A molecular dynamics study of the interaction of D-peptide amyloid inhibitors with their target sequence reveals a potential inhibitory pharmacophore conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.     

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.     

Incremental truncation as a strategy in the engineering of novel biocatalysts

The application and success of combinatorial approaches to protein engineering problems have increased dramatically. However, current directed evolution strategies lack a combinatorial methodology for creating libraries of hybrid enzymes which lack high homology or for creating libraries of highly homologous genes with fusions at regions of non-identity. To create such hybrid enzyme libraries, we have developed a series of combinatorial approaches that utilize the incremental truncation of genes, gene fragments or gene libraries. For incremental truncation, Exonuclease III is used to create a library of all possible single base-pair deletions of a given piece of DNA. Incremental truncation libraries (ITLs) have applications in protein engineering as well as protein folding, enzyme evolution, and the chemical synthesis of proteins. In addition, we are developing a methodology of DNA shuffling which is independent of DNA sequence homology.     

Designing gene libraries from protein profiles for combinatorial protein experiments

Protein combinatorial libraries provide new ways to probe the determinants of folding and to discover novel proteins. Such libraries are often constructed by expressing an ensemble of partially random gene sequences. Given the intractably large number of possible sequences, some limitation on diversity must be imposed. A non-uniform distribution of nucleotides can be used to reduce the number of possible sequences and encode peptide sequences having a predetermined set of amino acid probabilities at each residue position, i.e., the amino acid sequence profile. Such profiles can be determined by inspection, multiple sequence alignment or physically-based computational methods. Here we present a computational method that takes as input a desired sequence profile and calculates the individual nucleotide probabilities among partially random genes. The calculated gene library can be readily used in the context of standard DNA synthesis to generate a protein library with essentially the desired profile. The fidelity between the desired profile and the calculated one coded by these partially random genes is quantitatively evaluated using the linear correlation coefficient and a relative entropy, each of which provides a measure of profile agreement at each position of the sequence. On average, this method of identifying such codon frequencies performs as well or better than other methods with regard to fidelity to the original profile. Importantly, the method presented here provides much better yields of complete sequences that do not contain stop codons, a feature that is particularly important when all or large fractions of a gene are subject to combinatorial mutation.     

Chemical strategies for controlling protein folding and elucidating the molecular mechanisms of amyloid formation and toxicity

It has been more than a century since the first evidence linking the process of amyloid formation to the pathogenesis of Alzheimer's disease. During the last three decades in particular, increasing evidence from various sources (pathology, genetics, cell culture studies, biochemistry, and biophysics) continues to point to a central role for the pathogenesis of several incurable neurodegenerative and systemic diseases. This is in part driven by our improved understanding of the molecular mechanisms of protein misfolding and aggregation and the structural properties of the different aggregates in the amyloid pathway and the emergence of new tools and experimental approaches that permit better characterization of amyloid formation in vivo. Despite these advances, detailed mechanistic understanding of protein aggregation and amyloid formation in vitro and in vivo presents several challenges that remain to be addressed and several fundamental questions about the molecular and structural determinants of amyloid formation and toxicity and the mechanisms of amyloid-induced toxicity remain unanswered. To address this knowledge gap and technical challenges, there is a critical need for developing novel tools and experimental approaches that will not only permit the detection and monitoring of molecular events that underlie this process but also allow for the manipulation of these events in a spatial and temporal fashion both in and out of the cell. This review is primarily dedicated in highlighting recent results that illustrate how advances in chemistry and chemical biology have been and can be used to address some of the questions and technical challenges mentioned above. We believe that combining recent advances in the development of new fluorescent probes, imaging tools that enabled the visualization and tracking of molecular events with advances in organic synthesis, and novel approaches for protein synthesis and engineering provide unique opportunities to gain a molecular-level understanding of the process of amyloid formation. We hope that this review will stimulate further research in this area and catalyze increased collaboration at the interface of chemistry and biology to decipher the mechanisms and roles of protein folding, misfolding, and aggregation in health and disease.     

