Carboxymethylated-κ-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation

Reduced and carboxymethylated-κ-casein (RCM-κ-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 (Aβ), which is associated with Alzheimer’s disease. In this study, a series of flavonoids, many known to be inhibitors of Aβ fibril formation, were screened for their ability to inhibit RCM-κ-CN fibrilisation, and the results were compared with literature data on Aβ inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-κ-CN fibril formation. IC₅₀ values were between 10- and 200-fold higher with RCM-κ-CN than literature results for Aβ fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-κ-CN assay make it an economic alternative first screen for Aβ inhibitory activity, especially for use with large compound libraries.     

Two distinct aggregation pathways in transthyretin misfolding and amyloid formation

Misfolding and amyloid formation of transthyretin (TTR) is implicated in numerous degenerative diseases. TTR misfolding is greatly accelerated under acidic conditions, and thus most of the mechanistic studies of TTR amyloid formation have been conducted at various acidic pH values (2–5). In this study, we report the effect of pH on TTR misfolding pathways and amyloid structures. Our combined solution and solid-state NMR studies revealed that TTR amyloid formation can proceed via at least two distinct misfolding pathways depending on the acidic conditions. Under mildly acidic conditions (pH 4.4), tetrameric native TTR appears to dissociate to monomers that maintain most of the native-like β-sheet structures. The amyloidogenic protein undergoes a conformational transition to largely unfolded states at more acidic conditions (pH 2.4), leading to amyloid with distinct molecular structures. Aggregation kinetics is also highly dependent upon the acidic conditions. TTR quickly forms moderately ordered amyloids at pH 4.4, while the aggregation kinetics is dramatically reduced at a lower pH of 2.4. The effect of the pathogenic mutations on aggregation kinetics is also markedly different under the two different acidic conditions. Pathogenic TTR variants (V30M and L55P) aggregate more aggressively than WT TTR at pH 4.4. In contrast, the single-point mutations do not affect the aggregation kinetics at the more acidic condition of pH 2.4. Given that the pathogenic mutations lead to more aggressive forms of TTR amyloidoses, the mildly acidic condition might be more suitable for mechanistic studies of TTR misfolding and aggregation.     

Inhibiting toxic aggregation of amyloidogenic proteins: A therapeutic strategy for protein misfolding diseases

Background

The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases.

Scope of review

In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof.

Major conclusions

Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity.

General significance

Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.     

Amyloidogenic determinants are usually not buried

Background  

Amyloidoses are a group of usually fatal diseases, probably caused by protein misfolding and subsequent aggregation into amyloid fibrillar deposits. The mechanisms involved in amyloid fibril formation are largely unknown and are the subject of current, intensive research. In an attempt to identify possible amyloidogenic regions in proteins for further experimental investigation, we have developed and present here a publicly available online tool that utilizes five different and independently published methods, to form a consensus prediction of amyloidogenic regions in proteins, using only protein primary structure data.     

Comparison of the chaperon activity of glycerol and α-casein on amyloid formation of κ-casein in the presence of glycine and arginine

Amyloids are insoluble fibers which arise from inappropriately folded versions of proteins and have been associated with the pathology of many neurodegenerative diseases. α-Casein is one of the major components of the casein family which is known to show chaperone-like activity. Glycerol is a polyol compound which acts as a chemical chaperone to increase protein stability and inhibit protein aggregation. In this study, the effect of arginine and glycine on the chaperone ability of α-casein and glycerol against order aggregation of κ-casein was investigated and compared. We found that these additives reduced the chaperone ability of α-casein against the amyloid formation of κ-casein, especially in the presence of arginine. Importantly, our results show that the chaperone action of glycerol is enhanced in the presence of both arginine and glycine. Accordingly, our results suggest that these small molecules associated with glycerol, especially glycine, should be considered as a mechanism for the treatment of amyloid disease.     

Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several μmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.     

Mutations can cause light chains to be too stable or too unstable to form amyloid fibrils

Light chain (AL) amyloidosis is an incurable human disease, where the amyloid precursor is a misfolding‐prone immunoglobulin light‐chain. Here, we identify the role of somatic mutations in the structure, stability and in vitro fibril formation for an amyloidogenic AL‐12 protein by restoring four nonconservative mutations to their germline (wild‐type) sequence. The single restorative mutations do not affect significantly the native structure, the unfolding pathway, and the reversibility of the protein. However, certain mutations either decrease (H32Y and H70D) or increase (R65S and Q96Y) the protein thermal stability. Interestingly, the most and the least stable mutants, Q96Y and H32Y, do not form amyloid fibrils under physiological conditions. Thus, Q96 and H32 are key residues for AL‐12 stability and fibril formation and restoring them to the wild‐type residues preclude amyloid formation. The mutants whose equilibrium is shifted to either the native or unfolded states barely sample transient partially folded states, and therefore do not form fibrils. These results agree with previous observations by our laboratory and others that amyloid formation occurs because of the sampling of partially folded states found within the unfolding transition (Blancas‐Mejia and Ramirez‐Alvarado, Ann Rev Biochem 2013;82:745–774). Here we provide a new insight on the AL amyloidosis mechanism by demonstrating that AL‐12 does not follow the established thermodynamic hypothesis of amyloid formation. In this hypothesis, thermodynamically unstable proteins are more prone to amyloid formation. Here we show that within a thermal stability range, the most stable protein in this study is the most amyloidogenic protein.     

