Hydroxy-Porphyrin as an Effective,Endogenous Molecular Clamp during Early Stages of Amyloid Fibrillization

Amyloid fibril formation of proteins is of great concern in neurodegenerative disease and can be detrimental to the storage and stability of biologics. Recent evidence suggests that insulin fibril formation reduces the efficacy of type II diabetes management and may lead to several complications. To develop anti-amyloidogenic compounds of endogenous origin, we have utilized the hydrogen bond anchoring, π stacking ability of porphyrin, and investigated its role on the inhibition of insulin amyloid formation. We report that hydroxylation and metal removal from the heme moiety yields an excellent inhibitor of insulin fibril formation. Thioflavin T, tyrosine fluorescence, Circular Dichorism (CD) spectroscopy, Field emission scanning electron microscopy (FESEM) and molecular dynamics (MD) simulation studies suggest that hematoporphyrin (HP) having hydrogen bonding ability on both sides is a superior inhibitor compared to hemin and protoporphyrin (PP). Experiments with hen egg white lysozyme (HEWL) amyloid fibril formation also validated the efficacy of endogenous porphyrin based small molecules. Our results will help to decipher a general therapeutic strategy to counter amyloidogenesis.     

Peptide‐binding specificity of the prosurfactant protein C Brichos domain analyzed by electrospray ionization mass spectrometry

The C‐terminal domain of lung surfactant protein C (CTC) precursor (proSP‐C) is involved in folding of the transmembrane segment of proSP‐C. CTC includes a Brichos domain with homologs in cancer‐ and dementia‐associated proteins. Mutations in the Brichos domain cause misfolding of proSP‐C and hence amyloid fibril formation in interstitial lung disease. Electrospray ionization mass spectrometry (ESI‐MS) with collision‐induced dissociation (CID) experiments was applied to study non‐covalent interactions between human recombinant CTC or its Brichos domain, and SP‐C analogs, homotripeptides and peptides designed to model amyloid fibril formation. The results show that the Brichos domain contains the peptide‐binding function of CTC. In titration experiments, apparent dissociation constants (K_D) were in the micromolar range where triple‐valine showed the lowest K_D and triple‐tyrosine the highest. Non‐hydrophobic peptides failed to form complexes with Brichos. CID revealed that complexes with aromatic peptide ligands are more stable in the gas phase than complexes with non‐aromatic ligands. The Brichos domain was also shown to bind fibril‐forming peptides containing aromatic/hydrophobic residues. Copyright © 2009 John Wiley & Sons, Ltd.     

The organization of aromatic side groups in an amyloid fibril probed by solid-state 2H and 19F NMR spectroscopy

Some 25 diseases are associated with proteins and peptides that assemble into amyloid fibrils composed of beta-strands connected by hydrogen bonds oriented parallel to the fiber long axis. There is mounting evidence that amyloid formation involves specific interactions between amino acid side groups, which bring together beta-sheets to form layers with buried and exposed faces. This work demonstrates how a combination of solid-state 2H and 19F NMR experiments can provide constraints on fibril architecture by probing the environment and spatial organisation of aromatic side groups. It is shown that phenylalanine rings within fibrils formed by a decapeptide fragment of the islet amyloid polypeptide, amylin, are highly motionally restrained and are situated within 6.5 A of one another. Taken together with existing structural constraints for this peptide, these results are consistent with a fibril architecture that comprises layers of two or more beta-sheets, with the aromatic residues facing into the inter-sheet space and possibly engaged in pi-pi interactions. The methods presented will be of general utility in exploring the architecture of fibrils of larger, full-length peptides and proteins, including amylin itself.     

