Islet amyloid: phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis

Islet amyloid polypeptide (IAPP) contributes to the pathogenesis of type II diabetes by depositing as cytotoxic amyloid fibers in the endocrine pancreas. Fiber formation occurs with a marked conformational change from an unstructured precursor. Using real-time quantitative kinetic methods, fibrillogenesis was characterized as a function of protein, denaturant, and seed concentration. Several observations are in sharp contrast to the expectations for nucleation-dependent polymerization. First, the half-time of conversion for both de novo and seeded kinetics were found to be independent of protein concentration. Second, while elongation kinetics scale linearly with protein concentration, they are relatively insensitive to changes in the total seed concentration. Third, seeded bypass of de novo fiber formation kinetics shows a lag phase. The seeded lag phase is eliminated by a time delay before the introduction of seed to a de novo reaction. Last, conversion is highly cooperative, with the time required for 10-90% conversion occurring much faster than the lag time. At a minimum, four kinetic steps are required to describe these observations: activation, fiber independent nucleation, fiber-dependent nucleation, and elongation. Furthermore, we invoke a phase transition in which protein initially forms an off-pathway dispersion. This single construct allows us to model both the concentration independence of the de novo reaction time and the first-order concentration dependence of the elongation kinetics. Marked acceleration of this reaction by hexafluoro-2-propanol reinforces this view by altering the relative solubility of the two phases and/or by stabilizing hydrogen-bonded structures in the transition states of the reaction pathway.     

Organofluorine inhibitors of amyloid fibrillogenesis

The design and application of an effective, new class of organofluorine inhibitors of amyloid fibrillogenesis are described. Based on experimental evidence a core structure containing indol-3-yl, trifluoromethyl, hydroxyl, and carboxylic acid ester functions has been designed. Several substituted derivatives of this core structure have been synthesized, using various indole derivatives. While all inhibitor candidates have shown considerable effect (20-70% inhibition) in structure-activity relationship studies (inhibitor/Abeta = 10 ratio), several compounds have demonstrated excellent activity (93-96% inhibition). Using concentration dependence studies, the activity of the most active molecules have been quantified. These inhibitors practically completely block the fibril formation of Abeta(1)(-)(40), as shown by maximum inhibition values (IC(max) = 98-100%). The median inhibitor concentration values (IC(50) = 0.23-0.53 mol(inhibitor)/mol(A)(beta)) demonstrate favorable stoichiometry for the inhibition. The respective elimination of the functional groups from the core structure has resulted in a partial or complete loss of activity, indicating the significant role of each group. Experiments with these derivatives suggest the particular importance of the acidic hydroxyl group during peptide-inhibitor interaction.     

Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.     

Monomer-dependent secondary nucleation in amyloid formation

Secondary nucleation of monomers on the surface of an already existing aggregate that is formed from the same kind of monomers may lead to autocatalytic amplification of a self-assembly process. Such monomer-dependent secondary nucleation occurs during the crystallization of small molecules or proteins and self-assembled materials, as well as in protein self-assembly into fibrous structures. Indications of secondary nucleation may come from analyses of kinetic experiments starting from pure monomers or monomers supplemented with a low concentration of pre-formed aggregates (seeds). More firm evidence requires additional experiments, for example those employing isotope labels to distinguish new aggregates arising from the monomer from those resulting from fragmentation of the seed. In cases of amyloid formation, secondary nucleation leads to the formation of toxic oligomers, and inhibitors of secondary nucleation may serve as starting points for therapeutic developments. Secondary nucleation displays a high degree of structural specificity and may be enhanced by mutations or screening of electrostatic repulsion.     

Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis, its detailed molecular mechanisms remain unknown. This study is aimed at exploring the inhibitory activity of curcumin against the fibrillation of hen lysozyme. We found that the formation of amyloid fibrils at pH 2.0 in vitro was inhibited by curcumin in a dose-dependent manner. Moreover, quenching analysis confirmed the existence of an interaction between curcumin and lysozyme, and Van't Hoff analysis indicated that the curcumin–lysozyme interaction is predominantly governed by Van Der Waals force or hydrogen bonding. Curcumin was also found to acquire disaggregating ability on preformed lysozyme fibrils. Finally, we observed that curcumin pre-incubated at 25 °C for at least 7 days inhibited lysozyme fibrillogenesis better than untreated curcumin and the enhanced inhibition against HEWL fibrillation might be attributed to the presence of dimeric species.     

Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT^red) while oxidized dithiothreitol (DTT^ox) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT^red was added within 8 days of incubation. Our results showed that the initial addition of DTT^red interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT^red-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.     

