NMR-detected hydrogen exchange and molecular dynamics simulations provide structural insight into fibril formation of prion protein fragment 106-126

PrP106-126, a peptide corresponding to residues 107-127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. Although PrP106-126 has been shown to form amyloid-like fibrils in vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106-126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen-deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain approximately 50% beta-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106-126 in water assumes a stable structure consisting of two four-stranded parallel beta-sheets that are tightly packed against each other by methyl-methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations.     

Polyanion induced fibril growth enables the development of a reproducible assay in solution for the screening of fibril interfering compounds, and the investigation of the prion nucleation site.

The misfolded conformer of the prion protein (PrP) that aggregates into fibrils is believed to be the pathogenic agent in transmissible spongiform encephalopathies. In order to find fibril interfering compounds a screening assay in solution would be the preferred format to approximate more closely to physical conditions and enable the performance of kinetic studies. However, such an assay is hampered by the high irreproducibility because of the stochastic nature of the fibril formation process. According to published fibril models, the fibrillar core may be composed of stacked parallel beta-strands. In these models positive charge repulsion may reduce the chance of favorable stacking and cause the irreproducibility in the fibril formation. This study shows that the charge compensation by polyanions induced a very strong fibril growth which made it possible to develop a highly reproducible fibril interference assay. The stimulating effect of the polyanions depended on the presence of the basic residues Lys(106), Lys(110) and His(111). The assay was validated by comparison of the 50% fibril inhibition levels of peptide huPrP106-126 by six tetracyclic compounds. With this new assay, the fibrillogenic core (GAAAAGAVVG) of peptide huPrP106-126 was determined and for the first time it was possible to test the inhibition potentials of peptide analogues. Also it was found that variants of peptide huPrP106-126 with proline substitutions at positions Ala(115), Ala(120), or Val(122) inhibited the fibril formation of huPrP106-126.     

Prion protein preamyloid and amyloid deposits in Gerstmann-Str?ussler-Scheinker disease,Indiana kindred.

Gerstmann-Sträussler-Scheinker disease (GSS) is a familial neurological disorder pathologically characterized by amyloid deposition in the cerebrum and cerebellum. In GSS, the amyloid is immunoreactive to antisera raised against the prion protein (PrP) 27-30, a proteinase K-resistant peptide of 27-30 kDa that is derived by limited proteolysis from an abnormal isoform of a neuronal sialoglycoprotein of 33-35 kDa designated PrPSc. Polyclonal antibodies raised against synthetic peptides homologous to residues 15-40 (P2), 90-102 (P1), and 220-232 (P3) of the amino acid sequence deduced from hamster PrP cDNA were used to investigate immunohistochemically the distribution of PrP and PrP fragments in the brains of two patients from the Indiana kindred of GSS. Two types of anti-PrP-immunoreactive deposits were found: (i) amyloid deposits, which were exclusively labeled by anti-P1 antiserum to residues 90-102 of PrP, and (ii) preamyloid deposits, which were labeled by all anti-PrP antisera but did not exhibit the tinctorial and optical properties of amyloid. The latter appeared as diffuse immunostaining of the neuropil that targeted to areas in which amyloid deposits were most abundant. They were partially resistant to proteinase K digestion and consisted ultrastructurally of amorphous, flaky, electron-dense material. These findings substantiate our previous observation that the major amyloid component in the GSS Indiana kindred is an internal fragment of PrP and indicate that full-length abnormal isoforms of PrP and/or large PrP fragments accumulate in brain regions most affected by amyloid deposition. These findings support the view that in the GSS Indiana kindred a stepwise degradation of PrP occurs in situ in the process of amyloid fibril formation.     

Polyanion induced fibril growth enables the development of a reproducible assay in solution for the screening of fibril interfering compounds,and the investigation of the prion nucleation site

The misfolded conformer of the prion protein (PrP) that aggregates into fibrils is believed to be the pathogenic agent in transmissible spongiform encephalopathies. In order to find fibril interfering compounds a screening assay in solution would be the preferred format to approximate more closely to physical conditions and enable the performance of kinetic studies. However, such an assay is hampered by the high irreproducibility because of the stochastic nature of the fibril formation process. According to published fibril models, the fibrillar core may be composed of stacked parallel β-strands. In these models positive charge repulsion may reduce the chance of favorable stacking and cause the irreproducibility in the fibril formation. This study shows that the charge compensation by polyanions induced a very strong fibril growth which made it possible to develop a highly reproducible fibril interference assay. The stimulating effect of the polyanions depended on the presence of the basic residues Lys¹⁰⁶, Lys¹¹⁰ and His¹¹¹. The assay was validated by comparison of the 50% fibril inhibition levels of peptide huPrP106-126 by six tetracyclic compounds. With this new assay, the fibrillogenic core (GAAAAGAVVG) of peptide huPrP106-126 was determined and for the first time it was possible to test the inhibition potentials of peptide analogues. Also it was found that variants of peptide huPrP106-126 with proline substitutions at positions Ala¹¹⁵, Ala¹²⁰, or Val¹²² inhibited the fibril formation of huPrP106-126.     

