Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils: IMPLICATIONS FOR CANCER

Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.     

The effect of amino acid substitution in the imperfect repeat sequences of α-synuclein on fibrillation

Human α-synuclein is the causative protein of several neurodegenerative diseases, such as Parkinson's disease (PD) and dementia with Lewy Bodies (DLB). The N-terminal half of α-synuclein contains seven imperfect repeat sequences. One of the PD/DLB-causing point mutations, E46K, has been reported in the imperfect repeat sequences of α-synuclein, and is prone to form amyloid fibrils. The presence of seven imperfect repeats in α-synuclein raises the question of whether or not mutations corresponding to E46K in the other imperfect KTKE(Q)GV repeats have similar effects on aggregation and fibrillation, as well as their propensities to form α-helices. To investigate the effect of E(Q)/K mutations in each imperfect repeat sequence, we substituted the amino acid corresponding to E46K in each of the seven repeated sequences with a Lys residue. The mutations in the imperfect KTKE(Q)GV repeat sequences of the N-terminal region were prone to decrease the lag time of fibril formation. In addition, AFM imaging suggested that the Q24K mutant formed twisted fibrils, while the other mutants formed spherical aggregates and short fibrils. These observations indicate that the effect of the mutations on the kinetics of fibril formation and morphology of fibrils varies according to their location.     

Metals accelerate the formation and direct the structure of amyloid fibrils of NAC

Non-beta amyloid component of Alzheimer's disease amyloid or NAC is a highly amyloidogenic peptide consisting of 35 amino acids which was first identified associated with senile plaques in the Alzheimer's disease brain. It is a fragment of the presynaptic protein alpha-synuclein and, as such, it is implicated in the aetiologies of both Alzheimer's (AD) and Parkinson's (PD) disease. Metals are involved in the aggregation of amyloidogenic peptides such as beta amyloid (Abeta), British amyloid peptide (ABri) and alpha-synuclein though nothing is yet known about how they might influence the aggregation of NAC. We show herein that NAC will form beta-pleated conformers at a peptide concentration of only 2.0 microM and that metals, and Zn(II) and Cu(II) in particular, accelerate the formation of these fibrils. Cu(II) and Zn(II) did not influence the diameter or general structure of the fibrils which were formed though many more shorter fibrils were observed in their presence and these shorter fibrils were highly thioflavin T positive and they were efficient catalysts of the redox cycling of added Fe(II). By way of contrast, beta-pleated conformers of NAC which were formed in the presence of Al(III) showed much lower levels of thioflavin T fluorescence and were poorer catalysts of the redox cycling of added Fe(II) and these properties were commensurate with an increased abundance of a novel amyloid morphology which consisted of twisted fibrils with a periodicity of about 100 nm. These spirals of twisted fibrils were especially abundant in the presence of added Al(III) and it is speculated that NAC binding of Al(III) may be important in their formation and subsequent stability.     

Cyclin-dependent Kinase 5 Phosphorylation of Familial Prion Protein Mutants Exacerbates Conversion into Amyloid Structure

Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion.     

Identification of the core structure of lysozyme amyloid fibrils by proteolysis

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive beta-sheet structure and a substantial element of non beta-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the beta-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.     

Structural studies reveal that the diverse morphology of beta(2)-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of beta(2)-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the beta-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular beta-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular beta-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.     

Identification of the region of non-Aβ component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity

The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3-18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Circular dichroism indicates that none of the peptides displays beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.     

Generation of reactive oxygen species and activation of NF-kappaB by non-Abeta component of Alzheimer's disease amyloid

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.     

