Selective Inhibition of Aggregation and Toxicity of a Tau‐Derived Peptide using Its Glycosylated Analogues

Protein glycosylation is a ubiquitous post‐translational modification that regulates the folding and function of many proteins. Misfolding of protein monomers and their toxic aggregation are the hallmark of many prevalent diseases. Thus, understanding the role of glycans in protein aggregation is highly important and could contribute both to unraveling the pathology of protein misfolding diseases as well as providing a means for modifying their course for therapeutic purposes. Using β‐O‐linked glycosylated variants of the highly studied Tau‐derived hexapeptide motif VQIVYK, which served as a simplified amyloid model, we demonstrate that amyloid formation and toxicity can be strongly attenuated by a glycan unit, depending on the nature of the glycan itself. Importantly, we show for the first time that not only do glycans hinder self‐aggregation, but the glycosylated peptides are capable of inhibiting aggregation of the non‐modified corresponding amyloid scaffold.     

Multifunctional 8‐Hydroxyquinoline‐Appended Cyclodextrins as New Inhibitors of Metal‐Induced Protein Aggregation

Mounting evidence suggests a pivotal role of metal imbalances in protein misfolding and amyloid diseases. As such, metal ions represent a promising therapeutic target. In this context, the synthesis of chelators that also contain complementary functionalities to combat the multifactorial nature of neurodegenerative diseases is a highly topical issue. We report two new 8‐hydroxyquinoline‐appended cyclodextrins and highlight their multifunctional properties, including their Cu^II and Zn^II binding abilities, and capacity to act as antioxidants and metal‐induced antiaggregants. In particular, the latter property has been applied in the development of an effective assay that exploits the formation of amyloid fibrils when β‐lactoglobulin A is heated in the presence of metal ions.     

Amyloid‐β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect

Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β‐rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer′s disease amyloid‐β peptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP‐amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases.     

Reactive Amphiphilic Conjugated Polymers for Inhibiting Amyloid β Assembly

Protein misfolding and aberrant aggregations are associated with multiple prevalent and intractable diseases. Inhibition of amyloid assembly is a promising strategy for the treatment of amyloidosis. Reported here is the design and synthesis of a reactive conjugated polymer, a poly(p‐phenylene vinylene) derivative, functionalized with p‐nitrophenyl esters (PPV‐NP) and it inhibits the assembly of amyloid proteins, degrades preformed fibrils, and reduces the cytotoxicity of amyloid aggregations in living cells. PPV‐NP is attached to the proteins through hydrophobic interactions and irreversible covalent linkage. PPV‐NP also exhibited the capacity to eliminate Aβ plaques in brain slices in ex vivo assays. This work represents an innovative attempt to inhibit protein pathogenic aggregates, and may offer insights into the development of therapeutic strategies for amyloidosis.     

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In Vivo

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post‐translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.     

Peptide‐Based Molecular Strategies To Interfere with Protein Misfolding,Aggregation, and Cell Degeneration

Protein misfolding into amyloid fibrils is linked to more than 40 as yet incurable cell‐ and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. So far, however, only one of the numerous anti‐amyloid molecules has reached patients. This Minireview gives an overview of molecular strategies and peptide chemistry “tools” to design, develop, and discover peptide‐based molecules as anti‐amyloid drug candidates. We focus on two major inhibitor rational design strategies: 1) the oldest and most common strategy, based on molecular recognition elements of amyloid self‐assembly, and 2) a more recent approach, based on cross‐amyloid interactions. We discuss why peptide‐based amyloid inhibitors, in particular their advanced generations, can be promising leads or candidates for anti‐amyloid drugs as well as valuable tools for deciphering amyloid‐mediated cell damage and its link to disease pathogenesis.     

Confined Water in Amyloid‐Competent Oligomers of the Prion Protein

Conformational switching of the prion protein into the abnormal form involves the formation of (obligatory) molten‐oligomers that mature into ordered amyloid fibrils. The role of water in directing the course of amyloid formation remains poorly understood. Here, we show that the mobility of the water molecules within the on‐pathway oligomers is highly retarded. The water relaxation time within the oligomers was estimated to be ≈1 ns which is about three orders of magnitude slower than the bulk water and resembles the characteristics of (trapped) nano‐confined water. We propose that the coalescence of these obligatory oligomers containing trapped water is entropically favored because of the release of ordered water molecules in the bulk milieu and results in the sequestration of favorable inter‐chain amyloid contacts via nucleated conformational conversion. The dynamic role of water in protein aggregation will have much broader implications in a variety of protein misfolding diseases.     

Size Effect of Graphene Oxide on Modulating Amyloid Peptide Assembly

Protein misfolding and abnormal assembly could lead to aggregates such as oligomer, proto‐fibril, mature fibril, and senior amyloid plaques, which are associated with the pathogenesis of many amyloid diseases. These irreversible amyloid aggregates typically form in vivo and researchers have been endeavoring to find new modulators to invert the aggregation propensity in vitro, which could increase understanding in the mechanism of the aggregation of amyloid protein and pave the way to potential clinical treatment. Graphene oxide (GO) was shown to be a good modulator, which could strongly control the amyloidosis of Aβ (33–42). In particular, quartz crystal microbalance (QCM), circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) measurements revealed the size‐dependent manner of GO on modulating the assembly of amyloid peptides, which could be a possible way to regulate the self‐assembled nanostructure of amyloid peptide in a predictable manner.     