Molecular determinants for amyloid fibril formation: lessons from lung surfactant protein C

Amyloid fibrils are polymers composed of proteins in beta-sheet conformation, which are found in at least 20 diseases for which no cure is available. These diseases include Alzheimer's disease and spongiform encephalopathies, in which the amyloid beta-peptide and the prion protein (PrP), respectively, form amyloid. All fibrils are morphologically very similar although the native structures of the corresponding proteins are widely different. Proteins that are not known to form fibrils in vivo can do so under conditions where unfolding intermediates are well populated. This indicates that fibril formation can arise from most, if not all, polypeptide chains under certain conditions, and that nature has found ways to avoid fibrillogenic protein conformations. In support of this, it was recently found that unpaired beta-strands present in native proteins are prevented from forming intermolecular beta-sheets, by strategic placement of prolines and charged residues for example. Structural studies of the lung surfactant-associated protein C (SP-C) have revealed determinants for amyloid fibril formation. The poly-valine alpha-helix of SP-C spontaneously converts to beta-sheet aggregates in vitro and SP-C amyloid fibrils are found in pulmonary alveolar proteinosis. A beta, PrP, and SP-C harbour an alpha-helix which is strongly predicted to form a beta-strand, and in all cases investigated so far such alpha-helix/beta-sheet discordance correlates with the ability to form beta-sheet aggregates and fibrils.     

Beta-sheet core of human prion protein amyloid fibrils as determined by hydrogen/deuterium exchange

Propagation of transmissible spongiform encephalopathies is associated with the conversion of normal prion protein, PrP(C), into a misfolded, oligomeric form, PrP(Sc). Although the high-resolution structure of the PrP(C) is well characterized, the structural properties of PrP(Sc) remain elusive. Here we used MS analysis of H/D backbone amide exchange to examine the structure of amyloid fibrils formed by the recombinant human PrP corresponding to residues 90-231 (PrP90-231), a misfolded form recently reported to be infectious in transgenic mice overexpressing PrP(C). Analysis of H/D exchange data allowed us to map the systematically H-bonded beta-sheet core of PrP amyloid to the C-terminal region (staring at residue approximately 169) that in the native structure of PrP monomer corresponds to alpha-helix 2, a major part of alpha-helix 3, and the loop between these two helices. No extensive hydrogen bonding (as indicated by the lack of significant protection of amide hydrogens) was detected in the N-terminal part of PrP90-231 fibrils, arguing against the involvement of residues within this region in stable beta-structure. These data provide long-sought experimentally derived constraints for high-resolution structural models of PrP amyloid fibrils.     

A kinetic model for amyloid formation in the prion diseases: importance of seeding.

The transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by amyloid formation in the brain. The major amyloid protein is the prion protein (PrP). PrP and the beta-amyloid protein of Alzheimer disease share a similar sequence that, in both cases, may be responsible for the initiation of protein aggregation in vivo. We report here that a peptide based on this sequence in PrP (PrP96-111M) forms amyloid fibrils. The existence of a kinetic barrier to amyloid formation by this peptide was demonstrated, suggesting that formation of an ordered nucleus is the rate-determining step for aggregation. Seeding was demonstrated to occur with PrP96-111M amyloid fibrils but not with amyloid fibrils of a related peptide. This effect is consistent with the proposal that the aggregation of PrP, which characterizes TSE, involves a nucleation event analogous to the seeding of a crystallization.     

Amyloid peptide channels: blockade by zinc and inhibition by Congo red (amyloid channel block).

Amyloid peptides are the major constituents of amyloid deposits in various amyloid diseases including Alzheimer's disease, type II diabetes mellitus, prion diseases and others. The hallmark of amyloid is the binding of the dye, Congo red, which creates characteristic staining due to the dye's ability to bind the beta sheet aggregates referred to as amyloid. Previous reports have demonstrated that several cytotoxic, amyloidogenic peptides can form ion channels in planar phospholipid bilayer membranes and have suggested that these channels may represent the pathogenic mechanism of cell and tissue destruction in amyloid disease. Furthermore, zinc and Congo red can ameliorate or prevent the pathogenic effect of certain amyloidpeptides. We report here that zinc at micromolar concentrations caused a reversible blockade of islet amyloid polypeptide (IAPP, amylin) and PrP 106-126 channels whereas calcium and magnesium did not. Congo red completely inhibited channel formation if preincubated with amyloid peptides, but had no effect on IAPP or PrP 106-126 channels once formed. These results suggest a requirement for aggregation for the formation of amyloid peptide channels and are consistent with the "channel hypothesis" of amyloid disease. They also suggest potential avenues for ameliorative therapy of these illnesses.     

