Structural polymorphism of Alzheimer Aβ and other amyloid fibrils

Deposits of amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer’s disease, Creutzfeldt-Jakob disease and type II diabetes. Amyloid fibrils formed from different polypeptides contain a common cross-β spine. Nevertheless, amyloid fibrils formed from the same polypeptide can occur in a range of structurally different morphologies. The heterogeneity of amyloid fibrils reflects different types of polymorphism: (i) variations in the protofilament number, (ii) variations in the protofilament arrangement and (iii) different polypeptide conformations. Amyloid fibril polymorphism implies that fibril formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.     

Structural polymorphism of Alzheimer A�� and other amyloid fibrils

Deposits of amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer disease, Creutzfeldt-Jakob disease and type II diabetes. Amyloid fibrils formed from different polypeptides contain a common cross-β spine. Nevertheless, amyloid fibrils formed from the same polypeptide can occur in a range of structurally different morphologies. The heterogeneity of amyloid fibrils reflects different types of polymorphism: (1) variations in the protofilament number, (2) variations in the protofilament arrangement and (3) different polypeptide conformations. Amyloid fibril polymorphism implies that fibril formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.Key words: Alzheimer disease, aggregation, neurodegeneration, prion, protein folding     

Structural dynamics of the amyloid β-protein monomer folding nucleus

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.     

Lipid composition influences the release of Alzheimer's amyloid β-peptide from membranes

The behavior of the amyloid β-peptide (Aβ) within a membrane environment is integral to its toxicity and the progression of Alzheimer's disease. Ganglioside GM1 has been shown to enhance the aggregation of Aβ, but the underlying mechanism is unknown. Using atomistic molecular dynamics simulations, we explored the interactions between the 40-residue alloform of Aβ (Aβ(40) ) and several model membranes, including pure palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylserine (POPS), an equimolar mixture of POPC and palmitoyloleoylphosphatidylethanolamine (POPE), and lipid rafts, both with and without GM1, to understand the behavior of Aβ(40) in various membrane microenvironments. Aβ(40) remained inserted in POPC, POPS, POPC/POPE, and raft membranes, but in several instances exited the raft containing GM1. Aβ(40) interacted with GM1 largely through hydrogen bonding, producing configurations containing β-strands with C-termini that, in some cases, exited the membrane and became exposed to solvent. These observations provide insight into the release of Aβ from the membrane, a previously uncharacterized process of the Aβ aggregation pathway.     

The redox chemistry of the Alzheimer's disease amyloid β peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid β peptide (Aβ), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Aβ from the brain. Aβ can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Aβ will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Aβ oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Aβ levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Aβ species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.     

Evolutionary origin of a Kunitz-type trypsin inhibitor domain inserted in the amyloid β precursor protein of Alzheimer's disease

Summary The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid precursor protein (APP). It is known that APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhibitors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present APP gene diverged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in APP have an evolutionarily close relationship with the inter--trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membrane-bound serine protease in human T4⁺ lymphocytes.Offprint requests to: T. Gojobori     

Structural stability of amyloid fibrils of β2-microglobulin in comparison with its native fold

Among various amyloidogenic proteins, β₂-microglobulin (β2-m) responsible for dialysis-related amyloidosis is a target of extensive study because of its clinical importance and suitable size for examining the formation of amyloid fibrils in comparison with protein folding to the native state. The structure and stability of amyloid fibrils have been studied with various physicochemical methods, including H/D exchange of amyloid fibrils combined with dissolution of fibrils by dimethylsulfoxide and NMR analysis, thermodynamic analysis of amyloid fibril formation by isothermal calorimetry, and analysis of the effects of pressure on the structure of amyloid fibrils. The results are consistent with the view that amyloid fibrils are a main-chain-dominated structure with larger numbers of hydrogen bonds and pressure-accessible cavities in the interior, in contrast to the side-chain-dominated native structure with the optimal packing of amino acid residues. We consider that a main-chain dominated structure provides the structural basis for various conformational states even with one protein. When this feature is combined with another unique feature, template-dependent growth, propagation and maturation of the amyloid conformation, which cannot be predicted with Anfinsen's dogma, take place.     

Molecular biology of Alzheimer's amyloid—Dutch variant

Hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) (or familial cerebral amyloid angiopathy) and familial Alzheimer's disease (FAD) share several properties. Both are autosomal dominant forms of cerebral amyloidosis characterized by beta-amyloid (A beta) deposition. In HCHWA-D the A beta is predominantly found in blood vessels and in early parenchymal plaques, whereas in AD parenchymal A beta deposits in the form of senile plaques and neurofibrillary tangles are a more prominent finding. Point mutations in the amyloid precursor protein (APP) have recently been described, in both conditions. A G to C transversion at codon 618 (extracellular portion of APP695), producing a single amino acid substitution of glutamine instead of glutamine acid, occurs in HCHWA-D; whereas mutations at codon 642 in the intramembrane region of APP695 (phenylalanine, isoleucine, or glycine instead of valine) are associated with early onset FAD. This suggests that the site of particular mutations in the APP gene and the type of amino acid substitution in the APP holoprotein are more important in determining clinicopathological phenotype and age at which A beta is deposited. Thus FAD and HCHWA-D can be regarded as two sides of the same coin.     

