Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.     

Consensus prediction of amyloidogenic determinants in amyloid fibril-forming proteins

We combine the results of three prediction algorithms on a test set of 21 amyloidogenic proteins to predict amyloidogenic determinants. Two prediction algorithms are recently developed prediction algorithms of amyloidogenic stretches in protein sequences, whereas the third is a secondary structure prediction algorithm capable of identifying 'conformational switches' (regions that have both the propensity for alpha-helix and beta-sheet). Surprisingly, the results of prediction agree well and also agree with experimentally investigated amyloidogenic regions. Furthermore, they suggest several previously not identified amino acid stretches as potential amyloidogenic determinants. Most predicted (and experimentally observed) amyloidogenic determinants reside on the protein surface of relevant solved crystal structures. It appears that a consensus prediction algorithm is more objective than individual prediction methods alone.     

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.     

Identification of amyloid fibril-forming segments based on structure and residue-based statistical potential

MOTIVATION: Experimental evidence suggests that certain short protein segments have stronger amyloidogenic propensities than others. Identification of the fibril-forming segments of proteins is crucial for understanding diseases associated with protein misfolding and for finding favorable targets for therapeutic strategies. RESULT: In this study, we used the microcrystal structure of the NNQQNY peptide from yeast prion protein and residue-based statistical potentials to establish an algorithm to identify the amyloid fibril-forming segment of proteins. Using the same sets of sequences, a comparable prediction performance was obtained from this study to that from 3D profile method based on the physical atomic-level potential ROSETTADESIGN. The predicted results are consistent with experiments for several representative proteins associated with amyloidosis, and also agree with the idea that peptides that can form fibrils may have strong sequence signatures. Application of the residue-based statistical potentials is computationally more efficient than using atomic-level potentials and can be applied in whole proteome analysis to investigate the evolutionary pressure effect or forecast other latent diseases related to amyloid deposits. AVAILABILITY: The fibril prediction program is available at ftp://mdl.ipc.pku.edu.cn/pub/software/pre-amyl/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Prediction of amyloid aggregation in vivo

Many human diseases owe their pathology, to some degree, to the erroneous conversion of proteins from their soluble state into fibrillar, β-structured aggregates, often referred to as amyloid fibrils. Neurodegenerative diseases, such as Alzheimer and spongiform encephalopathies, as well as type 2 diabetes and both localized and systemic amyloidosis, are among the conditions that are associated with the formation of amyloid fibrils. Several mathematical tools can rationalize and even predict important parameters of amyloid fibril formation. It is not clear, however, whether such algorithms have predictive powers for in vivo systems, in which protein aggregation is affected by the presence of other biological factors. In this review, we briefly describe the existing algorithms and use them to predict the effects of mutations on the aggregation of specific proteins, for which in vivo experimental data are available. The comparison between the theoretical predictions and the experimental data obtained in vivo is shown for each algorithm and experimental data set, and statistically significant correlations are found in most cases.     

The amyloid state of proteins in human diseases

Amyloid fibers and oligomers are associated with a great variety of human diseases including Alzheimer's disease and the prion conditions. Here we attempt to connect recent discoveries on the molecular properties of proteins in the amyloid state with observations about pathological tissues and disease states. We summarize studies of structure and nucleation of amyloid and relate these to observations on amyloid polymorphism, prion strains, coaggregation of pathogenic proteins in tissues, and mechanisms of toxicity and transmissibility. Molecular studies have also led to numerous strategies for biological and chemical interventions against amyloid diseases.     

Interaction of the serum amyloid A proteins with phospholipid

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.     

