The Extracellular Molecular Chaperone Clusterin Inhibits Amyloid Fibril Formation and Suppresses Cytotoxicity Associated with Semen-Derived Enhancer of Virus Infection (SEVI).

Clusterin is a glycoprotein present at high concentrations in many extracellular fluids, including semen. Its increased expression accompanies disorders associated with extracellular amyloid fibril accumulation such as Alzheimer's disease. Clusterin is an extracellular molecular chaperone which prevents the misfolding and amorphous and amyloid fibrillar aggregation of a wide variety of unfolding proteins. In semen, amyloid fibrils formed from a 39-amino acid fragment of prostatic acid phosphatase, termed Semen-derived Enhancer of Virus Infection (SEVI), potentiate HIV infectivity. In this study, clusterin potently inhibited the in vitro formation of SEVI fibrils, along with dissociating them. Furthermore, clusterin reduced the toxicity of SEVI to pheochromocytoma-12 cells. In semen, clusterin may play an important role in preventing SEVI amyloid fibril formation, in dissociating SEVI fibrils and in mitigating their enhancement of HIV infection.

Amyloid fibril formation by αS1- and ß-casein implies that fibril formation is a general property of casein proteins.

Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, αS2- and κ-casein, is their propensity in vitro to form amyloid fibrils, the highly ordered protein aggregates associated with many age-related, including neurological, diseases. In this study, we explored whether amyloid fibril-forming propensity is a general feature of casein proteins by examining the other cow caseins (αS1 and ß) as well as ß-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow αS1- and ß-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, αS1- and ß-casein underwent conversion to amyloid fibrils over the course of ten days, as shown by thioflavin T binding, transmission electron microscopy, and X-ray fibre diffraction. At the lower temperature of 37°C where fibril formation was more limited, camel ß-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel ß-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of ß-casein. These findings highlight the persistence of amyloidogenic sequences within caseins, which likely contribute to their functional, heterotypic self-assembly; in all mammalian milks, at least two caseins coalesce to form casein micelles, implying that caseins diversified partly to avoid dysfunctional amyloid fibril formation.

Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2-casein.

Bovine milk αS2-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala81 to Lys113 is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of αS2-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of αS2-casein is a functional, yet amyloidogenic, structure.

Small heat shock proteins: multifaceted proteins with important implications for life.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.

Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.

The growing world of small heat shock proteins: from structure to functions.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).

The Amyloid Fibril-Forming Properties of the Amphibian Antimicrobial Peptide Uperin†3.5.

The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.

A multi-pathway perspective on protein aggregation: implications for control of the rate and extent of amyloid formation.

The nucleation-growth model has been used extensively for characterizing in vitro amyloid fibril formation kinetics and for simulating the relationship between amyloid and disease. In the majority of studies amyloid has been considered as the dominant, or sole, aggregation end product, with the presence of other competing non-amyloid aggregation processes, for example amorphous aggregate formation, being largely ignored. Here, we examine possible regulatory effects that off-pathway processes might exert on the rate and extent of amyloid formation - in particular their potential for providing false positives and negatives in the evaluation of anti-amyloidogenic agents. Furthermore, we investigate how such competing reactions might influence the standard interpretation of amyloid aggregation as a two-state system. We conclude by discussing our findings in terms of the general concepts of supersaturation and system metastability - providing some mechanistic insight as to how these empirical phenomena may manifest themselves in the amyloid arena.

Small heat-shock proteins: important players in regulating cellular proteostasis.

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.

SEVI, the semen enhancer of HIV infection along with fragments from its central region, form amyloid fibrils that are toxic to neuronal cells.

Semen-derived enhancer of viral infection (SEVI) is the term given to the amyloid fibrils formed by a 39-amino acid fragment (PAP248-286) of prostatic acidic phosphatase (PAP) found in human semen. SEVI enhances human immunodeficiency virus (HIV) infectivity by four to five orders of magnitude (Münch et al., 2007). Here, we show by various biophysical techniques including Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy that fragments encompassing the central region of SEVI, i.e. PAP248-271 and PAP257-267, form fibrils of similar morphology to SEVI. Our results show that the central region, residues PAP267-271, is crucially important in promoting SEVI fibril formation. Furthermore, SEVI and fibrillar forms of these peptide fragments are toxic to neuronal pheochromocytoma 12 cells but not to epithelial colon carcinoma cells. These findings imply that although SEVI assists in the attachment of HIV-1 to immune cells, it may not facilitate HIV entry by damaging the epithelial cell layer that presents a barrier to the HIV.

The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity.

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-ß peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation.

Many protein misfolding diseases, for example, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimer's disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

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.

Methionine oxidation enhances κ-casein amyloid fibril formation.

The effects of protein oxidation, for example of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer's disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met(95) and Met(106)) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. ß-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein's greater exposed surface hydrophobicity. No significant changes to κ-casein's intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of ß-casein to prevent this aggregation, may affect casein-casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA.

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support.

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.

A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, αA- and αB-crystallin.

The principal lens proteins αA- and αB-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, αA- and αB-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. αB-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization. The structure of either αA- or αB-crystallin is not known nor is the mechanism of their chaperone action. Our earlier (1)H NMR spectroscopic studies determined that mammalian sHsps have a highly dynamic, polar and unstructured region at their extreme C-terminus (summarized in Carver (1999) Prog. Ret. Eye Res. 18, 431). This C-terminal extension acts as a solubilizing agent for the relatively hydrophobic protein and the complex it makes with its target proteins during chaperone action. In this study, αA- and αB-crystallin were (15)N-labelled and their (1)H-(15)N through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. (1)H-(15)N spin-lattice (T(1)) and spin-spin (T(2)) relaxation times were measured for αA- and αB-crystallin in the absence and presence of a bound target protein, reduced α-lactalbumin. (1)H-(15)N Nuclear Overhauser Effect (NOE) values provide an accurate measure, on a residue-by-residue basis, of the backbone flexibility of polypeptides. From measurement of these NOE values, it was determined that the flexibility of the extension in αA- and αB-crystallin increased markedly at the extreme C-terminus. By contrast, upon chaperone interaction of αA-crystallin with reduced α-lactalbumin, flexibility was maintained in the extension but was distributed evenly across all residues in the extension. Two mutants of αB-crystallin in its C-terminal region: (i) I159A and I161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One 2, e1046). Comparison of (1)H-(15)N NOE values for these mutants with wild type αB-crystallin revealed alteration in flexibility of the extension, particularly at the extremity of K175L αB-crystallin, which may affect chaperone ability.

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation.

Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration; for example, the sHsp alphaB-crystallin (alphaB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of alphaB-c to suppress the aggregation of a polyQ protein. We found that alphaB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of alphaB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that alphaB-c potently inhibits the first stage of ataxin-3 aggregation; however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that alphaB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an example of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.

Carboxymethylated-kappa-casein: a convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation.

Reduced and carboxymethylated-kappa-casein (RCM-kappa-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Abeta), which is associated with Alzheimer's disease. In this study, a series of flavonoids, many known to be inhibitors of Abeta fibril formation, were screened for their ability to inhibit RCM-kappa-CN fibrilisation, and the results were compared with literature data on Abeta inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-kappa-CN fibril formation. IC(50) values were between 10- and 200-fold higher with RCM-kappa-CN than literature results for Abeta fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-kappa-CN assay make it an economic alternative first screen for Abeta inhibitory activity, especially for use with large compound libraries.

The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds.

Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation and inhibition of amyloid fibrils in the presence of anti-amyloidogenic compounds such as polyphenols. However, in this study, it was shown, using three polyphenolics (curcumin, quercetin and resveratrol), that ThT fluorescence should be used with caution in the presence of such exogenous compounds. The strong absorptive and fluorescent properties of quercetin and curcumin were found to significantly bias the ThT fluorescence readings in both in situ real-time ThT assays and single time-point dilution ThT-type assays. The presence of curcumin at concentrations as low as 0.01 and 1 mum was sufficient to interfere with the ThT fluorescence associated with fibrillar amyloid-beta(1-42) (0.5 mum) and fibrillar reduced and carboxymethylated kappa-casein (50 mum), respectively. The ThT fluorescence associated with fibrillar amyloid-beta(1-42) was also biased using higher concentrations of resveratrol, a polyphenol that is not spectroscopically active at the wavelengths of ThT fluorescence, implying that there can be direct interactions between ThT and the exogenous compound and/or competitive binding with ThT for the fibrils. Thus, in all cases where ThT is used in the presence of an exogenous compound, biases for amyloid-associated ThT fluorescence should be tested, regardless of whether the additive is spectroscopically active. Simple methods to conduct these tests were described. The Congo red spectral shift assay is demonstrated as a more viable spectrophotometric alternative to ThT, but allied methods, such as transmission electron microscopy, should also be used to assess fibril formation independently of dye-based assays. Structured digital abstract: * MINT-7259867: RCMkappa-CN (uniprotkb:P02668) and RCMkappa-CN (uniprotkb:P02668) bind (MI:0407) by electron microscopy (MI:0040) * MINT-7258930: RCMkappa-CN (uniprotkb:P02668) and RCMkappa-CN (uniprotkb:P02668) bind (MI:0407) by fluorescence technologies (MI:0051) * MINT-7259878: Amyloid beta (uniprotkb:P05067) and Amyloid beta (uniprotkb:P05067) bind (MI:0407) by fluorescence technologies (MI:0051).

(-)-epigallocatechin-3-gallate (EGCG) maintains kappa-casein in its pre-fibrillar state without redirecting its aggregation pathway.

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alpha-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMkappa-CN (reduced and carboxymethylated kappa-casein) and thereby protect pheochromocytoma-12 cells from RCMkappa-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMkappa-CN [the IC(50) for 50 microM RCMkappa-CN is 13+/-1 microM]. Biophysical studies reveal that EGCG prevents RCMkappa-CN fibril formation by stabilising RCMkappa-CN in its native-like state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMkappa-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent pi-pi stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated.