Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild‐type rats

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation‐prone proteins such as αSN, which is central in the development of Parkinson''s disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT‐positive aggregates that contain higher‐order β‐sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross‐seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.     

Inhibition of amyloid fibrillization of hen egg-white lysozymes by rifampicin and p-benzoquinone

It has been reported that more than 20 different human proteins can fold abnormally, resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current research, utilizing hen egg-white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p-benzoquinone followed a concentration-dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti-aggregating and a disaggregating activity, rifampicin, in comparison with p-benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p-benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.     

In silico prediction of novel residues involved in amyloid primary nucleation of human I56T and D67H lysozyme

Background

Amyloidogenic proteins are most often associated with neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, but there are more than two dozen human proteins known to form amyloid fibrils associated with disease. Lysozyme is an antimicrobial protein that is used as a general model to study amyloid fibril formation. Studies aimed at elucidating the process of amyloid formation of lysozyme tend to focus on partial unfolding of the native state due to the relative instability of mutant amyloidogenic variants. While this is well supported, the data presented here suggest the native structure of the variants may also play a role in primary nucleation.

Results

Three-dimensional structural analysis identified lysozyme residues 21, 62, 104, and 122 as displaced in both amyloidogenic variants compared to wild type lysozyme. Residue interaction network (RIN) analysis found greater clustering of residues 112–117 in amyloidogenic variants of lysozyme compared to wild type. An analysis of the most energetically favored predicted dimers and trimers provided further evidence for a role for residues 21, 62, 104, 122, and 112–117 in amyloid formation.

Conclusions

This study used lysozyme as a model to demonstrate the utility of combining 3D structural analysis with RIN analysis for studying the general process of amyloidogenesis. Results indicated that binding of two or more amyloidogenic lysozyme mutants may be involved in amyloid nucleation by placing key residues (21, 62, 104, 122, and 112–117) in proximity before partial unfolding occurs. Identifying residues in the native state that may be involved in amyloid formation could provide novel drug targets to prevent a range of amyloidoses.

     

Hacking the Code of Amyloid Formation: The Amyloid Stretch Hypothesis

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by nonamyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our conclusions together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.     

Hacking the Code of Amyloid Formation

Many research efforts in the last years have been directed towards understanding the factors determining protein misfolding and amyloid formation. Protein stability and amino acid composition have been identified as the two major factors in vitro. The research of our group has been focused on understanding the relationship between amino acid sequence and amyloid formation. Our approach has been the design of simple model systems that reproduce the biophysical properties of natural amyloids. An amyloid sequence pattern was extracted that can be used to detect amyloidogenic hexapeptide stretches in proteins. We have added evidence supporting that these amyloidogenic stretches can trigger amyloid formation by non-amyloidogenic proteins. Some experimental results in other amyloid proteins will be analyzed under the conclusions obtained in these studies. Our studies together with evidences from other groups suggest that amyloid formation is the result of the interplay between a decrease of protein stability, and the presence of highly amyloidogenic regions in proteins. As many of these results have been obtained in vitro, the challenge for the next years will be to demonstrate their validity in in vivo systems.     

Micelle formation by a fragment of human islet amyloid polypeptide

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid plaques found in the pancreatic islets of persons with type 2 diabetes mellitus. HIAPP belongs to the group of amyloidogenic proteins, characterized by their aggregation and deposition as fibrillar amyloid in various body tissues. The aggregation of amyloidogenic proteins is thought to occur via a common pathway, but currently no unifying kinetic model exists. In previous work, we presented a model of amyloid fibril formation formulated from our observations of the aggregation of an amyloidogenic fragment of hIAPP, amino acids 20-29. Our model is based on nucleation-dependent aggregation, modified by the formation of off-pathway hIAPP micelles. In the present study we confirm the presence of peptide micelles, and experimentally determine the critical micelle concentration in solutions of hIAPP fragments using three different techniques: conductivity, pH, and fluorescence. All three techniques yield a critical micelle concentration of 3-3.5 micro M peptide. Furthermore, based on changes in the fluorescence intensity of a labeled peptide fragment as well as a decrease in solution pH as a result of deprotonation of the amino terminus, we conclude that the amino terminus of the fragment undergoes a significant change of environment upon micellization.     