Infrared spectroscopy of human amyloid fibrils and immunoglobulin proteins

The presence of the antiparallel-β-pleated sheet coformation io isolated human amyloid protein fibrils has been confirmed by infrared spectroscopy. In most amyloid samples, this conformation was enhanced by acidic solution conditions. Infrared spectroscopy (Amide I and Amide V absorption bands) and x-ray diffraction methods were also used to examine the immunoglobulin molecule for solid state-β-structure. It was found that both heavy chains and Bence Jones proteins exhibited some β-pleated sheet content upon acid and/or heat treatment. Furthermore, pepsin digests comprising either the variable-rich region (Fd′) of the immunloglobulin heavy chain or in particular, filamentous variable segments of κ and λ Bence Jones proteins were, as isolated, very similar to amyloid in β-structure content. Data from other immunoglobulin-derived sample did not exhibit extensive β-pleated sheet content. On the other hand, most amyliod and immunoglobulin-derived samples did display some β-structure when cast from 50% HCOOH solution. Under these conditions, however, filamentous light chain-variable segments exhibited well-defined infrared patterns rich in antiparallel-β-pleated sheet structure and gave a “cross-β” x-ray diffraction pattern.     

Prediction of diffusion coefficients of proteins

A correlation for predicting the diffusion coefficients of proteins at standard conditions is proposed by adapting the Stokes-Einstein equation to a model for the equivalent hydrodynamic sphere. The radius of gyration, which accounts for the size and shape of protein molecules in solution, was used in deriving the new correlation. The correlation successfully predicted the diffusion coefficients of proteins, for which experimental data were available, with a higher degree of accuracy than achieved by previous correlation methods.     

Sonication of proteins causes formation of aggregates that resemble amyloid

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high beta-content, and proteins with significant native alpha-helical structure show increased beta-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5-20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.     

AMYPdb: A database dedicated to amyloid precursor proteins

Background  

Misfolding and aggregation of proteins into ordered fibrillar structures is associated with a number of severe pathologies, including Alzheimer's disease, prion diseases, and type II diabetes. The rapid accumulation of knowledge about the sequences and structures of these proteins allows using of in silico methods to investigate the molecular mechanisms of their abnormal conformational changes and assembly. However, such an approach requires the collection of accurate data, which are inconveniently dispersed among several generalist databases.     

Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins

Interest in amyloidogenesis has exploded in recent years, as scientists recognize the role of amyloid protein aggregates in degenerative diseases such as Alzheimer's and Parkinson's disease. Assembly of proteins or peptides into mature amyloid fibrils is a multistep process initiated by conformational changes, during which intermediate aggregation states such as oligomers, protofibrils, and filaments are sampled. Although once it was assumed that the mature fibril was the biologically toxic species, more recently it has been widely speculated that soluble intermediates are the most damaging. Because of its relevance to mechanism of disease, the paths traversed during fibrillogenesis, and the kinetics of the process, are of considerable interest. In this review we discuss various kinetic models used to describe amyloidogenesis. Although significant advances have been made, construction of rigorous, detailed, and experimentally validated quantitative models remains a work in progress. We briefly review recent literature that illustrates the interplay between kinetics and amyloid-membrane interactions: how do different intermediates interact with lipid bilayers, and how does the lipid bilayer affect kinetics of amyloidogenesis?     

Prediction of indirect interactions in proteins

Background  

Both direct and indirect interactions determine molecular recognition of ligands by proteins. Indirect interactions can be defined as effects on recognition controlled from distant sites in the proteins, e.g. by changes in protein conformation and mobility, whereas direct interactions occur in close proximity of the protein's amino acids and the ligand. Molecular recognition is traditionally studied using three-dimensional methods, but with such techniques it is difficult to predict the effects caused by mutational changes of amino acids located far away from the ligand-binding site. We recently developed an approach, proteochemometrics, to the study of molecular recognition that models the chemical effects involved in the recognition of ligands by proteins using statistical sampling and mathematical modelling.     