New strategy for the generation of specific D-peptide amyloid inhibitors

The conversion of a soluble protein into β-sheet-rich oligomeric structures and further fiber formation are critical steps in the pathogenesis of the group of human diseases known as amyloidoses. Drugs that interfere with this process may thus be able to prevent and/or cure these diseases. Recent results have shown that short amino acid stretches can provide most of the driving force needed to trigger amyloid formation of a protein. These evidence suggest that compounds that specifically bind to peptides synthesized upon the sequence of such amyloidogenic protein stretches might also be able to inhibit amyloid formation of the corresponding full-length protein and, likely, amyloid-induced cytotoxicity as well. Here we present a general strategy to obtain d-peptides that specifically interact with protein amyloid stretches. The screening of a d-peptide combinatorial library for inhibitors of an amyloidogenic peptide designed de novo has allowed us to extract a set of empirical rules for the design of d-peptide inhibitors of any six-residue amyloidogenic stretch. d-peptides generated on these bases prevent amyloid formation and disassemble preformed fibrils of different amyloid hexapeptides identified in human amyloid proteins. In addition, they are also specific for their target sequence. The d-peptide designed here for the Alzheimer's Aβ_1-42 peptide not only inhibits and disassembles amyloid material but also reduces Aβ_1-42 amyloid-induced cytotoxicity in cell culture.     

Utilizing natural diversity to evolve protein function: applications towards thermostability

Protein evolution relies on designing a library of sequences that capture meaningful functional diversity in a limited number of protein variants. Several approaches take advantage of the sequence space already explored through natural selection by incorporating sequence diversity available from modern genomes (and their ancestors) when designing these libraries. The success of these approaches is, partly, owing to the fact that modern sequence diversity has already been subjected to evolutionary selective forces and thus the diversity has already been deemed 'fit to survive'. Five of these approaches will be discussed in this review to highlight how protein engineers can use evolutionary sequence history/diversity of homologous proteins in unique ways to design protein libraries.     

Low-Level Expression of a Folding-Incompetent Protein in Escherichia coli: Search for the Molecular Determinants of Protein Aggregation In Vivo

Aggregation of peptides and proteins into insoluble amyloid fibrils or related intracellular inclusions is the hallmark of many degenerative diseases, including Alzheimer's disease, Parkinson's disease, and various forms of amyloidosis. In spite of the considerable progress carried out in vitro in elucidating the molecular determinants of the conversion of purified and isolated proteins into amyloid fibrils, very little is known on factors governing this process in the complex environment of living organisms. Taking advantage of increasing evidence that bacterial inclusion bodies consist of amyloid-like aggregates, we have expressed in Escherichia coli both wild type and 21 single-point mutants of the N-terminal domain of the E. coli protein HypF. All variants were expressed as folding-incompetent units in a controlled manner, at low and comparable levels. Their solubilities were measured by quantifying the protein amount contained in the soluble and insoluble fractions by Western blot analysis. A significant negative correlation was found between the solubility of the variants in E. coli and their intrinsic propensity to form amyloid fibrils, predicted using an algorithm previously validated experimentally in vitro on a number of unfolded peptides and proteins, and considering hydrophobicity, β-sheet propensity, and charge as major sequence determinants of the aggregation process. These findings show that the physicochemical parameters previously recognized to govern amyloid formation by fully or partially unfolded proteins are largely applicable in vivo and pave the way for the molecular exploration of a process as complex as protein aggregation in living organisms.     

