Mutational analysis of the propensity for amyloid formation by a globular protein

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.     

Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils

Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.     

Tuning amyloidogenic conformations through cosolvents and hydrostatic pressure: when the soft matter becomes even softer

Compact packing, burial of hydrophobic side-chains, and low free energy levels of folded conformations contribute to stability of native proteins. Essentially, the same factors are implicated in an even higher stability of mature amyloid fibrils. Although both native insulin and insulin amyloid are resistant to high pressure and influence of cosolvents, intermediate aggregation-prone conformations are susceptible to either condition. Consequently, insulin fibrillation may be tuned under hydrostatic pressure or-- through cosolvents and cosolutes-- by preferential exclusion or binding. Paradoxically, under high pressure, which generally disfavors aggregation of insulin, an alternative "low-volume" aggregation pathway, which leads to unique circular amyloid is permitted. Likewise, cosolvents are capable of preventing, or altering amyloidogenesis of insulin. As a result of cosolvent-induced perturbation, distinct conformational variants of fibrils are formed. Such variants, when used as templates for seeding daughter generations, reproduce initial folding patterns regardless of environmental biases. By the close analogy, this suggests that the "prion strains" phenomenon may mirror a generic, common feature in amyloids. The susceptibility of amyloidogenic conformations to pressure and cosolvents is likely to arise from their "frustration", as unfolding results in less-densely packed side-chains, void volumes, and exposure of hydrophobic groups. The effects of cosolvents and pressure are discussed in the context of studies on other amyloidogenic protein models, amyloid polymorphism, and "strains".     

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics,Thermodynamics, and Structure

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.     

Amyloid Formation by Human Carboxypeptidase D Transthyretin-like Domain under Physiological Conditions

Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.     

Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement

Protein aggregation and amyloid fibrillation can lead to several serious diseases and protein drugs ineffectiveness; thus, the detection and inhibition of these processes have been of great interest. In the present study, the inhibition of insulin amyloid fibrillation by laser irradiation was investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD), and thioflavin T (ThT) fluorescence. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1 M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1 M NaCl for 45 h incubation. Moreover, our CD results showed that the laser-irradiated insulin solution maintained mainly an α-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in β-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.     

Potential applications of stress solutes from extremophiles in protein folding diseases and healthcare

Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer’s Aβ peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aβ and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.     

Effect of EGCG On Fe(III)‐induced conformational transition of silk fibroin,a model of protein related to neurodegenerative diseases

The abnormal aggregation of amyloid proteins is reported to play a critical role in the etiology of neurodegenerative disorders. Studies have shown that excessive ferric irons are associated with the misfolding of amyloid proteins, and that (‐)‐epigallocatechin gallate (EGCG) is a good metallic ion chelator with inhibitory effect on the aggregation of amyloid proteins. EGCG has been thus considered as a potential drug candidate for the treatment of neurodegenerative diseases. However, the mechanism of action for EGCG in inhibition of aggregation of amyloid proteins is still remaining unclear. Silk fibroin (SF) shares similarities with amyloid proteins in some amino acid sequences and fibrillation kinetics. In this work, therefore, we used SF as a model of protein to investigate the effects of Fe(III) and EGCG on conformational transition by using turbidity assay, thioflavin T (ThT) fluorescence spectroscopy, Raman spectroscopy, and atomic force microscope (AFM). We demonstrated that low concentration of Fe(III) ions promoted the formation of β‐sheet conformers, while high concentration of Fe(III) ions inhibited further aggregation of SF. EGCG could significantly inhibit the conformational transition of SF when induced by Fe(III), and decrease the amount of β‐sheet conformers dose‐dependently. The findings provide important information regarding to EGCG as a potential agent for the prevention and treatment of neurodegenerative diseases. Fe(III) can accelerate the conformation transition of silk fibrion (SF) from random coil into β‐sheet, while (‐)‐epigallocatechin gallate (EGCG) inhibits Fe(III)‐induced β‐sheet aggregation of SF., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 100–107, 2016     

Effects of dithiothreitol on the amyloid fibrillogenesis of hen egg-white lysozyme