Extracellular phosphorylation of the amyloid β-peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β-peptides (Aβ) in the brain. Aβ derives by sequential proteolytic processing of the amyloid precursor protein by β- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aβ domain promote the formation of neurotoxic Aβ assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aβ and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aβ undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ assemblies that represent nuclei for fibrillization. Phosphorylated Aβ was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aβ. Phosphorylation of Aβ could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.     

Formation of amyloid fibrils from β-amylase

Fibril formation has been considered a significant feature of amyloid proteins. However, it has been proposed that fibril formation is a common property of many proteins under appropriate conditions. We studied the fibril formation of β-amylase, a non-amyloid protein rich in α-helical structure, because the secondary structure of β-amylase is similar to that of prions. With the conditions for the fibril formation of prions, β-amylase proteins were converted into amyloid fibrils. The features of β-amylase proteins and fibrils are compared to prion proteins and fibrils. Furthermore, the cause of neurotoxicity in amyloid diseases is discussed.     

Surfactant-induced conformational transition of amyloid β-peptide

Accumulating evidence suggests that Aβ_1–42–membrane interactions may play an important role in the pathogenesis of Alzheimer’s disease. However, the mechanism of this structural transition remains unknown. In this work, we have shown that submicellar concentrations of sodium dodecyl sulfate (SDS) can provide a minimal platform for Aβ_1–42 self-assembly. To further investigate the relation between Aβ_1–42 structure and function, we analyzed peptide conformation and aggregation at various SDS concentrations using circular dichroism (CD), Fourier transform infrared spectroscopy, and gel electrophoresis. These aggregates, as observed via atomic force microscopy, appeared as globular particles in submicellar SDS with diameters of 35–60 nm. Upon sonication, these particles increased in disc diameter to 100 nm. Pyrene I ₃/I ₁ ratios and 1-anilinonaphthalene-8-sulfonic acid binding studies indicated that the peptide interior is more hydrophobic than the SDS micelle interior. We have also used Forster resonance energy transfer between N-terminal labeled pyrene and tyrosine (10) of Aβ_1–42 in various SDS concentrations for conformational analysis. The results demonstrate that SDS at submicellar concentrations accelerates the formation of spherical aggregates, which act as niduses to form large spherical aggregates upon sonication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proteomics of β2-microglobulin amyloid fibrils

Knowledge on the chemical structure of β2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of β2-microglobulin extracted from non-osteotendinous tissues. β2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to β2-microglobulin derived from fibrils but is not observed in the β2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar β2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1–2% of the intact molecule.     

Structural basis of C-terminal β-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.     

Structural insights into mechanisms for inhibiting amyloid β42 aggregation by non-catechol-type flavonoids

The prevention of 42-mer amyloid β-protein (Aβ42) aggregation is promising for the treatment of Alzheimer’s disease. We previously described the site-specific inhibitory mechanism for Aβ42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aβ42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aβ42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The ¹H–¹⁵N SOFAST-HMQC NMR of Aβ42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular β-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aβ42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aβ42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aβ42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aβ42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aβ42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aβ42 aggregation.     

Backpropagation neural network analysis applied to β-sheet breakers used against Alzheimer's amyloid aggregation

Structure-activity relationships of β-sheet inhibitors against Alzheimer's Aβ(1–42) amyloid aggregation were studied by backpropagation neural network analysis. It was found that the total and electrostatic energies of geometry-optimized conformations of the ligands, and the hydration energy, simulate the biological potency.     

Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.     

Effects of amyloid β-protein on hippocampal long-term potentiation

The accumulation of amyloid β-protein (Aβ) plaques is identified as a major pathological feature of Alzheimer's disease (AD). Recent studies show that soluble species of Aβ are involved in the early memory dysfunction long before neurodegenerative changes. However, the mechanism underlying the neurotoxicity of soluble Aβ is still unclear. Long-term potentiation (LTP) has been thought as an important cellular model of synaptic plasticity for many years. The studies on the hippocampal LTP and Aβ, especially those using AD transgenic models, provided more evidence for the Aβ-induced dysfunction of learning and memory. Based on the recent researches on AD, this article reviewed the effects of Aβ, especially soluble Aβ and its active fragments, on the hippocampal LTP. The possible mechanisms by which Aβ impairs hippocampal LTP are also discussed.