Nucleation-dependent conformational conversion of the Y145Stop variant of human prion protein: structural clues for prion propagation

One of the most intriguing disease-related mutations in human prion protein (PrP) is the Tyr to Stop codon substitution at position 145. This mutation results in a Gerstmann-Straussler-Scheinker-like disease with extensive PrP amyloid deposits in the brain. Here, we provide evidence for a spontaneous conversion of the recombinant polypeptide corresponding to the Y145Stop variant (huPrP23-144) from a monomeric unordered state to a fibrillar form. This conversion is characterized by a protein concentration-dependent lag phase and has characteristics of a nucleation-dependent polymerization. Atomic force microscopy shows that huPrP23-144 fibrils are characterized by an apparent periodicity along the long axis, with an average period of 20 nm. Fourier-transform infrared spectra indicate that the conversion is associated with formation of beta-sheet structure. However, the infrared bands for huPrP23-144 are quite different from those for a synthetic peptide PrP106-126, suggesting conformational non-equivalence of beta-structures in the disease-associated Y145Stop variant and a frequently used short model peptide. To identify the region that is critical for the self-seeded assembly of huPrP23-144 amyloid, experiments were performed by using the recombinant polypeptides corresponding to prion protein fragments 23-114, 23-124, 23-134, 23-137, 23-139, and 23-141. Importantly, none of the fragments ending before residue 139 showed a propensity for conformational conversion to amyloid fibrils, indicating that residues within the 138-141 region are essential for this conversion.     

Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid.

By comparing the amino acid sequences of 11 mammalian and 1 avian prion proteins (PrP), structural analyses predicted four alpha-helical regions. Peptides corresponding to these regions of Syrian hamster PrP were synthesized, and, contrary to predictions, three of the four spontaneously formed amyloids as shown by electron microscopy and Congo red staining. By IR spectroscopy, these amyloid peptides exhibited secondary structures composed largely of beta-sheets. The first of the predicted helices is the 14-amino acid peptide corresponding to residues 109-122; this peptide and the overlapping 15-residue sequence 113-127 both form amyloid. The most highly amyloidogenic peptide is AGAAAAGA, which corresponds to Syrian hamster PrP residues 113-120 and is conserved across all species for which the PrP sequence has been determined. Two other predicted alpha-helices corresponding to residues 178-191 and 202-218 form amyloids and exhibit considerable beta-sheet structure when synthesized as peptides. These findings suggest the possibility that the conversion of the cellular isoform of PrP to the scrapie isoform of PrP involves the transition of one or more putative PrP alpha-helices into beta-sheets and that prion diseases are disorders of protein conformation.     

De novo designed peptide-based amyloid fibrils

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimer's disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in beta-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric beta-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into beta-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric beta-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed beta-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is +/-1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils.     

Vascular variant of prion protein cerebral amyloidosis with tau-positive neurofibrillary tangles: the phenotype of the stop codon 145 mutation in PRNP.

Deposition of PrP amyloid in cerebral vessels in conjunction with neurofibrillary lesions is the neuropathologic hallmark of the dementia associated with a stop mutation at codon 145 of PRNP, the gene encoding the prion protein (PrP). In this disorder, the vascular amyloid in tissue sections and the approximately 7.5-kDa fragment extracted from amyloid are labeled by antibodies to epitopes located in the PrP sequence including amino acids 90-147. Amyloid-laden vessels are also labeled by antibodies against the C terminus, suggesting that PrP from the normal allele is involved in the pathologic process. Abundant neurofibrillary lesions are present in the cerebral gray matter. They are composed of paired helical filaments, are labeled with antibodies that recognize multiple phosphorylation sites in tau protein, and are similar to those observed in Alzheimer disease. A PrP cerebral amyloid angiopathy has not been reported in diseases caused by PRNP mutations or in human transmissible spongiform encephalopathies; we propose to name this phenotype PrP cerebral amyloid angiopathy (PrP-CAA).     

Paired beta-sheet structure of an Abeta(1-40) amyloid fibril revealed by electron microscopy

Alzheimer's disease is a neurodegenerative disorder that is characterized by the cerebral deposition of amyloid fibrils formed by Abeta peptide. Despite their prevalence in Alzheimer's and other neurodegenerative diseases, important details of the structure of amyloid fibrils remain unknown. Here, we present a three-dimensional structure of a mature amyloid fibril formed by Abeta(1-40) peptide, determined by electron cryomicroscopy at approximately 8-A resolution. The fibril consists of two protofilaments, each containing approximately 5-nm-long regions of beta-sheet structure. A local twofold symmetry within each region suggests that pairs of beta-sheets are formed from equivalent parts of two Abeta(1-40) peptides contained in each protofilament. The pairing occurs via tightly packed interfaces, reminiscent of recently reported steric zipper structures. However, unlike these previous structures, the beta-sheet pairing is observed within an amyloid fibril and includes significantly longer amino acid sequences.     