Effects of p-benzoquinone and melatonin on amyloid fibrillogenesis of hen egg-white lysozyme

More than 20 different human proteins can fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained far from complete. In this work, utilizing hen egg-white lysozymes as a model system, two objectives were pursued: (1) to search for suitable conditions for producing amyloid fibrils and (2) to investigate inhibitory activities of two potential molecules against lysozyme fibril formation. Via numerous spectroscopic analyses and electron microscopy, our results showed that the formation of lysozyme amyloid fibrils at pH 2.0 was considerably increased by the addition of salt. Moreover, the inhibition of lysozyme amyloid formation by either p-benzoquinone or melatonin followed a concentration-dependent fashion. Furthermore, p-benzoquinone, in comparison with melatonin, served as a more effective inhibitor against amyloid fibril formation of lysozyme. We believe that a better understanding of how hen egg-white lysozymes aggregate will not only aid in deciphering the molecular mechanism of amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.     

Small molecule inhibitors of lysozyme amyloid aggregation

Protein amyloid aggregation is associated with a number of important human pathologies, but the precise mechanisms underlying the toxicity of amyloid aggregates are still incompletely understood. In this context, drugs capable of blocking or interfering with the aggregation of amyloidogenic proteins should be considered in strategies aimed at the development of novel therapeutic agents. Human lysozyme variants have been shown to form massive amyloid deposits in the livers and kidneys of individuals affected by hereditary systemic amyloidosis. Currently, there are no clinical treatments available to prevent or reverse formation of such amyloid deposits. We have recently described a number of di- and trisubstituted aromatic compounds that block the formation of soluble oligomers and amyloid fibrils of the β-amyloid peptide (Aβ) and protect hippocampal neurons in culture from Aβ-induced toxicity. Here, we show that some of those compounds inhibit the formation and disrupt preformed amyloid fibrils from both human and hen egg white lysozyme. These results suggest that these small molecule compounds may serve as prototypes for the development of drugs for the prevention or treatment of different types of amyloidoses.     

Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme

To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.     

Understanding and controlling amyloid aggregation with chirality

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.     

Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants

Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism.     

Generic inhibition of amyloidogenic proteins by two naphthoquinone-tryptophan hybrid molecules

Amyloid formation is associated with several human diseases including Alzheimer's disease (AD), Parkinson's disease, Type 2 Diabetes, and so forth, no disease modifying therapeutics are available for them. Because of the structural similarities between the amyloid species characterizing these diseases, (despite the lack of amino acid homology) it is believed that there might be a common mechanism of toxicity for these conditions. Thus, inhibition of amyloid formation could be a promising disease-modifying therapeutic strategy for them. Aromatic residues have been identified as crucial in formation and stabilization of amyloid structures. This finding was corroborated by high-resolution structural studies, theoretical analysis, and molecular dynamics simulations. Amongst the aromatic entities, tryptophan was found to possess the most amyloidogenic potential. We therefore postulate that targeting aromatic recognition interfaces by tryptophan could be a useful approach for inhibiting the formation of amyloids. Quinones are known as inhibitors of cellular metabolic pathways, to have anti- cancer, anti-viral and anti-bacterial properties and were shown to inhibit aggregation of several amyloidogenic proteins in vitro. We have previously described two quinone-tryptophan hybrids which are capable of inhibiting amyloid-beta, the protein associated with AD pathology, both in vitro and in vivo. Here we tested their generic properties and their ability to inhibit other amyloidogenic proteins including α-synuclein, islet amyloid polypeptide, lysozyme, calcitonin, and insulin. Both compounds showed efficient inhibition of all five proteins examined both by ThT fluorescence analysis and by electron microscope imaging. If verified in vivo, these small molecules could serve as leads for developing generic anti-amyloid drugs.     

