Conversion between parallel and antiparallel ?-sheets in wild-type and Iowa mutant A?40 fibrils.
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ABSTRACT:
SUBMITTER: Xi W
PROVIDER: S-EPMC5785298 | biostudies-literature | 2018 Jan
REPOSITORIES: biostudies-literature
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Project description:Wild-type, full-length (40- and 42-residue) amyloid β-peptide (Aβ) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-β structures in which the β-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aβ (D23N-Aβ(1-40)), which is associated with early onset neurodegeneration, indicate that D23N-Aβ(1-40) fibrils can contain either parallel or antiparallel β-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aβ(1-40) fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aβ(1-40) fibril structure. This model reveals how both parallel and antiparallel cross-β structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aβ(1-40) fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aβ(1-40) fibrils are cytotoxic. Thus, our antiparallel D23N-Aβ(1-40) fibril model represents a specific "toxic intermediate" in the aggregation process of a disease-associated Aβ mutant.
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Project description:Fibrillization or conformational change of alpha-synuclein is central in the pathogenesis of alpha-synucleinopathies, such as Parkinson disease. We found that the A30P mutant accelerates nucleation-dependent fibrillization of wild type (WT) alpha-synuclein. Electron microscopy observation and ultracentrifugation experiments revealed that shedding of fragments occurs from A30P fibrils and that these fragments accelerate fibrillization by serving as seeds. Immunochemical analysis using epitope-specific antibodies and biochemical analyses of protease-resistant cores demonstrated that A30P fibrils have a distinct conformation. Interestingly, WT fibrils formed with A30P seeds exhibited the same character as A30P fibrils, as did A30P fibrils formed with WT seeds, indicating that the A30P mutation affects the conformation and fibrillization of both WT and A30P. These effects of A30P mutation may explain the apparent conflict between the association of A30P with Parkinson disease and the slow fibrillization of A30P itself and therefore provide new insight into the molecular mechanisms of alpha-synucleinopathies.
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Project description:Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue-long β-amyloid peptide (Aβ40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aβ42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel β-sheet, whereas previous studies by other researchers have reported parallel β-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.
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Project description:Recent experiments indicate a connection between the structure of amyloid aggregates and their cytotoxicity as related to neurodegenerative diseases. Of particular interest is the Iowa Mutant, which causes early-onset of Alzheimer's disease. While wild-type Amyloid ?-peptides form only parallel beta-sheet aggregates, the mutant also forms meta-stable antiparallel beta sheets. Since these structural variations may cause the difference in the pathological effects of the two A?-peptides, we have studied in silico the relative stability of the wild type and Iowa mutant in both parallel and antiparallel forms. We compare regular molecular dynamics simulations with such where the viscosity of the samples is reduced, which, we show, leads to higher sampling efficiency. By analyzing and comparing these four sets of all-atom molecular dynamics simulations, we probe the role of the various factors that could lead to the structural differences. Our analysis indicates that the parallel forms of both wild type and Iowa mutant aggregates are stable, while the antiparallel aggregates are meta-stable for the Iowa mutant and not stable for the wild type. The differences result from the direct alignment of hydrophobic interactions in the in-register parallel oligomers, making them more stable than the antiparallel aggregates. The slightly higher thermodynamic stability of the Iowa mutant fibril-like oligomers in its parallel organization over that in antiparallel form is supported by previous experimental measurements showing slow inter-conversion of antiparallel aggregates into parallel ones. Knowledge of the mechanism that selects between parallel and antiparallel conformations and determines their relative stability may open new avenues for the development of therapies targeting familial forms of early-onset Alzheimer's disease.
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Project description:The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1-83) of apoA-I containing this mutation deposit as amyloid fibrils in patients' tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis.
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Project description:Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.
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Project description:Cerebral Amyloid Angiopathy (CAA), which involves amyloid deposition in blood vessels leading to fatal cerebral hemorrhage and recurring strokes, is present in the majority Alzheimer's disease cases. Familial mutations in the amyloid β peptide is correlated to higher risks of CAA, and are mostly comprised of mutations at residues 22 and 23. While the structure of the wild type Aβ peptide has been investigated in great detail, less is known about the structure of mutants involved in CAA and evolutions thereof. This is particularly true for mutations at residue 22, for which detailed molecular structures, as typically determined from Nuclear Magnetic Resonance (NMR) spectroscopy or electron microscopy, do not exist. In this report, we have used nanoscale infrared (IR) spectroscopy augmented with Atomic Force Microscopy (AFM-IR) to investigate structural evolution of the Aβ Dutch mutant (E22Q) at the single aggregate level. We show that that in the oligomeric stage, the structural ensemble is distinctly bimodal, with the two subtypes differing with respect to population of parallel β-sheets. Fibrils on the other hand are structurally homogeneous, with early-stage fibrils distinctly anti parallel in character, which develop parallel β-sheets upon maturation. Furthermore, the antiparallel structure is found to be a persistent feature across different stages of aggregation.