Peptide Detection of Fungal Functional Amyloids in Infected Tissue

Many fungal cell adhesion proteins form functional amyloid patches on the surface of adhering cells. The Candida albicans Agglutinin-like sequence (Als) adhesins are exemplars for this phenomenon, and have amyloid forming sequences that are conserved between family members. The Als5p amyloid sequence mediates amyloid fibril formation and is critical for cell adhesion and biofilm formation, and is also present in the related adhesins Als1p and Als3p. We have developed a fluorescent peptide probe containing the conserved Als amyloid-forming sequence. This peptide bound specifically to yeast expressing Als5p, but not to cells lacking the adhesin. The probe bound to both yeast and hyphal forms of C. albicans. Δals1/Δals3 single and double deletion strains exhibited reduced fluorescence, indicating that probe binding required expression of these proteins. Additionally, the Als peptide specifically stained fungal cells in abscesses in autopsy sections. Counterstaining with calcofluor white showed colocalization with the amyloid peptide. In addition, fungi in autopsy sections derived from the gastrointestinal tract showed colocalization of the amyloid-specific dye thioflavin T and the fluorescent peptide. Collectively, our data demonstrate that we can exploit amyloid sequence specificity for detection of functional amyloids in situ.     

A toy model for predicting the rate of amyloid formation from unfolded protein

We develop a toy model for predicting the rate of amyloid formation from an unfolded polypeptide. The model assumes irreversible amyloid growth, employs a collision encounter scheme and uses a Gaussian chain approximation to describe the polypeptide sequence. A principal feature of the model is its dependence on a number of key sequence residues whose correct placement, geometric arrangement and orientation in relation to their interacting partners define the success, or otherwise, of the amyloid formation reaction. Although not realistic at the molecular level, the model captures some essential features of the system and is therefore useful from a heuristic standpoint. For the case of amyloid formation from an unstructured state, the model suggests that the major determinants of the rate of fibril formation are the length of the sequence separating the critical amino acids promoting amyloid formation and the positional placement of the critical residues within the sequence. Our findings suggest also that the sequence distance between the key interacting amino acid residues may play a role in defining the maximum width of a fibril and that the addition of non-interacting segments of long structure-less polypeptide chain to an amyloidogenic peptide may act to inhibit fibril formation. We discuss these findings with reference to the placement of critical sequence residues within the polypeptide chain, the design of polypeptides with lower amyloid formation propensities and the development of aggregation inhibitors as potential therapeutics for protein depositional disorders.     

DNA sequence evidence for polymorphic forms of human serum amyloid A (SAA)

Serum amyloid A (SAA) is an acute-phase reactant and precursor to amyloid A protein, the major constituent of the fibril deposits of reactive amyloidosis. The factors determining whether the 104-amino acid SAA molecule is converted into the 76-amino acid amyloid A protein and deposited as fibrils are not known. As an initial step toward investigating the possibility that a particular primary structure of SAA is involved in amyloid formation, we have cloned and determined the nucleotide sequence of human SAA-specific cDNAs. The first clone, selected using an oligonucleotide probe, was shown to encode the signal peptide and amino-terminal region of SAA. The cDNA of this clone served as probe in the selection of two distinct, full-length SAA cDNAs, initially differentiated by the presence (pSAA21) or absence (pSAA82) of a PstI site in the coding sequence. The complete nucleotide sequence of pSAA82 cDNA was determined. Since there appear to be multiple human SAA alleles, it is conceivable that their differential expression is important to amyloid formation.     

Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis

The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-beta-peptide (Abeta42). Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Abeta aggregation, we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Abeta42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Abeta42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive. Cells expressing such fusions do not fluoresce. To isolate variants of Abeta42 with reduced tendencies to aggregate, we constructed and screened libraries of Abeta42-GFP fusions in which the sequence of Abeta42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Abeta42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Abeta42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Abeta42 aggregation. Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Abeta amyloidogenesis.     