Amino acid sequence determinants and molecular chaperones in amyloid fibril formation

Amyloid consists of cross-β-sheet fibrils and is associated with about 25 human diseases, including several neurodegenerative diseases, systemic and localized amyloidoses and type II diabetes mellitus. Amyloid-forming proteins differ in structures and sequences, and it is to a large extent unknown what makes them convert from their native conformations into amyloid. In this review, current understanding of amino acid sequence determinants and the effects of molecular chaperones on amyloid formation are discussed. Studies of the nonpolar, transmembrane surfactant protein C (SP-C) have revealed amino acid sequence features that determine its amyloid fibril formation, features that are also found in the amyloid β-peptide in Alzheimer’s disease and the prion protein. Moreover, a proprotein chaperone domain (CTC_Brichos) that prevents amyloid-like aggregation during proSP-C biosynthesis can prevent fibril formation also of other amyloidogenic proteins.     

The extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment.     

Reduced global cooperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining alpha-helices that form the core of the alpha-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, and the associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils.     

Inhibition of amyloid fibril formation and disassembly of pre-formed fibrils by natural polyphenol rottlerin

Natural polyphenols, curcumin, rottlerin and EGCG were selected for initial computational modeling of protein-ligand interaction patterns. The docking calculations demonstrated that these polyphenols can easily adjust their conformational shape to fit well into the binding sites of amyloidogenic proteins. The experimental part of the study focused on the effect of rottlerin on fibrillation of three distinct amyloidogenic proteins, namely insulin, lysozyme and Aβ_1–40 peptide. Different experimental protocols such as fluorescence spectroscopy, circular dichroism and atomic force microscopy, demonstrated that amyloid fibril formation of any of the three proteins is inhibited by low micromolar rottlerin concentrations. Most likely, the inhibition of amyloid formation proceeded via interaction of rottlerin with amyloidogenic regions of the studied proteins. Moreover, rottlerin was also effective in pre-formed fibrils disassembly, suggesting that interactions of rottlerin with fibrils were capable to interrupt the fibril-stabilizing bonds of β-sheets. The apparent IC₅₀ and DC₅₀ values were calculated in the range of 1.3–36.4 μM and 15.6–25.8 μM, respectively. The strongest inhibiting/disassembling effect of rottlerin was observed on Aβ_1–40 peptide. The cytotoxicity assay performed on the Neuro 2a cells indicated time-dependent cell morphology changes but rottlerin affected the cell viability only at concentration above 50 μM. The results of this study suggest that chemical modifications on rottlerin could be tested in the future as a promising strategy for the modulation of amyloidogenic proteins aggregation.     

Impact of membrane curvature on amyloid aggregation

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs.We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-β peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic β cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed.     

Amyloidogenic sequences in native protein structures

Numerous short peptides have been shown to form β‐sheet amyloid aggregates in vitro. Proteins that contain such sequences are likely to be problematic for a cell, due to their potential to aggregate into toxic structures. We investigated the structures of 30 proteins containing 45 sequences known to form amyloid, to see how the proteins cope with the presence of these potentially toxic sequences, studying secondary structure, hydrogen‐bonding, solvent accessible surface area and hydrophobicity. We identified two mechanisms by which proteins avoid aggregation: Firstly, amyloidogenic sequences are often found within helices, despite their inherent preference to form β structure. Helices may offer a selective advantage, since in order to form amyloid the sequence will presumably have to first unfold and then refold into a β structure. Secondly, amyloidogenic sequences that are found in β structure are usually buried within the protein. Surface exposed amyloidogenic sequences are not tolerated in strands, presumably because they lead to protein aggregation via assembly of the amyloidogenic regions. The use of α‐helices, where amyloidogenic sequences are forced into helix, despite their intrinsic preference for β structure, is thus a widespread mechanism to avoid protein aggregation.     

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.