Natural protective amyloids

Amyloidoses are a group of diseases including neurodegenerative diseases like Alzheimer's disease and also type II diabetes, spongiform encephalopathies and many others, believed to be caused by protein aggregation and subsequent amyloid fibril formation. However, occasionally, living organisms exploit amyloid fibril formation, a property inherent into amino acid sequences, and perform specific physiological functions from amyloids, in differing biological contexts. Some of these functional amyloids are natural protective amyloids. Here, we review recent evidence on silkmoth chorion protein synthetic peptide-analogues that documents the function of silkmoth chorion, the major component of the eggshell, a structure with extraordinary physiological and mechanical properties, as a natural protective amyloid. Also, we briefly discuss the reported function of other natural, protective amyloids like fish chorion, the protein Pmel17 which forms amyloid fibrils that act as templates and accelerate the covalent polymerization of reactive small molecules into melanin, the hydrophobins and the antifreeze protein from winter flounder. Molecular self-assembly is becoming an increasingly popular route to new supramolecular structures and molecular materials and the inspiration for such structures is commonly derived from self-assembling systems in biology. Therefore, a careful examination of these studies may set the basis for the exploration of new routes for the formation of novel biocompatible polymeric structures with exceptional physico-chemical properties, for potentially new biomedical and industrial applications.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105–115) Amyloid Protofibrils

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end‐capping effects have been conducted to understand amyloid fibril development. However, atomic‐level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end‐capping conditions.     

The effect of nanoparticles on amyloid aggregation depends on the protein stability and intrinsic aggregation rate

Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst.20107 (2), 521-532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolymeric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For mutants with a low intrinsic stability and high intrinsic aggregation rate, we find the opposite--a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.     

Inhibitory effect of safranal and crocin,two principle compounds of <Emphasis Type="Italic">Crocus sativus</Emphasis>, on fibrillation of lysozyme

A key feature in more than twenty amyloid-related diseases is the aggregation of intra-and/or extracellular misfolded proteins as amyloid fibrils. Therefore, preventing or reversing amyloid aggregation by using of small molecules is considered as useful approaches to the treatment of these diseases. We have evaluated the ability of safranal and crocin, to inhibit amyloid self-assembly of hen egg white lysozyme (HEWL), as an in vitro model system. Structural properties of HEWL in the presence of these compounds were investigated individually using thioflavin T, anilinonaphthalene-8-sulfonic acid fluorescence assays, far-UV circular dichroism and scanning electron microscopy as well as docking method. Our results showed that incubation of HEWL with either crocin or safranal at various concentrations leads a significant inhibition in the rate of amyloid formation. Docking analysis revealed crocin and safranal interact with the central hydrophobic region of lysozyme through van der Waals interaction. Hydroxyl group in crocin through hydrogen bonds connected to the several hydrophilic amino acids of lysozyme, while in safranal there are just one aldehyde group that through hydrogen bonds connected to aspartic acid in lysozyme. It can be concluded that both hydrophobic and hydrophilic groups contribute to lower lysozyme fibril accumulation.     

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein–protein interactions. We studied aromatic residues in the two structurally homologous cross-β amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of “breathing motions” over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-β architecture.     

Directing the secondary structure of polypeptides at will: from helices to amyloids and back again?

An ageing society faces an increasing number of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Creutzfeld-Jacob disease. The deposition of amyloid fibrils is a pathogenic factor causing the destruction of neuronal tissue. Amyloid-forming proteins are mainly alpha-helical in their native conformation, but undergo an alpha-helix to beta-strand conversion before or during fibril formation. Partially unfolded or misfolded beta-sheet fragments are discussed as direct precursors of amyloids. To potentially cure neurodegenerative diseases we need to understand the complex folding mechanisms that shift the equilibrium from the functional to the pathological isoform of the proteins involved. This paper describes a novel approach that allows us to study the interplay between peptide primary structure and environmental conditions for peptide and protein folding in its whole complexity on a molecular level. This de novo designed peptide system may achieve selective inhibition of fibril formation.     