Switch-peptides as folding precursors in self-assembling peptides and amyloid fibrillogenesis

The study of conformational transitions of peptides has obtained considerable attention recently because of their importance as a molecular key event in a variety of degenerative diseases. However, the study of peptide self-assembly into beta-sheets and amyloid beta (Abeta) fibrils is strongly hampered by their difficult synthetic access and low solubility. We have recently developed a new concept termed switch-peptides that allows the controlled onset of polypeptide folding and misfolding at physiologic conditions. As a major feature, the folding process is initiated by chemically or enzyme triggered O,N-acyl migration in flexible and soluble folding precursors containing Ser- or Thr-derived switch (S)-elements. The elaborated methodologies are exemplified for the in situ conversion of NPY- and Cyclosporine A-derived prodrugs, as well as for the onset and reversal of alpha and beta conformational transitions in Abeta peptides. In combining orthogonally addressable switch-elements, the consecutive switching on of S-elements gives new insights into the role of individual peptide segments (hot spots) in early processes of polypeptide self-assembly and fibrillogenesis. Finally, the well-known secondary structure disrupting effect of pseudoprolines (PsiPro) is explored for its use as a building block (S-element) in switch-peptides. To this end, synthetic strategies are described, allowing for the preparation of PsiPro-containing folding precursors, exhibiting flexible random-coil conformations devoid of fibril forming propensity. The onset of beta-sheet and fibril formation by restoring the native peptide chain in a single step classify PsiPro-units as the most powerful tool for inhibiting peptide self-assembly, and complement the present methodologies of the switch-concept for the study of fibrillogenesis.     

Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.     

Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis

We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers—exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the β-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a β-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy.     

Surface-enhanced nucleation of insulin amyloid fibrillation

Proteins can interact with biological surfaces such as cell membrane, chaperones, cornea, bone, arteries, veins, and heart cavities of the cardiovascular system and also with non-biological surfaces including dialysis membranes and tubing, catheters, invasive surgical instruments, needles, and artificial implants. Fibrillation of amyloid proteins is implicated in many human diseases, including Alzheimer’s, Parkinson’s, and type II diabetes. Here, we show that heterogeneous surfaces accelerate the human insulin nucleation process that is the rate-determining step during amyloid fibril formation. The observed shorter lag (nucleation) phase correlates both with surface wettability and surface roughness. Surfaces promote faster nucleation possibly by increasing the local concentration of protein molecules. A composite parameter combining both surface wettability and roughness suggests that the ideal surface for slower nucleation should be hydrophilic and smooth. These findings provide a basis for designing suitable biomaterials and biomedical devices, especially those to resist amyloidosis.     

Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone

Aggregation of proteins in tissues is associated with several diseases, including Alzheimer's disease. It is characterized by the accumulation of amyloid beta peptide (Abeta) in the extracellular spaces of the brain cells, resulting in neuronal death and other pathological changes. alpha-Crystallin, a small heat-shock protein in lens, and a peptide chaperone having the functional site sequence DFVIFLDVKHFSPEDLTVK of alphaA-crystallin may inhibit Abeta fibrillogenesis and toxicity. The peptide chaperone (mini-alphaA-crystallin), having an Abeta interacting domain and a complex solubilizing domain, was shown in previous studies to prevent aggregation of several proteins under denaturing conditions. In this in vitro study, using transmission electron microscopy and thioflavin T binding assay, we show that mini-alphaA-crystallin arrests the fibril formation of Abeta peptides. Mini-alphaA-crystallin also suppresses the toxic action of Abeta on rat pheochromocytoma (PC 12) cells. The wide chaperoning capability of the peptide and its ability to inhibit amyloid fibril formation and suppress toxicity suggest that mini-alphaA-crystallin may serve as a universal chaperone in controlling diseases of protein aggregation, including Alzheimer's disease.     

Examining the inhibitory actions of copolypeptides against amyloid fibrillogenesis of bovine insulin

Amyloid fibrillogenesis has been involved in at least 40 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been demonstrated to fibrillate in vitro. With bovine insulin as a model, the research presented here examines the influence of two simple, unstructured d,l-lysine-co-glycine (d,l-lys-co-gly) and d,l-lysine-co-L-phenylalanine (d,l-lys-co-phe) copolypeptides, on the in vitro fibril formation process of bovine insulin at pH 2.0 and 55 °C. Our results showed that amyloid fibrillogenesis of insulin may be suppressed by both copolypeptides in a concentration-dependent fashion. In addition, the copolypeptides with higher molar fractions of glycine or l-phenylalanine residue, which are considered to possess higher hydrophobic interacting capacities, demonstrated the superior inhibitory potency against insulin fibril formation. Our findings suggest that the association of insulin and copolypeptides, which is likely dominated by hydrophobic interactions and hydrogen bonding, may mitigate the extent of insulin fibrillogenesis. We believe the results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and amyloid-forming proteins.     