From consensus sequence peptide to high affinity ligand, a "library scan" strategy

A wide variety of proteins have been shown to recognize and bind to specific amino acid sequences on other proteins. These sequences can be readily identified using combinatorial peptide libraries. However, peptides containing these preferred sequences ("consensus sequence peptides") typically display only modest affinities for the consensus sequence-binding site on the intact protein. In this report, we describe a parallel synthesis strategy that transforms consensus sequence peptides into high affinity ligands. The work described herein has focused on the Lck SH2 domain, which binds the consensus peptide acetyl-Tyr(P)-Glu-Glu-Ile-amide with a K(D) of 1.3 micrometer. We employed a strategy that creates a series of spatially focused libraries that challenge specific subsites on the target protein with a diverse array of functionality. The final lead compound identified in this study displayed a 3300-fold higher affinity for the Lck SH2 domain than the starting consensus sequence peptide.     

Amyloid aggregates of the prion peptide PrP106-126 are destabilised by oxidation and by the action of dendrimers

The prion protein (PrP) peptide 106-126 forms amyloid aggregates in vitro and this sequence is speculated to be involved in the formation of amyloid fibrils by the abnormally folded PrP protein (PrPSc) found in spongiform encephalopathies. It is shown here by incubation experiments in water using Thioflavin T (ThT) as a fluorescent probe for amyloid formation that changes in C-terminal charge, oxidation state and conformational stabilisation lead to large changes in amyloid forming behaviour (amyloidogenicity) of this peptide. Amyloid formation is favoured by a charged C-terminus and is strongly inhibited by oxidation. Furthermore, cationic dendrimers are shown to perturb peptide fibrillation in a process dependent on the nature of the charged groups on the dendrimer surface.     

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.     

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

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

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.     

Optimized protocols for the isolation of specific protein-binding peptides or peptoids from combinatorial libraries displayed on beads

Many methods have been published by which combinatorial libraries may be screened for compounds capable of manipulating the function(s) of a target protein. One of the simplest approaches is to identify compounds in a library that bind the protein of interest, since these binding events usually occur on functionally important surfaces of the protein. These protein-binding compounds could also be of utility as protein capture agents in the construction of protein-detecting microarrays or related analytical devices and as reagents for the affinity purification of proteins from complex mixtures. In this article, we provide optimized methods for screening libraries of molecules displayed on the beads on which they were synthesized. This is a particularly convenient format for library screening for laboratories with limited budgets and modest robotics capabilities.     

Characterization of the Heterogeneity and Specificity of Interpolypeptide Interactions in Amyloid Protofibrils by Measurement of Site-Specific Fluorescence Anisotropy Decay Kinetics

The aggregation of proteins often results in highly ordered fibrillar structures. While significant insights have been obtained on structural aspects of amyloid fibrils, little is known about the structures of protofibrils, which are presumed to be the precursors of fibrils. An understanding of the molecular mechanism of the formation of protofibrils and fibrils requires information on the landscape of interpeptide interactions. This work addresses this question by using, as a model protein, barstar, which forms protofibrils and fibrils at low (< 3) pH. Use was made of the heterogeneity of aggregate populations encountered during fibril formation. Population heterogeneity was scored through rotational dynamics monitored by time-resolved fluorescence anisotropy of an environment-sensitive fluorophore, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), attached to specific locations in the protein. Firstly, it was observed that barstar, when labeled at certain locations with 1,5-IAEDANS, did not form mixed protofibrils with the corresponding unlabeled protein. Labeled and unlabeled proteins formed protofibrils as separate populations. A two-population model of fluorescence anisotropy decay kinetics exhibiting a ‘dip-and-rise’ behavior was the main readout in arriving at this conclusion. Additional support for this conclusion came from the fluorescence lifetime of the probe 1,5-IAEDANS. Subsequently, the location of the fluorophore was moved along the length of the protein in nine mutant proteins, and the capability to form mixed fibrils was assessed. The results revealed that about two-thirds of the protein sequence at the C-terminal end of the protein was intimately involved in the formation of ordered protofibrils, probably forming the core, while the remaining one-third of the protein (i.e., the N-terminal region) remained largely noninteractive and flexible. This methodology can be used as a general strategy to identify regions of a given protein sequence involved in interprotein interactions in amyloid protofibrils.     

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."