Capturing intermediate structures of Alzheimer's beta-amyloid, Abeta(1-40), by solid-state NMR spectroscopy

Molecular structures of diffusible amyloid intermediates, commonly observed in misfolding of amyloid proteins into fibrils, have attracted broad interest because the intermediates may be potent neurotoxins responsible for amyloid diseases such as Alzheimer's disease (AD) and because the intermediate structures provide an experimental basis for defining the misfolding pathway. However, owing to the intrinsically unstable and noncrystalline nature of the systems, traditional approaches such as X-ray crystallography and solution NMR have been ineffective for elucidating molecular-level structures of the amyloid intermediates. We present a novel approach using solid-state NMR (SSNMR) that permitted the first site-resolved structural measurement of an intermediate species in fibril formation for a 40-residue Alzheimer's beta-amyloid peptide, Abeta(1-40). In this approach, we combined detection of conformation and morphology changes by fluorescence spectroscopy and electron microscopy and quantitative structural examination for freeze-trapped intermediates by SSNMR. The results provide the initial evidence that a spherical amyloid intermediate of 15-30 nm in diameter exists prior to fibril formation of Abeta(1-40) and that the intermediate involves well-ordered beta-sheets in the C-terminal and hydrophobic core regions. The SSNMR-based approach presented here could be applied to intermediate species of diverse amyloid proteins.     

A designed protein interface that blocks fibril formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures.     

Natural Alkaloid Compounds as Inhibitors for Alpha-Synuclein Seeded Fibril Formation and Toxicity

The accumulation and aggregation of α-synuclein (α-syn) is the main pathologic event in Parkinson’s disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-Syn-seeded fibril formation and its induced toxicity occupy a major role in PD pathogenesis. Thus, assessing compounds that inhibit this seeding process is considered a key towards the therapeutics of synucleinopathies. Using biophysical and biochemical techniques and seeding-dependent cell viability assays, we screened a total of nine natural compounds of alkaloid origin extracted from Chinese medicinal herbs. Of these compounds, synephrine, trigonelline, cytisine, harmine, koumine, peimisine, and hupehenine exhibited in vitro inhibition of α-syn-seeded fibril formation. Furthermore, using cell viability assays, six of these compounds inhibited α-syn-seeding-dependent toxicity. These six potent inhibitors of amyloid fibril formation and toxicity caused by the seeding process represent a promising therapeutic strategy for the treatment of PD and other synucleinopathies.     

Peptide Self-Assembly into Amyloid Fibrils at Hard and Soft Interfaces—From Corona Formation to Membrane Activity

Peptides and proteins are exposed to a variety of interfaces in a physiological environment, such as cell membranes, protein nanoparticles (NPs), or viruses. These interfaces have a significant impact on the interaction, self-assembly, and aggregation mechanisms of biomolecular systems. Peptide self-assembly, particularly amyloid fibril formation, is associated with a wide range of functions; however, there is a link with neurodegenerative diseases, such as Alzheimer's disease. This review highlights how interfaces affect peptide structure and the kinetics of aggregation leading to fibril formation. In nature, many surfaces are nanostructures, such as liposomes, viruses, or synthetic NPs. Once exposed to a biological medium, nanostructures are coated with a corona, which then determines their activity. Both accelerating and inhibiting effects on peptide self-assembly have been observed. When amyloid peptides adsorb to a surface, they typically concentrate locally, which promotes aggregation into insoluble fibrils. Starting from a combined experimental and theoretical approach, models that allow for a better understanding of peptide self-assembly near hard and soft matter interfaces are introduced and reviewed. Research results from recent years are presented and relationships between biological interfaces, such as membranes and viruses, and amyloid fibril formation are proposed.     

