Prevention of Alzheimer's disease-associated Abeta aggregation by rationally designed nonpeptidic beta-sheet ligands

A new concept is introduced for the rational design of beta-sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a beta-sheet efficiently block the solvent-exposed beta-sheet portions in Abeta-(1-40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of Abeta aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a beta-sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10(-5) m. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of Abeta-(1-40) in the absence and in the presence of the ligands. From the comparable decrease in Abeta monomer concentration, we conclude that these beta-sheet ligands do not prevent the initial oligomerization of monomeric Abeta but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble Abeta aggregates formed in the presence of both inhibitors from 3- to 15-mers.     

Influence of fluorinated and hydrogenated nanoparticles on the structure and fibrillogenesis of amyloid beta-peptide

Peptide aggregation in amyloid fibrils is implicated in the pathogenesis of several diseases such as Alzheimer's disease. There is a strong correlation between amyloid fibril formation and a decrease in conformational stability of the native state. Amyloid-beta peptide (Abeta), the aggregating peptide in Alzheimer's disease, is natively unfolded. The deposits found in Alzheimer's disease are composed of Abeta fibrillar aggregates rich in beta-sheet structure. The influence of fluorinated complexes on the secondary structure and fibrillogenesis of Abeta peptide was studied by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). CD spectra show that complexes of polyampholyte and fluorinated dodecanoic acid induce alpha-helix structure in Abeta, but their hydrogenated analogous lead to beta-sheet formation and aggregation. The fluorinated nanoparticles with highly negative zeta potential and hydrophobic fluorinated core have the fundamental characteristics to prevent Abeta fibrillogenesis.     

Lipid membranes modulate the structure of islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is thought to play an important role in the pathogenesis of type II diabetes. Despite a growing body of evidence implicating membrane interaction in IAPP toxicity, the membrane-bound form has not yet been well characterized. Here we used circular dichroism (CD) and fluorescence spectroscopy to investigate the molecular details of the interaction of IAPP with lipid membranes of varying composition. In the presence of membranes containing negatively charged phosphatidylserine (PS), we observed significant acceleration in the formation of IAPP aggregates. This acceleration is strongly modulated by the PS concentration and ionic strength, and is also observed at physiologically relevant PS concentrations. CD spectra of IAPP obtained immediately after the addition of membranes containing PS revealed features characteristic of an alpha-helical conformation approximately approximately 15-19 residues in length. After a longer incubation with membranes, IAPP gave rise to CD spectra characteristic of a beta-sheet conformation. Taken together, our CD and fluorescence data indicate that conditions that promote weakly stable alpha-helical conformations may promote IAPP aggregation. The potential roles of IAPP-membrane interaction and the novel membrane-bound alpha-helical conformation in IAPP aggregation are discussed.     

Non-native aggregation of alpha-chymotrypsinogen occurs through nucleation and growth with competing nucleus sizes and negative activation energies

The kinetics and structural transitions of non-native aggregation of alpha-chymotrypsinogen (aCgn) were investigated over a wide range of temperature and initial protein concentration at pH 3.5, where high molecular weight aggregates remained soluble throughout the reaction. A comparison of thermodynamic, kinetic, and spectroscopic data shows that aggregation under non-native-favoring conditions proceeds through a molten globule unfolded monomer state, with a nucleation and growth mechanism. Formation of irreversible aggregates and conversion to beta-sheet secondary structures occur simultaneously without detectable intermediates, suggesting that beta-sheet formation may be a commitment step during the nucleation and growth stages. Analysis of the kinetics using a Lumry-Eyring with nucleated polymerization (LENP) model provides the predominant nucleus size and the product of the intrinsic nucleation and intrinsic growth time scales at each state point. We find that the nucleus size depends on both temperature and protein concentration, and in some cases there is competition between two distinct nucleus sizes. The observed rate coefficient (kobs) for aggregation displays a maximum as a function of temperature because of the competition between folding-unfolding thermodynamics and the intrinsic growth and nucleation rates; the latter contribution has a large, negative activation enthalpy that dominates kobs at elevated temperatures. Temperature-jump experiments reveal that aggregates depolymerize at high temperatures, indicating that they are lower in enthalpy than the free monomer. Overall, the results suggest more generally that non-native aggregation may proceed through more than one nucleus size and that intrinsic kinetics of nucleation and growth may have significant entropic barriers.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.     

Interactions of amyloid beta-peptide (1-40) with ganglioside-containing membranes

Interactions between amyloid beta-peptides (Abeta) and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. To gain insight into the molecular details of this association, we investigated the interactions of Abeta (1-40) with ganglioside-containing membranes by circular dichroism (CD) and Fourier transform infrared-polarized attenuated total reflection (FTIR-PATR) spectroscopy. The CD study revealed that at physiological ionic strength Abeta (1-40) specifically binds to ganglioside-containing membranes inducing a two-state, unordered --> beta-sheet transition above a threshold intramembrane ganglioside concentration, which depends on the host lipid bilayers used. Furthermore, differences in the number and position of sialic acid residues of the carbohydrate backbone significantly affected the conformational transition of the peptide. FTIR-PATR spectroscopy experiments demonstrated that Abeta (1-40) forms an antiparallel beta-sheet, the plane of which lies parallel to the membrane surface, inducing dehydration of lipid interfacial groups and perturbation of acyl chain orientation. These results suggest that Abeta (1-40) imposes negative curvature strain on ganglioside-containing lipid bilayers, disturbing the structure and function of the membranes.     

