and formation of amyloid fibrils, when the same amino acid sequence can form amyloid fibrils of different morphology. At present, according to the literature data, we can choose three ways of polymerization of insulin molecules depending on the nucleus size. The first suggests that fibrillogenesis can occur through assembly of insulin monomers. The second suggests that precursors of fibrils are dimers, and the third assumes that precursors of fibrils are oligomers. Additional experimental works and new methods of investigation and assessment of results are needed to clarify the general picture of insulin amyloid formation.     

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.     

Sampling the self-assembly pathways of KFFE hexamers

The formation of amyloid fibrils is often encountered in Alzheimer's disease, type II diabetes, and transmissible spongiform encephalopathies. In the last few years, however, mounting evidence has suggested that the soluble oligomers of amyloid-forming peptides are also cytotoxic agents. Understanding the early pathway steps of amyloid self-assembly at atomic detail might therefore be crucial for the development of specific inhibitors to prevent amyloidosis in humans. Using the activation-relaxation technique and a generic energy model, we study in detail the aggregation of a hexamer of KFFE peptide. Our simulations show that a monomer remains disordered, but that six monomers placed randomly in an open box self-associate to adopt, with various orientations, three possible distant low-energy structures. Two of these structures show a double-layer beta-sheet organization, in agreement with the structure of amyloid fibrils as observed by x-ray diffraction, whereas the third one consists of a barrel-like curved single-layer hexamer. Based on these results, we propose a bidirectional growth mode of amyloid fibril, involving alternate lateral and longitudinal growths.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly a-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90–231 and 121–231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non- thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.Key words: misfolding, aggregation, amyloid, prion, conformational conversion, fluorescence     

Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

Covalent structural changes in unfolded GroES that lead to amyloid fibril formation detected by NMR: insight into intrinsically disordered proteins

Co-chaperonin GroES from Escherichia coli works with chaperonin GroEL to mediate the folding reactions of various proteins. However, under specific conditions, i.e. the completely disordered state in guanidine hydrochloride, this molecular chaperone forms amyloid fibrils similar to those observed in various neurodegenerative diseases. Thus, this is a good model system to understand the amyloid fibril formation mechanism of intrinsically disordered proteins. Here, we identified a critical intermediate of GroES in the early stages of this fibril formation using NMR and mass spectroscopy measurements. A covalent rearrangement of the polypeptide bond at Asn(45)-Gly(46) and/or Asn(51)-Gly(52) that eventually yield β-aspartic acids via deamidation of asparagine was observed to precede fibril formation. Mutation of these asparagines to alanines resulted in delayed nucleus formation. Our results indicate that peptide bond rearrangement at Asn-Gly enhances the formation of GroES amyloid fibrils. The finding provides a novel insight into the structural process of amyloid fibril formation from a disordered state, which may be applicable to intrinsically disordered proteins in general.     

Strong inhibition of peptide amyloid formation by a fatty acid

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.     

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay

Abstract

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.