Role of metal ions in aggregation of intrinsically disordered proteins in neurodegenerative diseases

Neurodegenerative diseases constitute a set of pathological conditions originating from the slow, irreversible, and systematic cell loss within the various regions of the brain and/or the spinal cord. Depending on the affected region, the outcomes of the neurodegeneration are very broad and diverse, ranging from the problems with movements to dementia. Some neurodegenerative diseases are associated with protein misfolding and aggregation. Many proteins that misfold in human neurodegenerative diseases are intrinsically disordered; i.e., they lack a stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) functionally complement ordered proteins, being typically involved in regulation and signaling. There is accumulating evidence that altered metal homeostasis may be related to the progression of neurodegenerative diseases. This review examines the effects of metal ion binding on the aggregation pathways of IDPs found in neurodegenerative diseases.     

Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins

Understanding the energetic and structural basis of protein folding in a physiological context may represent an important step toward the elucidation of protein misfolding and aggregation events that take place in several pathological states. In particular, investigation of the structure and thermodynamic properties of partially folded intermediate states involved in productive folding or in misfolding/aggregation may provide insight into these processes and suggest novel approaches to prevent misfolding in living organisms. This goal, however, has remained elusive, because such intermediates are often transient and correspond to metastable states that are little populated under physiological conditions. Characterization of these states requires their stabilization by means of manipulation of the experimental conditions, involving changes in temperature, pH, or addition of different types of denaturants. In the past few years, hydrostatic pressure has been increasingly used as a thermodynamic variable in the study of both protein folding and misfolding/aggregation transitions. Compared with other chemical or physical denaturing agents, a unique feature of pressure is its ability to induce subtle changes in protein conformation, allowing the stabilization of partially folded states that are usually not significantly populated under more drastic conditions. Much of the recent work in this field has focused on the characterization of folding intermediates, because they seem to be involved in a variety of disease-causing protein misfolding and aggregation reactions. Here, we review recent examples of the use of hydrostatic pressure as a tool to gain insight into the forces and energetics governing the productive folding or the misfolding and amyloid aggregation of proteions.     

A double point mutation of SOD1 targeting net charge promotes aggregation under destabilizing conditions: Correlation of charge distribution and ALS-provoking mutation

A progressive neurodegenerative illness such as amyotrophic lateral sclerosis (ALS) is characterized by the misfolding and aggregation of human CuZn superoxide dismutase (hSOD1) into amyloid aggregates. Thus, designing strategies for the choice of WT-SOD1 and double mutant (G12D/G138E) with an increased net negative charge can be a good idea to elucidate the pathological mechanism of SOD1 in ALS under some destabilizing conditions. Consequently, we show evidence that protein charge, together with other destabilizing conditions, plays an important role in ALS disease. To achieve this purpose, we use methods, such as spectroscopy and transmission electron microscopy (TEM) to monitor the formation of amyloid aggregation. The specific activity of WT-SOD1 was approximately 1.72 times higher than that of the double mutant. Under amyloidogenic circumstances, structural properties such as local, secondary, oligomeric, and fibrillar structures were explored. The double mutant's far-UV CD spectra displayed a broad minimum peak in the region 213 to 218 nm, suggesting the production of β-rich amyloid fibrils. FTIR spectra of the double mutant samples at different incubation times showed a low-frequency peak around 1630–1640 cm^-1, attributed to a parallel β-sheet. Moreover, CR-binding assay and TEM analysis revealed and confirmed that mutation with an increased repulsive charge promotes the formation of fibrous aggregates. Consequently, ALS mutations with a higher repulsive charge are the apparent exceptions that validate the rule. This findings revealed that the double mutant increases protein aggregation through a novel mechanism, likely involving destabilization of structure and a change in the net negative charge.     

Glycation Accelerates Fibrillization of the Amyloidogenic W7FW14F Apomyoglobin

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.     

GPI anchoring facilitates propagation and spread of misfolded Sup35 aggregates in mammalian cells

Prion diseases differ from other amyloid‐associated protein misfolding diseases (e.g. Alzheimer's) because they are naturally transmitted between individuals and involve spread of protein aggregation between tissues. Factors underlying these features of prion diseases are poorly understood. Of all protein misfolding disorders, only prion diseases involve the misfolding of a glycosylphosphatidylinositol (GPI)‐anchored protein. To test whether GPI anchoring can modulate the propagation and spread of protein aggregates, a GPI‐anchored version of the amyloidogenic yeast protein Sup35NM (Sup35^GPI) was expressed in neuronal cells. Treatment of cells with Sup35NM fibrils induced the GPI anchor‐dependent formation of self‐propagating, detergent‐insoluble, protease‐resistant, prion‐like aggregates of Sup35^GPI. Live‐cell imaging showed intercellular spread of Sup35^GPI aggregation to involve contact between aggregate‐positive and aggregate‐negative cells and transfer of Sup35^GPI from aggregate‐positive cells. These data demonstrate GPI anchoring facilitates the propagation and spread of protein aggregation and thus may enhance the transmissibility and pathogenesis of prion diseases relative to other protein misfolding diseases.     