At least 25 human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillation, the detailed molecular mechanisms remain rather elusive and there are currently no effective cures for treating these amyloid diseases. The present study examined the effects of dithiothreitol on the fibrillation of hen egg-white lysozyme (HEWL). Our results revealed that the fibrillation of hen lysozyme was significantly inhibited by reduced dithiothreitol (DTT^red) while oxidized dithiothreitol (DTT^ox) had no anti-aggregating activity. Effective inhibitory activity against hen lysozyme fibrillation was observed only when DTT^red was added within 8 days of incubation. Our results showed that the initial addition of DTT^red interacted with HEWL, leading to a loss in conformational stability. It was concluded from our findings that DTT^red-induced attenuation of HEWL fibrillation may be associated with disulfide disruption and extensive structural unfolding of HEWL. Our data may contribute to rational design of effective therapeutic strategies for amyloid diseases.     

Detecting hidden sequence propensity for amyloid fibril formation

The preponderance of evidence implicates protein misfolding in many unrelated human diseases. In all cases, normal correctly folded proteins transform from their proper native structure into an abnormal beta-rich structure known as amyloid fibril. Here we introduce a computational algorithm to detect nonnative (hidden) sequence propensity for amyloid fibril formation. Analyzing sequence-structure relationships in terms of tertiary contact (TC), we find that the hidden beta-strand propensity of a query local sequence can be quantitatively estimated from the secondary structure preferences of template sequences of known secondary structure found in regions of high TC. The present method correctly pinpoints the minimal peptide fragment shown experimentally as the likely local mediator of amyloid fibril formation in beta-amyloid peptide, islet amyloid polypeptide (hIAPP), alpha-synuclein, and human acetylcholinesterase (AChE). It also found previously unrecognized beta-strand propensities in the prototypical helical protein myoglobin that has been reported as amyloidogenic. Analysis of 2358 nonhomologous protein domains provides compelling evidence that most proteins contain sequences with significant hidden beta-strand propensity. The present method may find utility in many medically relevant applications, such as the engineering of protein sequences and the discovery of therapeutic agents that specifically target these sequences for the prevention and treatment of amyloid diseases.     

Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 °C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4 mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.     

Trehalose and Magnesium Chloride Exert a Common Anti-amyloidogenic Effect Towards Hen Egg White Lysozyme

Many degenerative disorder such as Parkinsons, Alzheimers, Huntingtons disease, etc are caused due to the deposition of amyloid fibrils, formed due to the ordered aggregation of misfolded/unfolded proteins. Misfolded or unfolded proteins aggregate mostly through hydrophobic interactions which are unexposed in native state, but become exposed upon unfolding. To counteract amyloid related diseases, inhibition of the protein self assembly into fibril is a potential therapeutic strategy. The study aims at investigating the effect of selected compounds, namely trehalose and magnesium chloride hexahydrate towards inhibition and disaggregation of amyloid fibrils using Hen Egg White Lysozyme as a model. We further attempted to understand the mechanism of action with the help of various biophysical, microscopic as well as computational studies. A common mechanism of action was identified where the selected compounds exert their anti-amyloidogenic effects by altering HEWL conformations characterized by reduction in the beta sheet content and decrease in exposed hydrophobic surfaces. The altered conformation seems to have lesser amyloidogenic propensity leading to inhibition as well as disaggregation of amyloids.     

Aggregation and conformational studies on a pentapeptide derivative

Most of the disease causing proteins such as beta amyloid, amylin, and huntingtin protein, which are natively disordered, readily form fibrils consisting of beta-sheet polymers. Though all amyloid fibrils are made up of beta-sheet polymers, not all peptides with predominant beta-sheet content in the native state develop into amyloid fibrils. We hypothesize that stable amyloid like fibril formation may require mixture of different conformational states in the peptide. We have tested this hypothesis on amyloid forming peptide namely HCl(Ile)(5)NH(CH(2)CH(2)O)(3)CH(3) (I). We show peptide I, has propensity to form self-assembled structures of beta-sheets in aqueous solutions. When incubated over a period of time in aqueous buffer, I self assembled into beta sheet like structures with diameters ranging from 30 to 60 A that bind with amyloidophilic dyes like Congo red and Thioflavin T. Interestingly peptide I developed into unstable fibrils after prolonged aging at higher concentration in contrast with the general mature fibril-forming propensity of various amyloid petides known to date.     

Protein Misfolding and Aggregation in Ageing and Disease

Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.     

Structure-specific effects of protein topology on cross-beta assembly: studies of insulin fibrillation

Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.     

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.     

Complete observation of all structural,conformational, and fibrillation transitions of monomeric globular proteins at submicellar sodium dodecyl sulfate concentrations

Although considerable information is available regarding protein–sodium dodecyl sulfate (SDS) interactions, it is still unclear as to how much SDS is needed to denature proteins. The role of protein charge and micellar surfactant concentration on amyloid fibrillation is also unclear. This study reports on equilibrium measurements of SDS interaction with six model proteins and analyzes the results to obtain a general understanding of conformational breakdown, reorganization and restructuring of secondary structure, and entry into the amyloid fibrillar state. Significantly, all of these responses are entirely resolved at much lower than the critical micellar concentration (CMC) of SDS. Electrostatic interaction of the dodecyl sulfate anion (DS⁻) with positive surface potential on the protein can completely unfold both secondary and tertiary structures, which is followed by protein chain restructuration to α-helices. All SDS-denatured proteins contain more α-helices than the corresponding native state. SDS interaction stochastically drives proteins to the aggregated fibrillar state.     

Mechanism for the alpha-helix to beta-hairpin transition

The aggregation of alpha-helix-rich proteins into beta-sheet-rich amyloid fibrils is associated with fatal diseases, such as Alzheimer's disease and prion disease. During an aggregation process, protein secondary structure elements-alpha-helices-undergo conformational changes to beta-sheets. The fact that proteins with different sequences and structures undergo a similar transition on aggregation suggests that the sequence nonspecific hydrogen bond interaction among protein backbones is an important factor. We perform molecular dynamics simulations of a polyalanine model, which is an alpha-helix in its native state and observe a metastable beta-hairpin intermediate. Although a beta-hairpin has larger potential energy than an alpha-helix, the entropy of a beta-hairpin is larger because of fewer constraints imposed by the hydrogen bonds. In the vicinity of the transition temperature, we observe the interconversion of the alpha-helix and beta-sheet states via a random coil state. We also study the effect of the environment by varying the relative strength of side-chain interactions for a designed peptide-an alpha-helix in its native state. For a certain range of side-chain interaction strengths, we find that the intermediate beta-hairpin state is destabilized and even disappears, suggesting an important role of the environment in the aggregation propensity of a peptide.     

Amyloid fibril formation by native and modified bovine β-lactoglobulins proceeds through unfolded form of proteins: a comparative study

The misfolding and extracellular amyloid deposition of specific proteins are associated with a large family of human pathologies, often called protein conformational diseases. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Recently, β-lactoglobulin (β-lg) was driven toward amyloid aggregation under specific extreme conditions. In the present study, citraconylation was employed to neutralize the charges on accessible lysine residues of β-lg and different approaches such as turbidimetry, thermodynamic analysis, extrinsic fluorimetry and theoretical studies have been successfully used to compare the different behaviors of the native and modified proteins. Kinetic analyses of native β-lg aggregation showed a gradual development of turbidity, whereas the modified β-lg displayed an increased propensity toward aggregation. Our results clearly demonstrated that the stability of modified β-lg is markedly reduced, compared to the native one. Using of TANGO and WALTZ algorithms (as well as modelling softwares) which describe aggregation tendencies of different parts of a protein structure, we suggested critical importance of some of the lysine residues in the aggregation process. The results highlighted the critical role of protein stability and elucidated the underlying role of hydrophobic/electrostatic interactions in lactoglobulin-based experimental system.     

Quaternary conformational stability: The effect of reversible self‐association on the fibrillation of two insulin analogs

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far‐UV CD) and second derivative Fourier transform infrared (2D‐FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α‐helical in native insulin to predominantly β‐sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self‐associate. Composition gradient multi‐angle light scattering (CG‐MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant‐induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first‐order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359–2370. © 2011 Wiley Periodicals, Inc.