On the nucleation of amyloid beta-protein monomer folding

Neurotoxic assemblies of the amyloid beta-protein (Abeta) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Abeta assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Abeta(1-40) and Abeta(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Abeta(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a beta-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Abeta nucleates the intramolecular folding of Abeta monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Abeta(1-40) and Abeta(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Abeta(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Abeta(1-42)-specific C-terminal fold may hold promise.     

Temperature dependence of dielectric constant at 10 GHz of Na-DNA gels

Permittivity ε′ and dielectric loss ε″ of aqueous Na-DNA gels have been measured at 10 GHz in the temperature interval ?15 to + 45°C. The experimental results are analyzed in terms of a three-component equation (Na-DNA, interfacial water, bulk water) and yield a value of 35 water molecules/nucleotide interacting with DNA. According to theoretical and experimental data the presence of strongly bonded and weakly bonded water is considered. The modified water exhibits a mean dielectric relaxation time two-or threefold greater than that of bulk water.     

A comparison of amyloid fibrillogenesis using the novel fluorescent compound K114

Proteinaceous inclusions with amyloidogenic properties are a common link between many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Histological and in vitro studies of amyloid fibrils have advanced the understanding of protein aggregation, and provided important insights into pathogenic mechanisms of these neurodegenerative brain amyloidoses. The classical amyloid dyes Congo Red (CR) and thioflavin T and S, have been used extensively to detect amyloid inclusions in situ. These dyes have also been utilized to monitor the maturation of amyloid fibrils assembled from monomer subunits in vitro. Recently, the compound (trans,trans)-1-bromo-2,5-bis-(3- hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), derived from the structure of CR, was shown to bind to a wide range of amyloid inclusions in situ. More importantly it was also used to label brain amyloids in live animals. Herein, we show that an analogue of BSB, (trans,trans)-1-bromo-2,5-bis-(4-hydroxy)styrylbenzene (K114), recognizes amyloid lesions, and has distinctive properties which allowed the quantitative monitoring of the formation of amyloid fibrils assembled from the amyloid-beta peptide, alpha-synuclein, and tau.     

Kinetic factors may reshape the dependence of crystal nucleation rate on temperature in protein bulk solution

Here we provide an analysis of primary results obtained from a study of apoferritin crystal nucleation in compositionally invariant bulk solution at constant supersaturation ratio of the protein. The temperature dependence of the stationary crystal nucleation rate in the protein bulk solution is obtained with the help of experimentally determined probability for detection of at least one crystal per solution volume until a given time. The stationary crystal nucleation rate demonstrates unusual behavior with temperature. We emphasize that this is caused by kinetic factors that are often disregarded in the frame of the classical nucleation theory but can certainly affect the nucleation kinetics.     

Temperature dependence of the polarization and the dielectric constant near the paraelectric-ferroelectric transitions in BaTiO3

Temperature dependences of the spontaneous polarization and the dielectric constant are calculated near the paraelectric-ferroelectric (cubic-tetragonal) transition in BaTiO₃ using our mean field model. By expanding the free energy in terms of the spontaneous polarization (order parameter), expressions for the temperature dependence of the spontaneous polarization and the dielectric constant are derived. By considering the temperature dependence of the Raman frequencies for the lattice mode (～310 cm^?1) which is related to the spontaneous polarization, the experimental data from the literature is analyzed near the first order paraelectric-ferroelectric transition in BaTiO₃. The dielectric constant is then calculated as a function of temperature for the cubic-tetragonal transition in BaTiO₃. Our results show that the observed behavior of the spontaneous polarization in the ferroelectric phase (T<T_C) and that of the dielectric constant in both paraelectric (T>T_C) and ferroelectric phases, can be described adequately by the mean field model studied here for BaTiO₃.     

Renaturation of bacteriophage phiX174 DNA-RNA hybrid: RNA length effect and nucleation rate constant

A one to one RNA-DNA hybrid was synthesized using bacteriophage φX174 DNA as a template for the Escherichia coli RNA polymerase system. The renaturation rate for the hybrid was found to vary as the square root of the molecular weight of the RNA. The RNA molecular weights were determined using a standardized dimethylsulfoxide, cesium sulfate equilibrium density gradient. The optimum nucleation rate constant for the renaturation of the hybrid in 0.4 m-sodium chloride was found to be 1.3 × 10⁵, as compared with to 1.7 × 10⁵ for DNA renaturation.