A toxic monomeric conformer of the polyglutamine protein

Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a beta-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into beta-sheet-rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble beta-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic beta-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.     

An atomic model for the pleated beta-sheet structure of Abeta amyloid protofilaments.

Synchrotron x-ray studies on amyloid fibrils have suggested that the stacked pleated beta-sheets are twisted so that a repeating unit of 24 beta-strands forms a helical turn around the fibril axis (. J. Mol. Biol. 273:729-739). Based on this morphological study, we have constructed an atomic model for the twisted pleated beta-sheet of human Abeta amyloid protofilament. In the model, 48 monomers of Abeta 12-42 stack (four per layer) to form a helical turn of beta-sheet. Each monomer is in an antiparallel beta-sheet conformation with a turn located at residues 25-28. Residues 17-21 and 31-36 form a hydrophobic core along the fibril axis. The hydrophobic core should play a critical role in initializing Abeta aggregation and in stabilizing the aggregates. The model was tested using molecular dynamics simulations in explicit aqueous solution, with the particle mesh Ewald (PME) method employed to accommodate long-range electrostatic forces. Based on the molecular dynamics simulations, we hypothesize that an isolated protofilament, if it exists, may not be twisted, as it appears to be when in the fibril environment. The twisted nature of the protofilaments in amyloid fibrils is likely the result of stabilizing packing interactions of the protofilaments. The model also provides a binding mode for Congo red on Abeta amyloid fibrils. The model may be useful for the design of Abeta aggregation inhibitors.     

Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation

It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.     

Interpretation of concentration-dependence in aggregation kinetics

Aggregation processes are analyzed by two kinetic models, the random polymerization model and the nucleation-dependent polymerization model. A kinetic equation for the random polymerization model can be derived analytically, revealing the relation between the initial monomer concentration ([M]0), the rate constant (k(a)), time (t), the yield of detectable aggregate ([F]), and the critical aggregation number (m). However, time-course curves for the nucleation-dependent polymerization model can be obtained by numerical calculation. It is found that lag time (t(d)) and half-time (t1/2) are proportional to [M](-1) in the random polymerization model, while t(d) and t1/2 are proportional to [M1](-s) (1 < s < n; n is nucleus size) at the lower concentration and are less dependent on [M1] at the higher concentration in the nucleation-dependent polymerization model.     

HSP90-like artificial chaperone activity based on indole beta-cyclodextrin

Indole beta-cyclodextrin (beta-1) was found to be able to prevent aggregation of citrate synthase (CS) on heating condition. As a result, beta-1 showed anti-CS aggregation in this system by regulating in early stage. The depression mechanism of beta-1 for aggregation of CS is as follows: the beta-1 formed a complex with hydrophobic parts of the beta-sheet structure of CS. From CD spectra, CS was changed own conformation was changed by beta-1 addition. So, it was concluded that beta-1 works as beta-sheet inducer in thermal condition. On the other hand, native beta-cyclodextrin (beta-CyD) shows small suppression capability for CS aggregation.     

Temperature-dependent beta-sheet formation in beta-amyloid Abeta(1-40) peptide in water: uncoupling beta-structure folding from aggregation

To probe the role of temperature in the conversion of soluble Alzheimer's beta-amyloid peptide (Abeta) to insoluble beta-sheet rich aggregates, we analyzed the solution conformation of Abeta(1-40) from 0 to 98 degrees C by far-UV circular dichroism (CD) and native gel electrophoresis. The CD spectra of 15-300 microg/ml Abeta(1-40) in aqueous solution (pH approximately 4.6) at 0 degrees C are concentration-independent and suggest a substantially unfolded and/or unusually folded conformation characteristic of Abeta monomer or dimer. Heating from 0 to 37 degrees C induces a rapid reversible coil to beta-strand transition that is independent of the peptide concentration and thus is not linked to oligomerization. Consequently, this transition may occur within the Abeta(1-40) monomer or dimer. Incubation at 37 degrees C leads to slow reversible concentration-dependent beta-sheet accumulation; heating to 85 degrees C induces further beta-sheet folding and oligomerization. Our results demonstrate the importance of temperature and thermal history for the conformation of Abeta.     

Sequence dependence of amyloid fibril formation: insights from molecular dynamics simulations

The clarification of the physico-chemical determinants underlying amyloid deposition is critical for our understanding of misfolding diseases. With this purpose we have performed a systematic all-atom molecular dynamics (MD) study of a series of single point mutants of the de novo designed amyloidogenic peptide STVIIE. Sixteen different 50ns long simulations using explicit solvent have been carried out starting from four different conformations of a polymeric six-stranded beta-sheet. The simulations have provided evidence for the influence of a small number of site-specific hydrophobic interactions on the packing and stabilization of nascent aggregates, as well as the interplay between side-chain interactions and the net charge of the molecule on the strand arrangement of polymeric beta-sheets. This MD analysis has also shed light into the origin of the position dependence on mutation of beta-sheet polymerization that was found experimentally for this model system. Our results suggest that MD can be applied to detect critical positions for beta-sheet aggregation within a given amyloidogenic stretch. Studies similar to the one presented here can guide site-directed mutations or the design of drugs that specifically disrupt the key stabilizing interactions of beta-sheet aggregates.     