Promotion of oxidative lipid membrane damage by amyloid beta proteins

Senile plaques in the cerebral parenchyma are a pathognomonic feature of Alzheimer's disease (AD) and are mainly composed of aggregated fibrillar amyloid beta (Abeta) proteins. The plaques are associated with neuronal degeneration, lipid membrane abnormalities, and chemical evidence of oxidative stress. The view that Abeta proteins cause these pathological changes has been challenged by suggestions that they have a protective function or that they are merely byproducts of the pathological process. This investigation was conducted to determine whether Abeta proteins promote or inhibit oxidative damage to lipid membranes. Using a mass spectrometric assay of oxidative lipid damage, the 42-residue form of Abeta (Abeta42) was found to accelerate the oxidative lipid damage caused by physiological concentrations of ascorbate and submicromolar concentrations of copper(II) ion. Under these conditions, Abeta42 was aggregated, but nonfibrillar. Ascorbate and copper produced H(2)O(2), but Abeta42 reduced H(2)O(2) concentrations, and its ability to accelerate oxidative damage was not affected by catalase. Lipids could be oxidized by H(2)O(2) and copper(II) in the absence of ascorbate, but only at significantly higher concentrations, and Abeta42 inhibited this reaction. These results indicate that the ability of Abeta42 to promote oxidative damage is more potent and more likely to be manifest in vivo than its ability to inhibit oxidative damage. In conjunction with prior results demonstrating that oxidatively damaged membranes cause Abeta42 to misfold and form fibrils, these results suggest a specific chemical mechanism linking Abeta42-promoted oxidative lipid damage to amyloid fibril formation.     

Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins

Interest in amyloidogenesis has exploded in recent years, as scientists recognize the role of amyloid protein aggregates in degenerative diseases such as Alzheimer's and Parkinson's disease. Assembly of proteins or peptides into mature amyloid fibrils is a multistep process initiated by conformational changes, during which intermediate aggregation states such as oligomers, protofibrils, and filaments are sampled. Although once it was assumed that the mature fibril was the biologically toxic species, more recently it has been widely speculated that soluble intermediates are the most damaging. Because of its relevance to mechanism of disease, the paths traversed during fibrillogenesis, and the kinetics of the process, are of considerable interest. In this review we discuss various kinetic models used to describe amyloidogenesis. Although significant advances have been made, construction of rigorous, detailed, and experimentally validated quantitative models remains a work in progress. We briefly review recent literature that illustrates the interplay between kinetics and amyloid-membrane interactions: how do different intermediates interact with lipid bilayers, and how does the lipid bilayer affect kinetics of amyloidogenesis?     

Structure of core domain of fibril-forming PHF/Tau fragments

Short peptide sequences within the microtubule binding domain of the protein Tau are proposed to be core nucleation sites for formation of amyloid fibrils displaying the paired helical filament (PHF) morphology characteristic of neurofibrillary tangles. To study the structure of these proposed nucleation sites, we analyzed the x-ray diffraction patterns from the assemblies formed by a variety of PHF/tau-related peptide constructs containing the motifs VQIINK (PHF6*) in the second repeat and VQIVYK (PHF6) in the third repeat of tau. Peptides included: tripeptide acetyl-VYK-amide (AcVYK), tetrapeptide acetyl-IVYK-amide (AcPHF4), hexapeptide acetyl-VQIVYK-amide (AcPHF6), and acetyl-GKVQIINKLDLSNVQKDNIKHGSVQIVYKPVDLSKVT-amide (AcTR4). All diffraction patterns showed reflections at spacings of 4.7 A, 3.8 A, and approximately 8-10 A, which are characteristic of an orthogonal unit cell of beta-sheets having dimensions a=9.4 A, b=6.6 A, and c=approximately 8-10 A (where a, b, and c are the lattice constants in the H-bonding, chain, and intersheet directions). The sharp 4.7 A reflections indicate that the beta-crystallites are likely to be elongated along the H-bonding direction and in a cross-beta conformation. The assembly of the AcTR4 peptide, which contains both the PHF6 and PHF6* motifs, consisted of twisted sheets, as indicated by a unique fanning of the diffuse equatorial scattering and meridional accentuation of the (210) reflection at 3.8 A spacing. The diffraction data for AcVYK, AcPHF4, and AcPHF6 all were consistent with approximately 50 A-wide tubular assemblies having double-walls, where beta-strands constitute the walls. In this structure, the peptides are H-bonded together in the fiber direction, and the intersheet direction is radial. The positive-charged lysine residues face the aqueous medium, and tyrosine-tyrosine aromatic interactions stabilize the intersheet (double-wall) layers. This particular contact, which may be involved in PHF fibril formation, is proposed here as a possible aromatic target for anti-tauopathy drugs.