Sarcomeric proteins of the titin family form amyloids

It was shown for the first time that skeletal muscle sarcomeric proteins of the titin family (X-, C- and H-proteins) are able to form in vitro amyloid aggregates of different types: granular aggregates, protofibrils, helically twisted ribbons, linear fibrils, and bundles of linear fibrils. Their amyloid nature was confirmed by electron, polarization, and fluorescence microscopy and by spectral methods. As opposed to other amyloidogenic proteins, X-, C-, and H-proteins easily form amyloids under mild conditions close to physiological ones (pH, ionic strength, temperature). Like amyloid fibrils of Abeta-peptide and tau protein in Alzheimer's disease, amyloid aggregates formed by X-, C-, and H-proteins are destroyed by the antibiotic tetracycline. Thus, new proteins-precursors of amyloids and possible participants of amyloidoses in muscles were discovered. Further study of in vitro amyloidogenesis of these proteins would help to find approaches to controlling this process in organs and tissues.     

A common beta-sheet architecture underlies in vitro and in vivo beta2-microglobulin amyloid fibrils

Misfolding and aggregation of normally soluble proteins into amyloid fibrils and their deposition and accumulation underlies a variety of clinically significant diseases. Fibrillar aggregates with amyloid-like properties can also be generated in vitro from pure proteins and peptides, including those not known to be associated with amyloidosis. Whereas biophysical studies of amyloid-like fibrils formed in vitro have provided important insights into the molecular mechanisms of amyloid generation and the structural properties of the fibrils formed, amyloidogenic proteins are typically exposed to mild or more extreme denaturing conditions to induce rapid fibril formation in vitro. Whether the structure of the resulting assemblies is representative of their natural in vivo counterparts, thus, remains a fundamental unresolved issue. Here we show using Fourier transform infrared spectroscopy that amyloid-like fibrils formed in vitro from natively folded or unfolded beta(2)-microglobulin (the protein associated with dialysis-related amyloidosis) adopt an identical beta-sheet architecture. The same beta-strand signature is observed whether fibril formation in vitro occurs spontaneously or from seeded reactions. Comparison of these spectra with those of amyloid fibrils extracted from patients with dialysis-related amyloidosis revealed an identical amide I' absorbance maximum, suggestive of a characteristic and conserved amyloid fold. Our results endorse the relevance of biophysical studies for the investigation of the molecular mechanisms of beta(2)-microglobulin fibrillogenesis, knowledge about which may inform understanding of the pathobiology of this protein.     

Amyloid fibril formation in the context of full-length protein: effects of proline mutations on the amyloid fibril formation of beta2-microglobulin

Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.     

Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants

High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.     

Chiral bifurcation in aggregating insulin: an induced circular dichroism study

The structural unambiguity of folding is lost when disordered protein molecules convert into β-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure. Bifurcation and chiral mesoscopic-scale organization of amyloid fibrils are new aspects of protein misfolding. Here we characterize bifurcation events accompanying insulin fibrillation upon intensive vortexing. Upon agitation, two types of insulin fibrils with opposite chiral senses are formed; however, predominance of either species is only stochastically determined. The uncertainty of fibrils’ chiral sense holds only for fibrils grown within the physiological temperature range, while above 50 °C, the bifurcation is no longer observed—fibrils’ chiral moieties become uniformly biased towards ligand probes, as revealed by the extrinsic Cotton effect of thioflavin T, Congo red, and molecular iodine. According to transmission electron microscopy and scanning electron microscopy data, chiral variants of insulin fibrils consist of fibrous superstructures, distinct from spherulites, formed by the protein in nonagitated solutions. Gradual dissociation of the fibrils in the presence of dimethyl sulfoxide is noncooperative and can be resolved into three distinct phases: decay of the higher-order chiral structures, breakdown of fibrils, and unfolding of intermolecular β-sheet. The chiral aggregates are also destabilized by elution of NaCl implying that Debye screening of charged β-sheets provided by chloride counterions is needed for sustaining their kinetic stability. At elevated temperatures, cross-seeding of agitated insulin samples with preformed fibrils revealed a chiral conflict that prevented the passing of structural features of mother seeds to daughter fibrils in a manner typical of amyloid “strains.”     

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.     

Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Kinetics of aggregation of synthetic beta-amyloid peptide.

beta-Amyloid peptide is the major protein component of senile plaques and cerebrovascular amyloid deposits in patients with Alzheimer's disease. The peptide deposits extracellularly in the form of amyloid fibrils, in a cross-beta conformation. beta-amyloid peptide is a 39- to 43-residue segment of a normal membrane precursor protein. In this work, a peptide homologous to the first 40 amino acids of beta-amyloid peptide, beta(1-40), was synthesized and characterized. beta(1-40) exhibited a sharp change in solubility near physiological pH and gel formation at concentrations of 3 mg/ml or greater. Circular dichroism indicated that beta(1-40) contained approximately two-thirds beta-structure, but no alpha-helical character. Quasi-elastic and classical light scattering measurements showed that beta(1-40) aggregated end-to-end in solution, reaching average molecular weights greater than 4 x 10(6) after 13 days. The aggregates were best modeled as rigid rods of 5 nm diameter, similar to the diameter of amyloid fibrils purified from plaques. A mathematical model based on diffusion-limited aggregation was developed to describe the kinetics of aggregation.     

Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation

Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.     

Structural studies reveal that the diverse morphology of β2-microglobulin aggregates is a reflection of different molecular architectures

Amyloid deposition accompanies over 20 degenerative diseases in human, including Alzheimer's, Parkinson's, and prion diseases. Recent studies revealed the importance of other type of protein aggregates, e.g., non-specific aggregates, protofibrils, and small oligomers in the development of such diseases and proved their increased toxicity for living cells in comparison with mature amyloid fibrils. We carried out a comparative structural analysis of different monomeric and aggregated states of β₂-microglobulin, a protein responsible for hemodialysis-related amyloidosis. We investigated the structure of the native and acid-denatured states, as well as that of mature fibrils, immature fibrils, amorphous aggregates, and heat-induced filaments, prepared under various in vitro conditions. Infrared spectroscopy demonstrated that the β-sheet compositions of immature fibrils, heat-induced filaments and amorphous aggregates are characteristic of antiparallel intermolecular β-sheet structure while mature fibrils are different from all others suggesting a unique overall structure and assembly. Filamentous aggregates prepared by heat treatment are of importance in understanding the in vivo disease because of their stability under physiological conditions, where amyloid fibrils and protofibrils formed at acidic pH depolymerize. Atomic force microscopy of heat-induced filaments represented a morphology similar to that of the low pH immature fibrils. At a pH close to the pI of the protein, amorphous aggregates were formed readily with association of the molecules in native-like conformation, followed by formation of intermolecular β-sheet structure in a longer time-scale. Extent of the core buried from the solvent in the various states was investigated by H/D exchange of the amide protons.     

Amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the Alzheimer's beta(25-35) amyloid fibril

Amyloid-induced aggregation and precipitation of soluble proteins were investigated in vitro using the amyloid fibrils of the beta(25--35) peptide, a cytotoxic fragment of the Alzheimer's beta-peptide at positions 25--35. The aggregation rate of firefly luciferase was found to be modulated by both a chaperone molecule DnaK and the beta(25--35) amyloid, but their effects were opposite in direction. The amyloid fibril drastically facilitated the luciferase aggregation, which may define a kind of anti-chaperone activity. The effect of the beta(25--35) amyloid to promote protein aggregation and precipitation was further demonstrated for a wide variety of target proteins. The amount of coprecipitation was well correlated with the predicted isoelectric point of the target proteins, indicating that the interaction between the beta(25--35) amyloid and the target was driven by an electrostatic force between them. This view was confirmed by the experiments using an electrically neutral mutant peptide, beta(25--35)KA. It was also found that clustering of the beta(25--35) peptide to form amyloid and the conformation of the target protein are additional factors that determine the strength of the amyloid-protein interaction. Spectroscopic and electron microscopic methods have revealed that the proteins coprecipitated with the beta(25--35) amyloid formed amorphous aggregates deposited together with the amyloid fibrils. The conformation of protein molecules left in the residual soluble fraction was also damaged in the amyloid-containing solution. As a summary, this study has proposed a scheme for events related to the nonspecific amyloid-protein interaction, which may play substantial roles in in vivo conditions.