Molecular dynamics study of amyloid formation of two Abl-SH3 domain peptides.

Molecular dynamics (MD) simulations were carried out for two-strand and ten-strand beta-sheets constructed from two peptides corresponding to the diverging turn of two homologous Abl-SH3 domains, DLSFMKGE (MK; from Drosophila) and DLSFKKGE (KK; from man), in explicit water at the temperatures of 30, 170/190 and 300 K. It was found that the 2 x MK beta-sheet is more stable than the 2 x KK beta-sheet, and that the 10 x MK beta-sheet is more stable than the 10 x KK beta-sheet; this suggests that the MK systems are fibril-creating and the KK systems are not. These results might explain why most SH3 domains possess two conserved basic residues at the diverging turn, which may act as gatekeepers in order to avoid aggregation.     

Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathways

As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes beta-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, Agg(HP), which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, Agg(HP) transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially beta-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, I(HP), is formed under high pressure. After pressure release, I(HP) relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, I(HP), constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids.     

Structural analysis of amyloid beta peptide fragment (25-35) in different microenvironments

Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.     

Formation of amyloid fibrils by bovine carbonic anhydrase

Amyloids are typically characterized by extensive aggregation of proteins where the participating polypeptides are involved in formation of intermolecular cross beta-sheet structures. Alternate structure attainment and amyloid formation has been hypothesized to be a generic property of a polypeptide, the propensities of which vary widely depending on the polypeptide involved and the physicochemical conditions it encounters. Many proteins that exist in the normal form in-vivo have been shown to form amyloid when incubated in partially denaturing conditions. The protein bovine carbonic anhydrase II (BCA II) when incubated in mildly denaturing conditions showed that the partially unfolded conformers assemble together and form ordered amyloid aggregates. The properties of these aggregates were tested using the traditional Congo-Red (CR) and Thioflavin-T (ThT) assays along with fluorescence microscopy, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. The aggregates were found to possess most of the characteristics ascribed to amyloid fibers. Thus, we report here that the single-domain globular protein, BCA II, is capable of forming amyloid fibrils. The primary sequence of BCA II was also analyzed using recurrence quantification analysis in order to suggest the probable residues responsible for amyloid formation.     

Interaction between amyloid beta-protein aggregates and membranes.

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes.     

Structure-based design and study of non-amyloidogenic, double N-methylated IAPP amyloid core sequences as inhibitors of IAPP amyloid formation and cytotoxicity.

Pancreatic amyloid is formed by the aggregation of the 37-residue islet amyloid polypeptide (IAPP) in type II diabetes patients and is cytotoxic. Pancreatic amyloid deposits are found in more than 95 % of type II diabetes patients and their formation is strongly associated with disease progression. IAPP amyloid forms via a conformational transition of soluble IAPP into aggregated beta-sheets. We recently identified IAPP(22-27) (NFGAIL) as a minimum length sequence sufficient to self-associate into beta-sheet-containing amyloid fibrils. Here, we have used the NFGAIL model of the IAPP amyloid core as a structural template to design non-amyloidogenic derivatives of amyloidogenic sequences of IAPP that are able to interact with the native sequences and inhibit amyloid formation. The design of the derivatives was based on a simple, structure-based minimalistic and selective N-methylation approach. Accordingly, a minimum number of two amide bonds on the same side of the beta-strand of the amyloid core was N-methylated. This was expected to eliminate the two intermolecular backbone NH to CO hydrogen bonds which are critical for the extension of the beta-sheet dimers into multimers and amyloid. Other beta-strand "contact sides" remained intact allowing for the derivatives to interact with the native sequences. Double N-methylated derivatives of amyloidogenic and cytotoxic partial IAPP sequences generated included F(N-Me)GA(N-Me)IL, NF(N-Me)GA(N-Me)IL, SNNF(N-Me)GA(N-Me)IL, and SNNF(N-Me)GA(N-Me)ILSS and were found to be devoid of beta-sheet structure, amyloidogenicity and cytotoxicity according to Fourier transform-infrared spectroscopy (FT-IR), Congo red (CR) staining, electron microscopy (EM), and cell viability tests. The derivatives were able to interact with the native sequences and inhibit amyloid formation as shown by circular dichroism spectroscopy (CD), FT-IR and EM. Moreover, SNNF(N-Me)GA(N-Me)ILSS inhibited cytotoxicity of SNNFGAILSS and is thus the first reported inhibitor of IAPP amyloid formation and cytotoxicity. Our results demonstrate the validity of the design approach for IAPP and suggest that it may find application in understanding the structural features of amyloid formation and in the development of inhibitors of amyloid formation and cytotoxicity of other amyloidogenic polypeptides as well.