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Project description:The ?E693 (Japanese) mutation of the ?-amyloid precursor protein leads to production of ?E22-A? peptides such as ?E22-A?(1-39). Despite reports that these peptides do not form fibrils, here we show that, on the contrary, the peptide forms fibrils essentially instantaneously. The fibrils are typical amyloid fibrils in all respects except that they cause only low levels of thioflavin T (ThT) fluorescence, which, however, develops with no lag phase. The fibrils bind ThT, but with a lower affinity and a smaller number of binding sites than wild-type (WT) A?(1-40). Fluorescence depolarization confirms extremely rapid aggregation of ?E22-A?(1-39). Size exclusion chromatography (SEC) indicates very low concentrations of soluble monomer and oligomer, but only in the presence of some organic solvent, e.g., 2% (v/v) DMSO. The critical concentration is approximately 1 order of magnitude lower for ?E22-A?(1-39) than for WT A?(1-40). Several lines of evidence point to an altered structure for ?E22-A?(1-39) compared to that of WT A?(1-40) fibrils. In addition to differences in ThT binding and fluorescence, PITHIRDS-CT solid-state nuclear magnetic resonance (NMR) measurements of ?E22-A?(1-39) are not compatible with the parallel in-register ?-sheet generally observed for WT A?(1-40) fibrils. X-ray fibril diffraction showed different D spacings: 4.7 and 10.4 Å for WT A?(1-40) and 4.7 and 9.6 Å for ?E22-A?(1-39). Equimolar mixtures of ?E22-A?(1-39) and WT A?(1-40) also produced fibrils extremely rapidly, and by the criteria of ThT fluorescence and electron microscopic appearance, they were the same as fibrils made from pure ?E22-A?(1-39). X-ray diffraction of fibrils formed from 1:1 molar mixtures of ?E22-A?(1-39) and WT A?(1-40) showed the same D spacings as fibrils of the pure mutant peptide, not the wild-type peptide. These findings are consistent with extremely rapid nucleation by ?E22-A?(1-39), followed by fibril extension by WT A?(1-40), and "conversion" of the wild-type peptide to a structure similar to that of the mutant peptide, in a manner reminiscent of the prion conversion phenomenon.
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Project description:We present solid-state NMR measurements of β-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-β peptide (Aβ(1-42)). We build upon our previous report of a β-strand spanned by residues 30-42, which arranges into an antiparallel β-sheet. New results presented here indicate that there is a second β-strand formed by residues 11-24. Contrary to expectations, NMR data indicate that this second β-strand is organized into a parallel β-sheet despite the co-existence of an antiparallel β-sheet in the same structure. In addition, the in-register parallel β-sheet commonly observed for amyloid fibril structure does not apply to residues 11-24 in the 150-kDa oligomer. Rather, we present evidence for an inter-strand registry shift of three residues that likely alternate in direction between adjacent molecules along the β-sheet. We corroborated this unexpected scheme for β-strand organization using multiple two-dimensional NMR and 13C-13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes.
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Project description:To help elucidate the role of secondary structure packing preferences in protein folding, here we present an analysis of the packing geometry observed between alpha-helices and between alpha-helices and beta-sheets in 1316 diverse, nonredundant protein structures. Finite-length vectors were fit to the alpha-carbon atoms in each of the helices and strands, and the packing angle between the vectors, Omega, was determined at the closest point of approach within each helix-helix or helix-sheet pair. Helix-sheet interactions were found in 391 of the proteins, and the distributions of Omega values were calculated for all the helix-sheet and helix-helix interactions. The packing angle preferences for helix-helix interactions are similar to those previously observed. However, analysis of helix-strand packing preferences uncovered a remarkable tendency for helices to align antiparallel to parallel regions of beta-sheets, independent of the topological constraints or prevalence of beta-alpha-beta motifs in the proteins. This packing angle preference is significantly diminished in helix interactions involving mixed and antiparallel beta-sheets, suggesting a role for helix-sheet dipole alignment in guiding supersecondary structure formation in protein folding. This knowledge of preferred packing angles can be used to guide the engineering of stable protein modules.
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