Structural regulation of a peptide-conjugated graft copolymer: a simple model for amyloid formation

The self-assembly of peptides and proteins into beta-sheet-rich high-order structures has attracted much attention as a result of the characteristic nanostructure of these assemblies and because of their association with neurodegenerative diseases. Here we report the structural and conformational properties of a peptide-conjugated graft copolymer, poly(gamma-methyl-L-glutamate) grafted polyallylamine (1) in a water-2,2,2-trifluoroethanol solution as a simple model for amyloid formation. Atomic force microscopy revealed that the globular peptide 1 self-assembles into nonbranching fibrils that are about 4 nm in height under certain conditions. These fibrils are rich in beta-sheets and, similar to authentic amyloid fibrils, bind the amyloidophilic dye Congo red. The secondary and quaternary structures of the peptide 1 can be controlled by manipulating the pH, solution composition, and salt concentration; this indicates that the three-dimensional packing arrangement of peptide chains is the key factor for such fibril formation. Furthermore, the addition of carboxylic acid-terminated poly(ethylene glycol), which interacts with both of amino groups of 1 and hydrophobic PMLG chains, was found to obviously inhibit the alpha-to-beta structural transition for non-assembled peptide 1 and to partially cause a beta-to-alpha structural transition against the 1-assembly in the beta-sheet form. These findings demonstrate that the amyloid fibril formation is not restricted to specific protein sequences but rather is a generic property of peptides. The ability to control the assembled structure of the peptide should provide useful information not only for understanding the amyloid fibril formation, but also for developing novel peptide-based material with well-defined nanostructures.     

Interactions of cyclodextrins with aromatic compounds studied by vibrational circular dichroism spectroscopy

The host/guest complexation between cyclodextrins (CDs) and aromatic compounds was studied by vibrational circular dichroism (VCD) spectroscopy in mid-IR region. Benzoic acid, 4-aminobenzoic acid, and 2,6-naphthalene-dicarboxylic acid acting as the guests with aromatic skeleton, cause the significant changes in VCD patterns of CD, which indicate that the secondary hydroxyl groups of the CDs are involved in the host/guest complexation. In addition, the intensities and dissymmetry factors (deltaA/A) of the VCD bands, which belong to skeletal CD vibrations, depend on the sizes of the guest molecules. Our results indicate that the formation of the CD inclusion complexes can be followed by VCD spectroscopy.     

The Prenucleation Equilibrium of the Parathyroid Hormone Determines the Critical Aggregation Concentration and Amyloid Fibril Nucleation

Nucleation and growth of amyloid fibrils were found to only occur in supersaturated solutions above a critical concentration (c_crit). The biophysical meaning of c_crit remained mostly obscure, since typical low values of c_crit in the sub-μM range hamper investigations of potential oligomeric states and their structure. Here, we investigate the parathyroid hormone PTH₈₄ as an example of a functional amyloid fibril forming peptide with a comparably high c_crit of 67±21 μM. We describe a complex concentration dependent prenucleation ensemble of oligomers of different sizes and secondary structure compositions and highlight the occurrence of a trimer and tetramer at c_crit as possible precursors for primary fibril nucleation. Furthermore, the soluble state found in equilibrium with fibrils adopts to the prenucleation state present at c_crit. Our study sheds light onto early events of amyloid formation directly related to the critical concentration and underlines oligomer formation as a key feature of fibril nucleation. Our results contribute to a deeper understanding of the determinants of supersaturated peptide solutions. In the current study we present a biophysical approach to investigate c_crit of amyloid fibril formation of PTH₈₄ in terms of secondary structure, cluster size and residue resolved intermolecular interactions during oligomer formation. Throughout the investigated range of concentrations (1 μM to 500 μM) we found different states of oligomerization with varying ability to contribute to primary fibril nucleation and with a concentration dependent equilibrium. In this context, we identified the previously described c_crit of PTH₈₄ to mark a minimum concentration for the formation of homo-trimers/tetramers. These investigations allowed us to characterize molecular interactions of various oligomeric states that are further converted into elongation competent fibril nuclei during the lag phase of a functional amyloid forming peptide.     

Identification of amyloidogenic peptide sequences using a coarse-grained physicochemical model

Cross-beta amyloid is implicated in over 20 human diseases. Experiments suggest that specific sequence elements within amyloidogenic proteins play a major role in seeding amyloid formation. Identifying these seeding sequences is important for rationalizing the molecular mechanisms of amyloid formation and for elaborating therapeutic strategies that target amyloid. Theoretical techniques play an important role in facilitating the identification and structural characterization of putative seeding sequences; most amyloid species are not amenable to high resolution experimental structure techniques. In this study we have combined a coarse-grained physicochemical protein model with a highly efficient Monte Carlo sampling technique to identify amyloidogenic sequences in four proteins for which respective experimental peptide fragmentation data exist. Peptide sequences were defined as amyloidogenic if the ensemble structure predicted for three interacting peptides described a stable and regular three-stranded beta-sheet. For such peptides, free energies were calculated to provide a measure of amyloid propensity. The overall agreement between the experimental and predicted data is good, and we correctly identify several self-recognition motifs proposed to define the cross-beta amyloid fibril architectures of two of the proteins. Our results compare very favorably with those obtained using atomistic molecular dynamics methods, though our simulations are 30-40 times faster.     

Effects of mutation on the amyloidogenic propensity of apolipoprotein C-II(60-70) peptide

Using experimental and computational methods we identified the effects of mutation on the structure and dynamics of the amyloidogenic peptide apoC-II(60-70), in monomeric and oligomeric states. Methionine (Met60) substitutions to hydrophilic Gln, hydrophobic Val, and methionine sulfoxide residues were investigated and the results compared with observations of fibril formation by the wild-type, Met60Gln, Met60Val, and oxidised Met60 (oxi-Met) apoC-II(60-70) peptides. ThT fluorescence measurements showed fibril formation by all peptides, however with different kinetics. The wild-type and Met60Val peptides formed fibrils fastest, while oxi-Met and Met60Gln peptides exhibited significantly longer lag phases. Molecular dynamics simulations showed that the mutated monomers exhibited structural features consistent with fibril-forming propensity, such as β-hairpin conformation and a hydrophobic core. However, important differences to the wild-type were also noted, such as increased structural flexibility (oxi-Met and Met60Gln systems) and a broader distribution of the aromatic angle orientation, which could contribute to the different fibrillation kinetics observed in these peptides. Our results also showed that the critical nucleus size for fibril formation by apoC-II(60-70) may not be very large, since tetrameric oligomers in anti-parallel configuration were very stable within the 100 ns of simulations. The single-point mutations Met60Val and Met60Gln had no significant effect on the structural stability of the tetramer. The rate of fibril formation by apoC-II(60-70) peptides was generally much faster compared to longer apoC-II(56-76) peptides. Also, the effects of amino acid modifications on the kinetics of peptide fibril formation differ from the effects observed for apoC-II(56-76) and full-length apoC-II, suggesting that additional mechanisms are involved in fibril formation by mature apoC-II.     

Peroxidic perfluoropolyether for the covalent binding of perfluoropolyether chains on carbon black surface

Peroxidic perfluoropolyethers (PFPEs) are suitable tools for the covalent linkage of fluorinated groups on substrates containing aromatic moieties. Thus the thermal decomposition process of such fluorinated peroxides allowed the covalent linkage of PFPE radicals to the polycyclic aromatic structure of a graphitic carbon black. Contact angle measurements on molded pellets made with modified carbon black powders revealed a gradual enhancement of the hydrophobicity, which follows the increase of the fluorine content on the surface according to XPS experiments. BET analyses also revealed variations of the surface area of carbonaceous samples. Products and by-products were also evaluated by mass balances of decomposed portions of PFPE residues, respectively, PFPE chains bonded on carbon black and PFPE fluids obtained by homocoupling side-reactions. Modified carbonaceous materials were analyzed by solid state ¹⁹F-MAS NMR and the results are in agreement with the proposed radical mechanism.