Cyclodextrins as Protective Agents of Protein Aggregation: An Overview

Cyclodextrins are extensively used in different fields (e.g., catalysis, chromatography, pharma, supramolecular chemistry, bioorganic chemistry, and bioinorganic chemistry), and their applications have been widely reviewed. Their main application in the field of pharmaceutical is as a drug carrier. This review overviews, for the first time, the use of cyclodextrins and their derivatives as antiaggregant agents in a number of proteins (e.g., amyloid‐β, insulin, recombinant human growth hormone, prion protein, transthyretin, and α‐synuclein) and some multimeric enzymes. There are many diseases that are correlated to protein misfolding and amyloid formation processes affecting numerous organs and tissues. There are over 30 different amyloid proteins and a number of corresponding diseases. Alzheimer's disease is the most common neurodegenerative disease. Treatment of these diseases is still a goal to reach, and many molecules are studied in this perspective. Cyclodextrins have also been studied, and they show great potential; as such, further studies could be very promising. This review aims to be a stimulus for the design of new cyclodextrin derivatives to obtain multifunctional systems with antiaggregant activity.     

Tyrosine side chains as an electrochemical probe of stacked beta-sheet protein conformations

The in vivo formation of beta-pleated protein aggregates underlies a number of fatal neurodegenerative disorders, such as Alzheimer disease. Since molecular mechanisms of protein misfolding and aggregation remain poorly understood, this has been calling for many diverse biophysical tools capable of addressing different dynamic and conformational aspects of the phenomenon. The two model polypeptides used in this study are poly(l-tyrosine) and insulin. According to FT-IR spectra, poly(l-tyrosine) produced two distinct types of films with dominant either disordered or antiparallel beta-sheet conformations depending on carrier solvent used for film's deposition. Electrochemical analysis of both the types of polypeptide films by the means of cyclic voltammetry and differential pulse voltammetry proved that different electrochemical behaviour of the tyrosine residues is determined by the conformation of polypeptide chains. We have rationalized this difference in terms of varying electrochemical accessibility of Tyr residues in each structure. We have also carried out spectral and electrochemical characterization of insulin beta-sheet-rich amyloid fibrils. It appears that the detectable electrochemical response of the protein stems from the presence of four tyrosine residues per insulin monomer. Since hydrophobic residues, among them tyrosines play an important role in the formation of protein amyloid fibrils, but, on a molecular level, may be also critical in explaining neurotoxic properties of aggregates, their electrochemical properties may become a very valuable complementary tool in biophysical studies on protein misfolding.     

Structural Effects of Multiple Pathogenic Mutations Suggest a Model for the Initiation of Misfolding of the Prion Protein

A molecular understanding of the prion diseases requires delineation of the origin of misfolding of the prion protein (PrP). An understanding of how different disease‐linked mutations affect the structure and dynamics of native monomeric PrP can provide a clue about how misfolding commences. In this study, hydrogen–deuterium exchange mass spectrometry was used to show that several disease‐linked mutant variants, which are thermodynamically destabilized, share a common structural perturbation in their native states: helix 1 is destabilized to an extent that correlates well with the destabilization of the native protein. The mutant variants misfold and form oligomers faster than does the wild‐type protein, at rates that increase exponentially with the extent to which helix 1 is destabilized in the native protein. It appears, therefore, that the loss of helix 1 structure marks the beginning of PrP misfolding and oligomerization.     

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In Vivo

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.     

Protein-Protein interactions leading to aggregation: Perspectives on mechanism, significance and control

Protein aggregates, whether amorphous or structured (amyloid), have attracted much attention in recent years and despite extensive efforts, the mechanism of their formation is poorly understood. While “natural” aggregation (polymerization) of monomers could improve the biological function of some proteins, it is usually the darker side of this phenomenon which is discussed in many studies: deleterious aggregation that could lead to loss of biological activity under in vitro conditions or cause misfolding diseases. In this review, protein aggregation has been overviewed, starting from some general concepts involved in its formation, followed by mentioning studies aimed at elucidation of its kinetics and mechanism, or characterization of intermediates that would be aggregation-prone, and finally, reporting some of the studies related to the design of methods that would control the process. Similarly, amyloid aggregates have been defined, and current methods used in their characterization have been briefly described, with an emphasis on in silico studies. Finally, identification and design of such molecules which may be effective in control of this process is discussed.     