Evidence for assembly of prions with left-handed beta-helices into trimers

Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross-beta structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP(Sc)) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right- or left-handed beta-helical models. From a study of 119 all-beta folds observed in globular proteins, we have now determined that, if PrP(Sc) follows a known protein fold, it adopts either a beta-sandwich or parallel beta-helical architecture. With increasing evidence arguing for a parallel beta-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed beta-helical fold. Left-handed beta-helices readily form trimers, providing a natural template for a trimeric model of PrP(Sc). This trimeric model accommodates the PrP sequence from residues 89-175 in a beta-helical conformation with the C terminus (residues 176-227), retaining the disulfide-linked alpha-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed beta-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed beta-helical model for PrP(Sc) may provide important clues to the structure of filaments found in some other neurodegenerative diseases.     

The fold of alpha-synuclein fibrils

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson's disease it is believed that the aggregation of alpha-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of alpha-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta1-strand interacts with beta2, beta2 with beta3, beta3 with beta4, and beta4 with beta5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximately 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.     

Abeta amyloid fibrils possess a core structure highly resistant to hydrogen exchange

We describe here experiments designed to characterize the secondary structure of amyloid fibrils of the Alzheimer's amyloid plaque peptide Abeta, using hydrogen-deuterium exchange measurements evaluated by mass spectrometry. The results show that approximately 50% of the amide protons of the polypeptide backbone of Abeta(1-40) resist exchange in aqueous, neutral pH buffer even after more than 1, 000 h of incubation at room temperature. We attribute this extensive, strong protection to H-bonding by residues in core regions of beta-sheet structure within the fibril. The backbone amide hydrogens exchange at variable rates, suggesting different degrees of protection within the fibril. These data suggest that it is unlikely that the entire Abeta sequence is involved in H-bonded secondary structure within the amyloid fibril. Future studies using the methods described here should reveal further details of Abeta fibril structure and assembly. These methods also should be amenable to studies of other amyloid fibrils and protein aggregates.     

Molecular dynamics simulations of spontaneous fibril formation by random-coil peptides

Assembly of normally soluble proteins into amyloid fibrils is a cause or associated symptom of numerous human disorders, including Alzheimer's and the prion diseases. We report molecular-level simulation of spontaneous fibril formation. Systems containing 12-96 model polyalanine peptides form fibrils at temperatures greater than a critical temperature that decreases with peptide concentration and exceeds the peptide's folding temperature, consistent with experimental findings. Formation of small amorphous aggregates precedes ordered nucleus formation and subsequent rapid fibril growth through addition of beta-sheets laterally and monomeric peptides at fibril ends. The fibril's structure is similar to that observed experimentally.     

Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation.

Islet amyloid polypeptide (IAPP), a putative polypeptide hormone, is a product of pancreatic beta-cells and the major constituent of the amyloid deposits seen mainly in islets of type 2 diabetic humans and diabetic cats. The connection between IAPP amyloid formation and diabetes is unknown, but a limited segment of the IAPP molecule, positions 20-29, seems responsible for the aggregation to fibrils. Differences in the amino acid sequence of this region probably determine whether or not islet amyloid can develop in a particular species. Amyloid fibril formation can be mimicked in vitro with the aid of synthetic peptides. With this technique we show that peptides corresponding to IAPP positions 20-29 of human and cat, species that develop IAPP-derived islet amyloid, form amyloid-like fibrils in vitro. The corresponding IAPP segment from three rodent species that do not develop IAPP-derived amyloid did not give rise to fibrils. Substitution of the human IAPP-(20-29) decapeptide with one or two amino acid residues from species without islet amyloid generally reduced the capacity to form fibrils. We conclude that the sequence Ala-Ile-Leu-Ser-Ser, corresponding to positions 25-29 of human IAPP, is strongly amyloidogenic and that a proline-for-serine substitution in position 28, as in several rodents, almost completely inhibits formation of amyloid fibrils.     

Molecular architecture of human prion protein amyloid: a parallel, in-register beta-structure

Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and alpha-helical prion protein, PrP(C), to the beta-sheet-rich PrP(Sc). This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP(C) monomer, structures of the pathogenic PrP(Sc) or synthetic PrP(Sc)-like aggregates remain elusive. Here we have used site-directed spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP(C). Our data show that, in contrast to earlier, largely theoretical models, the con formational conversion of PrP(C) involves major refolding of the C-terminal alpha-helical region. The core of the amyloid maps to C-terminal residues from approximately 160-220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of beta-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to beta-structure.