Intrinsically Semi-disordered State and Its Role in Induced Folding and Protein Aggregation

Intrinsically disordered proteins (IDPs) refer to those proteins without fixed three-dimensional structures under physiological conditions. Although experiments suggest that the conformations of IDPs can vary from random coils, semi-compact globules, to compact globules with different contents of secondary structures, computational efforts to separate IDPs into different states are not yet successful. Recently, we developed a neural-network-based disorder prediction technique SPINE-D that was ranked as one of the top performing techniques for disorder prediction in the biannual meeting of critical assessment of structure prediction techniques (CASP 9, 2010). Here, we further analyze the results from SPINE-D prediction by defining a semi-disordered state that has about 50 % predicted probability to be disordered or ordered. This semi-disordered state is partially collapsed with intermediate levels of predicted solvent accessibility and secondary structure content. The relative difference in compositions between semi-disordered and fully disordered regions is highly correlated with amyloid aggregation propensity (a correlation coefficient of 0.86 if excluding four charged residues and proline, 0.73 if not). In addition, we observed that some semi-disordered regions participate in induced folding, and others play key roles in protein aggregation. More specifically, a semi-disordered region is amyloidogenic in fully unstructured proteins (such as alpha-synuclein and Sup35) but prone to local unfolding that exposes the hydrophobic core to aggregation in structured globular proteins (such as SOD1 and lysozyme). A transition from full disorder to semi-disorder at about 30–40 Qs is observed in the poly-Q (poly-glutamine) tract of huntingtin. The accuracy of using semi-disorder to predict binding-induced folding and aggregation is compared with several methods trained for the purpose. These results indicate the usefulness of three-state classification (order, semi-disorder, and full-disorder) in distinguishing nonfolding from induced-folding and aggregation-resistant from aggregation-prone IDPs and in locating weakly stable, locally unfolding, and potentially aggregation regions in structured proteins. A comparison with five representative disorder-prediction methods showed that SPINE-D is the only method with a clear separation of semi-disorder from ordered and fully disordered states.     

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.     

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/α‐synuclein complex by Hip

The ATP‐dependent protein chaperone heat‐shock protein 70 (Hsp70) displays broad anti‐aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α‐synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation‐prone polypeptides. We show a nucleotide‐dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co‐aggregation phenomenon can be prevented in vitro by the co‐chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.     

Covalently attached fatty acyl chains alter the aggregation behavior of an amyloidogenic peptide derived from human β2‐microglobulin

Aggregation of a polypeptide chain into highly ordered amyloid aggregates is a complex process. Various factors, both extrinsic and intrinsic to the polypeptide chain, have been shown to perturb this process, leading to a drastic change in the amyloidogenic behavior, which is reflected in the polymorphism of amyloid aggregates at various levels of self‐assembly. In this paper, we have investigated the ability of covalently linked long‐chain fatty acids in modulating the self‐assembly of an aromatic amino acid‐rich highly amyloidogenic sequence derived from the amino acid region 59–71 of human β₂‐microglobulin by thioflavin T (ThT) fluorescence microscopy, circular dichroism, and fluorescence spectroscopy. Our results indicate that under identical conditions of dissolution and concentration, each peptide enhances the fluorescence of ThT. However, the aggregates are morphologically distinct. For the same peptide, the aggregate morphologies are dependent on peptide concentration. Further, an optimum concentration, which varies with solution ionic strength, is required for the formation of fibrillar aggregates. We show that covalent modification of this amyloidogenic sequence, with long‐chain fatty acids, affects the way the higher order amyloid structures assemble from the cross‐β units, in fatty acyl chain‐dependent and position‐dependent manner. Our data indicate that noncovalent interactions leading to amyloid fibril formation can be modulated by the hydrophobicity of covalently attached long‐chain fatty acids resulting in self‐assembly of the peptide chain to form nonfibrillar aggregates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

(−)-Epigallocatechin-3-Gallate (EGCG) Maintains κ-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-β, α-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 RCMκ-CN (reduced and carboxymethylated κ-casein) and thereby protect pheochromocytoma-12 cells from RCMκ-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMκ-CN [the IC₅₀ for 50 μM RCMκ-CN is 13 ± 1 μM]. Biophysical studies reveal that EGCG prevents RCMκ-CN fibril formation by stabilising RCMκ-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 RCMκ-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent π-π stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated.     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.