Maintenance of Amyloid β Peptide Homeostasis by Artificial Chaperones Based on Mixed‐Shell Polymeric Micelles

The disruption of Aβ homeostasis, which results in the accumulation of neurotoxic amyloids, is the fundamental cause of Alzheimer’s disease (AD). Molecular chaperones play a critical role in controlling undesired protein misfolding and maintaining intricate proteostasis in vivo. Inspired by a natural molecular chaperone, an artificial chaperone consisting of mixed‐shell polymeric micelles (MSPMs) has been devised with tunable surface properties, serving as a suppressor of AD. Taking advantage of biocompatibility, selectivity toward aberrant proteins, and long blood circulation, these MSPM‐based chaperones can maintain Aβ homeostasis by a combination of inhibiting Aβ fibrillation and facilitating Aβ aggregate clearance and simultaneously reducing Aβ‐mediated neurotoxicity. The balance of hydrophilic/hydrophobic moieties on the surface of MSPMs is important for their enhanced therapeutic effect.     

The Polyphenol EGCG Inhibits Amyloid Formation Less Efficiently at Phospholipid Interfaces than in Bulk Solution

Age-related diseases, like Alzheimer's disease and type 2 diabetes mellitus, are characterized by protein misfolding and the subsequent pathological deposition of fibrillized protein, also called amyloid. Several classes of amyloid-inhibitors have recently been tested, traditionally under bulk conditions. However, it has become apparent that amyloid fibrils and oligomers assemble and exert their cytotoxic effect at cellular membranes, rather than in bulk solution. Knowledge is therefore required of inhibitor activity specifically at the phospholipid membrane interface. Here we show, using surface-specific sum-frequency generation (SFG) spectroscopy and atomic force microscopy (AFM), that the commonly used (-)-epigallocatechin gallate (EGCG) is a much less efficient amyloid inhibitor at a phospholipid interface than in bulk solution. Moreover, EGCG is not able to disaggregate existing amyloid fibrils at a phospholipid interface, in contrast to its behavior in bulk. Our results show that interfaces significantly affect the efficiency of inhibition by EGCG inhibitors and should therefore be considered during the design and testing of amyloid inhibitors.     

Cyclic Peptides as Inhibitors of Amyloid Fibrillation

Many neurodegenerative diseases, like Parkinson’s, Alzheimer’s, or Huntington’s disease, occur as a result of amyloid protein fibril formation and cell death induced by this process. Cyclic peptides (CPs) and their derivatives form a new class of powerful inhibitors that prevent amyloid fibrillation and decrease the cytotoxicity of aggregates. The strategies for designing CPs are described, with respect to their amino acid sequence and/or conformational similarity to amyloid fibrils. The implications of CPs for the study and possible treatment of amyloid‐related diseases are discussed.     

How Single Site Mutations Can Help Understanding Structure Formation of Amyloid β1−40

Amyloid fibrils represent the structural endpoint on the energetic (mis)folding landscape of very many proteins. Physiologically, amyloid fibrils are observed as a characteristic hallmark in misfolding diseases often associated with degenerative and neurodegenerative disorders. In the beginning of the scientific discussion, the focus is laid on the fibrillar state, but over the time it becomes increasingly clear that low molecular weight and transient aggregates are of crucial importance for pathological mechanisms. Structural studies find different intra- and intermolecular contacts for the most well-studied peptide amyloid β (Aβ) depending on the stage of fibrillation. In particular, the contact between residues phenylalanine 19 (F19) and leucine 34 (L34) seems to be highly conserved, suggesting that it must be of particular significance for Aβ misfolding and possibly the pathological properties of the peptide. This review aims to highlight the rational and the usefulness of point mutations in Aβ peptides and their impact on the critical interstrand contact F19−L34 depending on the stage of fibrillation. While the amyloid structure of Aβ is very robust against quite a few modifications, the toxicity of mutated Aβ molecules highly depends on the F19−L34 contact.     

Characterizing amyloid‐beta protein misfolding from molecular dynamics simulations with explicit water

Extracellular deposition of amyloid‐beta (Aβ) protein, a fragment of membrane glycoprotein called β‐amyloid precursor transmembrane protein (βAPP), is the major characteristic for the Alzheimer's disease (AD). However, the structural and mechanistic information of forming Aβ protein aggregates in a lag phase in cell exterior has been still limited. Here, we have performed multiple all‐atom molecular dynamics simulations for physiological 42‐residue amyloid‐beta protein (Aβ42) in explicit water to characterize most plausible aggregation‐prone structure (APS) for the monomer and the very early conformational transitions for Aβ42 protein misfolding process in a lag phase. Monitoring the early sequential conformational transitions of Aβ42 misfolding in water, the APS for Aβ42 monomer is characterized by the observed correlation between the nonlocal backbone H‐bond formation and the hydrophobic side‐chain exposure. Characteristics on the nature of the APS of Aβ42 allow us to provide new insight into the higher aggregation propensity of Aβ42 over Aβ40, which is in agreement with the experiments. On the basis of the structural features of APS, we propose a plausible aggregation mechanism from APS of Aβ42 to form fibril. The structural and mechanistic observations based on these simulations agree with the recent NMR experiments and provide the driving force and structural origin for the Aβ42 aggregation process to cause AD. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti‐amyloid and anti‐oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high‐throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation.